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Abstract
The brain basis of conscious experience is one of the great unsolved mysteries of science. How can a material object became aware of the world around it and of its very own awareness? Many scholars think this question is unanswerable. New approaches to this age-old mind-body problem have recently been encouraged by the development of PET and MRI and the powerful tools of modern neuroscience. We are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology.
A parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking NREM and REM sleep. At the level of the brain it is possible to record PET and MRI and by using an animal model to analyse cellular and molecular mechanisms. The advantage of this approach, which I call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). The brain basis of activation (A) input-output gaining (I) and modulation (M) can now be described in relation to the changes in consciousness associated with them. The data can be used to create a three-dimensional model (AIM) which describes the brain-mind trajectory in its state space.
Neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. Such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. However, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. Modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. Interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. Thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation.
Supported by NIH grant NS19640.
SL2-1-1 Frontal cortex and higher-order motor control: Preferential use of multiple premotor and prefrontal areas dependent on behavioral context Jun Tanji Brain Science Research Center, Tamagawa University, Machida, Tokyo, Japan
In the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. Although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. In fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. For instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. The involvement of the prefrontal cortex in movement planning has also been inferred. Up to a certain extent, it is true that the use of different areas have some factors in common. However, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. In this lecture, I will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area.
SL3-1-1 Neuronal functions and molecular motor, kinesin superfamily proteins, KIFs: From transport of synaptic proteins and mRNAs, to brain wiring, neuronal survival and higher brain function Nobutaka Hirokawa Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Japan
The intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. To elucidate this mechanism we have identified and characterized kinesin superfamily proteins, KIFs, using molecular cell biology, molecular genetics, biophysics, and structural biology. KIFs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (KIF1A/KIF1Bbeta), NMDA type(KIF17) and AMPA type(KIF5s) glutamate receptors and mRNAs(KIF5s) such as CaMKII  mRNA and Arc mRNA with a large RNA-transporting protein complex containing at least 42 RNA related proteins such as hn RNP-U, Staufen, and FMRPs. KIF2A is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. KIF3 suppresses tumorigenesis by transporting Ncadherin/beta catenin containing vesicles from Golgi to plasma membrane in the neuroepithelium. KIF4 controls the activity-dependent survival of postmitotic neurons by regulating PARP-1 activity in brain development. Thus, KIFs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning.
Tsukahara Award 2-1 What we can learn from the functional recovery after brain injury Tadashi Isa National Institute for Physiological Sciences, Okazaki, Japan It is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. Such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. In this talk, I will present our recent progress in two lines of studies using non-human primate models related to this issue. The first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. After the virtually complete lesion, relatively independent finger movements can be recovered in 1-3 months. We have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the PET imaging and reversible local inactivation technique. In the second, I will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (V1). After complete ablation of V1, the monkeys recover performance of visually guided saccades toward the blind field in 2 months. Saccades to the blind field have low sensitivity and less accurate. However, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. I will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery.
Takuji Iwasato RIKEN Brain Science Center (BSI), Wako-shi, Saitama, Japan
In the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer IV neurons and thalamocortical afferent (TCA) terminals. While barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. We developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. We have focused on the NMDA-type glutamate receptor (NMDAR) and calcium-stimulated adenylyl cyclases, as NMDAR-and cAMP-cascades are central to various types of neuronal plasticity, both in adulthood and during development. Series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic TCA terminals compared to those at postsynaptic cortical neurons.
Research funds: PRESTO (JST), KAKENHI 17023055, KAKENHI 15029261 SY1-1-01-2 Distinct roles of two 7-pass transmembrane cadherins in neurite growth control Tadashi Uemura 1,6 , Yasuyuki Shima 1,6 , Keisuke Sehara 2 , Manabu Nakayama 3 , Shinya Kawaguchi 4 , Mikio Hoshino 5 , Yoichi Nabeshima 5 , Tomoo Hirano 4,6 1 Graduate School of Biostudies, Kyoto University, Kyoto, Japan; 2 School of Science, Japan; 3 Kazusa DNA Research Center at Chiba, Japan; 4 Graduate School of Science, Japan; 5 Graduate School of Medicine, Japan; 6 CREST, JST, Japan Drosophila Flamingo (Fmi) and mammalian Celsr1-3, which are 7pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. We show that mammalian 7-pass transmembrane cadherins Celsr2 and Celsr3 are activated by their homophilic interactions and regulate neurite growth in a distinct manner. Both gene-silencing and co-culture assay showed that Celsr2 enhanced neurite growth whereas Celsr3 suppressed it. Our result suggested that Celsr2 had a stronger activity as G-protein coupled receptor than Celsr3 did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. Thus, neuron-neuron interactions modulate neurite growth differentially through this couple of 7-pass transmembrane cadherins.
Masatoshi Takeichi RIKEN Center for Developmental Biology, Kobe, Japan For synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. While a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. When cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. This is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. Another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of Ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin-1 is preferentially localized in axons; and nectin-3, in both axons and dendrites. We present evidence that the heterophilic binding between axonal nectin-1 and dendritic nectin-3 is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. Thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites.
Hitoshi Sakano Department of Biophysics and Biochemistry, University of Tokyo, Tokyo, Japan
We have studied how the olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) gene converge their axons to a specific set of glomeruli in the olfactory bulb (OB). Retrogradestaining of OSN axons indicated that the dorsal/ventral (D/V) arrangement of glomeruli in the OB is correlated with the expression areas of corresponding ORs along the dorsomedial/ventrolateral axis in the OE. In contrast, the anterior/posterior (A/P) arrangement of glomeruli appears to be independent of the epithelial locations of OSNs and more dependent on the expressed ORs. It was found that G proteinmediated cAMP signals regulate the positioning of glomeruli along the A/P axis in the OB. We also found that multiple sets of cell adhesion molecules, e.g., ephrin-As and Eph-As, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing OR species. We propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of OSN projection.
Research funds: CREST, JST, KAKENHI 14104026 SY1-1-01-5 Lamina-restricted guidance of hippocampal mossy fibers Hajime Fujisawa 1 , Fumikazu Suto 2 1 Nagoya University, Nagoya, Japan; 2 Institute of Genetics, Mishima, Japan
Axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. One important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. Projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of CA3 pyramidal cells. We here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. First, a neural repellent Sema6A is expressed in CA3 pyramidal cells and principally suppresses invasion of mossy fibers to CA3. Second, the repulsive signal of Sema6A is mediated by plexin-A4 expressing in mossy fibers. Third, the repulsive activities of Sema6A are attenuated by plexin-A2 in the proximal dendritic segments of CA3 pyramidal cells, resulting in the segments permissive for mossy fibers to invade.
Over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). The term cognitive control is an umbrella term for a set of putative control processes. These control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. The structure most closely associated with cognitive control is prefrontal cortex (PFC). PFC is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial PFC. I will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. Three fMRI studies will be presented, focusing on (1) working memory maintenance and manipulation, (2) rule representation and task-switching, and (3) relational reasoning. Based on these data, I will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research.
SY1-1-09-5 Towards manipulative neuroscience based on non-invasive brain decoding
In ATR Computational Neuroscience Laboratories, we proposed several computational models such as cerebellar internal models, MOSAIC, modular and hierarchical reinforcement-learning model. Some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. These computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. We named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. This new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. But, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed.
Research funds: NICT, KARC SY1-1-09-6 Neural mechanisms in Williams Syndrome-Insights from neuroimaging Andreas Meyer-Lindenberg NIMH, Bethesda, MD, USA Williams syndrome (WS), a rare disorder caused by hemizygous microdeletion of −25 genes on chromosome 7q11.23, has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. WS, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. Recent months have seen dramatic advances in uncovering the functional and structural neural substrates of WS and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. We will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome.
Daisuke Yamamoto Tohoku University Graduate School of Life Sciences, Japan
The fruitless locus of Drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. The fruitless primary transcript is subject to sexually dimorphic splicing mediated by Transformer, and encodes a group of proteins that are putative transcription factors of the BTB-Zn finger protein family. The male-specific Fruitless protein is expressed in small groups of CNS neurons of males, but not of females. Fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. By experiments that label individually the neurons that express Fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. Fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of Fruitless. Thus, the Fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons.
Hitoshi Okamoto RIKEN Brain Science Institute, Wako, Saitama, Japan
The emotional behavior depends on the evolutionarily most conserved neural circuits. Especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. Thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. With these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. We already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. To investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish.
Research funds: KAKENHI (17023501) SY1-1-17-3 A molecular biological approach for songbirds to study learned vocal communication Kazuhiro Wada, Erich Jarvis Duke University, USA Songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. However, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. To overcome this limitation, we generated a resource of full-length cDNAs for gene expression analyses and functional gene manipulation in songbirds. We constructed total 21 full-length cDNA libraries from brains in different behavioral and developmental conditions. With these cDNAs, we created a novel database and 18 K songbird cDNA array. We used the arrays to reveal a set of 33 genes regulated by singing behavior. Their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. With the full-length cDNAs, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. This resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior.
Research funds: Uehara Memorial fellowship to K.W. and NIH grant to E.J. -1-17-4 Stepping pattern learning using mice: Histochemical identification of activated neuronal circuits Takashi Kitsukawa Graduate school of Frontier Biosciences, Osaka University, Osaka, Japan Identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. Also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. An efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-Fos staining. I would like to introduce our work as an example of using this method. In order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. We double-stained brain sections from mice which performed this task with c-Fos and a neuronal marker such as Enkephalin, Substance P or Nitric Oxide Synthase, each of which denotes a particular neuronal type. Our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved.
Synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. Such spike-timingdependent plasticity (STDP) has attracted wide attentions because of its high computational power and physiologically plausible induction. We previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. However, how STDP is associated with structural changes has not been elucidated. We here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal CA1 pyramidal neurons. Our results indicate that STDP is intimately associated with bidirectional structural plasticity at the level of single spines.
Research funds: KAKENHI 17680033 SY1-2-02-4 Role of CaMKII as a structural protein that stabilizes actin cytoskeleton in dendritic spines Kenichi Okamoto, Radhakrishnan Narayanan, Yasunori Hayashi Massachusetts Institute of Technology, USA Actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between F-actin and G-actin. Tetanic stimulation causes a persistent shift of actin equilibrium towards F-actin which enlarges dendritic spines. But the mechanisms which maintain these changes remain elusive. We propose that CaMKII  acts as an actin stabilizing molecule to maintain spine structure. CaMKII  is not only an abundant F-actin binding protein, it can also make oligomers. We found that CaMKII  oligomer crosslinks F-actin and stabilizes actin depolymerization kinetics. In spines, CaMKII  oligomer slows down actin dynamics and CaMKII  is enriched in spines by actin polymerization. The suppression of endogenous CaMKII  alters spine shape to filopodia-like structures. These experiments suggest that CaMKII  plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. We also found that CaMKII  detaches from F-actin in an activity dependent manner. We will discuss how CaMKII  maintains actin equilibrium in activity dependent dendritic plasticity.
Ryohei Yasuda Duke University Medical Center, USA The small GTPase protein Ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. Using 2-photon fluorescence lifetime imaging microscopy in combination with a FRET-based Ras activity sensor, we visualized the activity of Ras signaling with high spatiotemporal resolution. Our studies indicate that calcium entry due to action potentials causes Ras to activate in a supra-linear manner (Yasuda et al., 2006) . Furthermore, in response to single spine stimulation using 2-photon glutamate uncaging, Ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. These results suggest that nonlinear filtering by Ras regulators as well as the spatial spreading of Ras and Ras regulators shape spatiotemporal patterns of Ras signaling.
Hiroshi Shibasaki Takeda General Hospital, Kyoto, Japan
Involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. Myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. Depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. Cortical myoclonus is short in duration (50 ms). By back averaging EEG or MEG time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by 20 ms for hand). It is mediated by fact-conducting corticospinal pathway. Cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. These findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus.
Mark Hallett NINDS, Bethesda, MD, USA There have been many recent advances in the understanding of the physiology of focal dystonia. Three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. This lecture will emphasize the abnormal inhibition. Abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. A loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. Cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. GABA content may be decreased as shown with magnetic resonance spectroscopy. A particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. Studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. Understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods.
Research funds: NIH Intramural program SY1-2-10-3 Basal ganglia-cortical systems reinforcing tonic motor activity in health and disease Peter Brown Sobell Department, Institute of Neurology, UK
The synchronisation of neuronal activity in the beta frequency (∼20 Hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. It is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. In this talk I will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. Thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. Beta synchrony is also greatly exaggerated in untreated Parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. Excessive dopaminergic stimulation, either during treatment for Parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in BG-cortical loops leading to excessive movement. Recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema.
Research funds: MRC SY1-2-10-4 Coding of reward value of actions and valuebased action selection in the basal ganglia
A damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in Parkinson disease. Recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. We recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. During a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. During handle-turns, another subset of neurons was activated. These results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. Roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed.
In vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. We designed a new PET reporter gene system with F-18 fluoroestradiol (FES) and human estrogen receptor ligand binding domain (hERL), which would work in various tissues with little physiological effect. We have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (hTP), a factor works for revascularization, as therapeutic gene and hERL. Cos7 cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of FES that the control side. This system was successfully applied to ES cell transplantation monitoring also. Inducible hERL expression system was stably transfected into mouse ES cells and viable ES cells could be detected in vivo using FES. These data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring.
Tetsuya Suhara Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan
The molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. Neurons and glias express various receptors and transporters and those can be a specific target of the imaging. The functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. Amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. Because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. On the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. Since there is no ideal animal model for psychiatric disorders, the abnormal dopamine D1 receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. The bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease.
Sumiko Mochida Department of physiology, Tokyo Medical University, Tokyo, Japan
In presynaptic terminals, packages of neurotransmitters, synaptic vesicles (SVs), are localized at specific sites in different stages by regulation of proteins complexes. The recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. For SVs, new proteins were found and the anatomy of vesicle structure was clarified. Readiness for transmitter release, SVs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. New kinase SAD-1 found at the SV and active zone or PKA phosphorylates specific proteins. SV exocytosis is triggered by conformational changes in the fusion proteins complex when Ca 2+ sensing protein was activated. Synchronies of SV fusion are maintained by a Ca 2+ sensing protein, synaptotagmin I. After transmitter release SVs are recycled: surprisingly, recycled SVs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. These new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release.
Shigeo Takamori 21st century COE program, Department of Neuorology and Neurological Science, Tokyo Medical and Dental University, Tokyo, Japan Synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. To achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. Since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as SNARE proteins and the rab GTPases and on neurotransmitter uptake such as vesicular transporters and vacuolar H + -ATPase. However, a detailed picture of a vesicle membrane with all of its constituents is not yet available. In the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. In particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. SY1-3-11-3 SAD: A novel kinase implicated in phosphoproteome at the presynaptic active zone Toshihisa Ohtsuka Department of Clinical and Molecular Pathology, Faculty of Medicine/Graduate School of Medicine, University of Toyama, Toyama, Japan SAD is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in C. elegans and mice. To test if SAD is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of SAD. Interestingly, SAD localizes both on synaptic vesicles and at the presynaptic active zones. Moreover, SAD, together with prominent active zone proteins CAST and Bassoon, is tightly associated with the active zone cytomatrix. In accord with its unique localization at the nerve terminals, SAD appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein RIM1. Thus, these results suggest that SAD regulates not only neural polarization during development but also neurotransmitter release at mature synapses.
Toshiaki Sakisaka 1 , Takeshi Baba 1 , Sumiko Mochida 2 , Yoshimi Takai 1 1 Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine, Osaka, Japan; 2 Depatment of Physiology, Tokyo Medical University, Tokyo, Japan Two types of factors are involved in Ca 2+ -dependent neurotransmitter release: the SNARE system and its regulators. The regulators of the SNARE system include many factors, such as the Rab3 system, Munc-13, Munc-18, Doc-2, and tomosyn. We have previously reported that tomosyn, a syntaxin-1-binding protein, works as a molecular clamp that controls free syntaxin-1 availability for the formation of the SNARE complex and thereby regulates synaptic vesicle exocytosis. Here we show that PKA-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin-1, resulting in enhanced the formation of the SNARE complex. Conversely, ROCK phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the SNARE complex. Thus, tomosyn is likely to be an upstream regulator of the SNARE system, whose activity is regulated via well known signal transduction pathways.
Tei-ichi Nishiki Department of Physiology, Okayama University, Okayama, Japan A synaptic vesicle membrane protein, synaptotagmin I is thought to be a Ca 2+ sensor for neurotransmitter release. However, the physiological contributions of its Ca 2+ -binding domains (C 2 A and C 2 B) are still unclear. We have studied the roles of aspartate (Asp) residues in the C 2 B Ca 2+ -binding sites. In synaptotagmin I deficient neurons, although synchronous release was abolished as previously reported (Cell 79, p. 717) , asynchronous release was significantly increased. This defect was completely rescued by expressing wild-type synaptotagmin I, indicating the crucial roles of synaptotagmin I for triggering synchronous release and suppressing asynchronous release. Synaptotagmin I with mutations in the second or third Asp inhibited synchronous release, but still could suppress asynchronous release. Thus, we conclude that synaptotagmin I maintains the synchrony of transmitter release in two ways. Ca 2+ binding to the C 2 B is essential for synchronizing release. Suppressing of asynchronous release seems not to require Ca 2+ binding to the C 2 B because mutation in the second Asp inhibits Ca 2+ binding, yet still allows the protein to suppress asynchronous release.
Yukiko Goda, Kevin J. Darcy, Kevin Staras, Lucy M. Collinson MRC Cell Biology Unit and LMCB, University College London, UK It has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. We tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. We found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. These newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. Our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses.
Research funds: MRC, NIH and NARSAD
Hiroshi Kunugi Department of Mental Disorder Research, National Institute of Neuroscience, NCNP, Japan Neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. In the past decade, we have systematically screened BDNF, NT-3 and its low-affinity receptor P75 genes for polymorphisms and their possible association with neuropsychiatric diseases. As a result, three polymorphisms of the BDNF gene (C270T in the 5 noncoding region, BDNF-linked polymorphic region, and Val66Met), three polymorphisms of the NT-3 gene (G-3004A, microsatellite in intron 1, and Gly-63Glu) and a missene polymorphism of the p75 gene (Ser205Leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or Alzheimer's disease, although some contradictive negative results have also been reported. Here I summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p75 in neuropsychiatric diseases.
Recently, a single nucleotide polymorphism (Val66Met) in the BDNF gene resulting in a prodomain substitution at position 66 from a valine (Val) to methionine (Met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. We have recently determined in vitro that BDNF Met aberrantly engages a specific Vps10 protein, sortilin, that is part of a highly specialized sorting machinery that regulate BDNF trafficking to secretory pathways. In order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant BDNF (BDNF Met/Met ). We have determined that there is a regulated secretion defect for BDNF Met , as well as altered hippocampal structure and function in these BDNF Met mice, in a manner similar to that reported in humans with this variant BDNF. Thus, this BDNF Met/Met mouse may provide an in vivo model system to inform human studies focused on associations of this variant BDNF with clinical disorders.
Research funds: NIH Grant# NS052819 SY1-3-19-6 Processing of BDNF and brain disorders Masami Kojima 1,2 1 AIST, Osaka, Japan; 2 SORST, JST, Saitama, Japan
The fact that pro-and mature neurotrophins elicit opposite effects through p75 neurotrophin receptor (p75NTR) and Trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. Here we examined the effects of two rare single nucleotide polymorphisms (SNPs); 372 (T/G) and 378 (T/G) of the human BDNF gene, causing amino acid substitution (R125M and R127L) near the cleavage site. Western blot analysis and two-side ELISA demonstrated that these SNPs prevented the cleavage, resulting in secretion of proBDNF, but not mature BDNF. These SNPs did not affect intracellular distribution or mode of secretion of the protein. Application of the uncleavable proBDNF (proBDNFML) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. Together, these results reveal structural determinants for the cleavage of proBDNF, and demonstrate distinct functions of proBDNF for different populations of neurons. We have now analyzed the brain functions of the mice expressing this form of BDNF.
SY1-4-04-1 Pleiotropic effects of gdnf in regulation of enteric nervous system development Hideki Enomoto Laboratory for Neuronal Differentiation and Regeneration, RIKEN Center for Developmental Biology, Kobe, Japan Formation of the enteric nervous system (ENS) is governed by multiple extracellular signals at a given time during development. Inactivation of the gene encoding GDNF, RET or GFR␣1 leads to nearly complete absence of enteric neurons during early development. Although this finding establishes GDNF as an essential extracellular signal acting at the initial stage in ENS development, little is known about whether and how ENCCs continue to depend on GDNF later in development. We have generated mice in which function of GFR␣1, the high affinity receptor for GDNF, is conditionally inactivated in a time-specific fashion. We will show how GDNF signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general.
Bone marrow stromal cells (MSCs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. We present using replicationincompetent HSV-1 vector that MSC population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. Three kinds of gene-transferred MSCs, HGF, IL2ss+FGF-2, and VEGF were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. Each growth factor gene-transferred MSCs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native Research funds: KAKENHI (14657350) SY1-4-04-4 HGF gene therapy for the treatment of spinal cord injury Masaya Nakamura 1 , Akio Iwanami 1 , Kazuya Kitamura 1,2 , Yoshiaki Toyama 1 , Hideyuki Okano 2 1 Department of Ophthalmology Surgery, Keio University, Tokyo, Japan; 2 Department of Physiology, Keio University, Japan Hepatocyte growth factor (HGF) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. In this study, we demonstrated the validity of HGF for treatment of spinal cord injury (SCI) in adult rats. First, we analyzed temporal expression of HGF and c-Met in the injured spinal cord. HGF-mRNA expression was relatively low in the acute phase of SCI compared with c-Met mRNA expression. Hypothesizing that HGF is insufficient immediately after SCI, we induced SCI at Th10 level in adult rat 3 days after injecting Herpes vector-mediated gene transfer of HGF (HGF group) or LacZ (control) into spinal cord. Motor function was evaluated by BBB score. 1 or 6 weeks after injury, histological analyses were performed. There were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and GAP43+fibers in HGF group compared to the control group. Animals of the HGF group showed better functional recovery than the controls. These findings suggest that HGF could have therapeutic effects for SCI.
Hiroshi Funakoshi, Toshikazu Nakamura Division of Molecular Regenerative Medicine, Osaka University Graduate School of Medicine, Osaka, Japan HGF was initially identified and molecularly cloned as a mitogen for primary hepatocytes (Nakamura et al., 1989) . Recently, HGF is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. Mutant c-Met/HGF receptor knock-in mouse reveals that HGF decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (Maina et al., 1999) . Therefore, it is possible to speculate that HGF could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. Here we show the examples of beneficial effects of HGF on model animals of different neural and neurodegenerative diseases, using several delivery methods for HGF including gene therapy approach. We also present the possible application of HGF in modifying the neurogenesis for the disease.
References Maina et al., 1999 . Nature Neuroscience. Nakamura et al., 1989 . Nature.
Hiroyuki Kato Center for Clinical Medicine and Research, International University of Health and Welfare, Nasushiobara, Japan
We examined stroke patients using fMRI at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. Normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. At the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. At the chronic stages, recovery of activation and/or persistent addition of activation were observed. Thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. The findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. Interestingly, the time period during which reorganization occurred was limited to first 1-2 months after stroke, suggesting the presence of a critical period.
In the cat, Illert et al. (1977) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the C3-C4 segments. In contrast, recently it has been shown that polysynaptic pyramidal EPSPs are only rarely observed in forelimb motoneurons of macaque monkeys and humans. We reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. (1) In the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal EPSPs after blockade of glycinergic inhibition by strychnine. Moreover, we recently identified C3-C4 propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. (2) In human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. Stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. Functions of the pathway both in physiological and pathological conditions will be discussed.
Bisphenol-A (BPA) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. We previously reported that prenatal and neonatal exposure to BPA potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. Many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. We found that in vitro treatment with BPA caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of GFAP. A low concentration of BPA significantly enhanced the Ca 2+ responses to dopamine in both neurons and astrocytes. These findings provide evidence that BPA induces dopaminergic changes in neurons and astrocytes. This phenomenon may, at least in part, contribute to the enhancement by BPA of the development of psychological dependence on drugs of abuse.
Mami Yamasaki, Yonehiro Kanemura The Department of Neurosurgery, Clinical research institute, Osaka National Hospital, National Hospital Organization, Osaka, Japan L1CAM(L1) is a member of the immunoglobulin superfamily of cell adhesion molecules. X-linked hydrocephalus, MASA syndrome and certain forms of X-linked spastic paraplegia are now known to be due to mutations in the gene for L1. Therefore, these syndromes have been reclassified as L1 syndrome. We performed a nation-wide L1gene analysis and identified LI gene mutations in 35 families with L1 syndrome. All the patients showed developmental delay in various degree. We discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. The loss of extracellular domain functions like L1-mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in L1 syndrome.
Research funds: KAKENHI (16390424), (16689025) SY1-5-05-4 Rett syndrome and developing brain Yoshiko Nomura Segawa Neurological Clinic for Children, Japan Rett syndrome (RTT) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. Hypofunction of the brainstem monoaminergic neurons is suggested. Pathology showed no degeneration. Methyl-CpG-binding protein 2 gene (MECP2) located at Xq28 is the causative gene. Types of mutation at different functional domains are correlated to clinical severities. X-inactivation also influences phenotypic variability. MeCP2 was thought as a global transcriptional repressor, but finding of BDNF as a target gene suggest its role in the neuronal activity-dependent gene regulation. Genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like 5 gene (CDKL5) manifest as atypical RTT. The mutations of MECP2 are found in other clinical conditions, such as X-linked mental retardation, Angelman syndrome, autism, and severe neonatal encephalopathy. Thus, the evaluation of RTT gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only RTT but also other neurodevelopmental disorders.
In our previous studies, we have proposed that ROS and/or ROS-mediated signal play(s) an essential role in 6-OHDA-induced, caspase-dependent apoptosis. In contrast, MPP+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. Subsequently, we have demonstrated that MPP+ induces release of cytochrome c but not activation of caspase and proposed that depletion of ATP and/or calcium-activated calpain-mediated degradation of procaspase-9 are responsible for the absence of subsequent activation of caspases. Furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to MPP+-mediated dopaminergic neuronal death. As such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in MPP+-induced cell death. Taken together, we reason that distinct set of proteases activation is involved in experimental models of PD. Therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death.
Satoshi Ogawa Department of Neuroanatimy, Kanazawa University of Medical School, Ishikawa, Japan
We discuss the role of ER-stress in neuronal cell death in SNpc by introducing two models. Upregulation of Pael-Receptor in the substantia nigra pars (SNpc) of mice induces endoplasmic reticulum (ER) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. The role of ER stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase Parkin and the ER chaperone ORP150, suggesting parkin dysfunction result in ER-stress mediated neuronal cell death. Conversely, transgenic rats overexpressing megsin (Tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid Schiff (PAS) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal CA3, and substantia nigrta. Enhanced ER stress was observed in dopamine neurons in SNpc, accompanied with loss of neuronal viability and motor coordination. In both subregions, PAS-positive inclusions were also positive with megsin. These data suggest that enhanced ER stress causes selective vulnerability in a set of neuronal populations.
Noradrenaline (NA) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. Accumulating evidence has suggested that the central NA system modulates consolidation and reconsolidation of long-term emotional memory. Here we show that the NA system is particularly important for retrieval of reconsolidated emotional memory. The mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. This memory deficit is restored by pharmacological stimulation of NA activity before the test and is also restored by intra-amygdala NA stimulation through ␣1or -adrenergic receptors. Moreover, intra-amygdala NA stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. Our findings indicate that the amygdalar NA system, primarily through ␣1and -adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace.
Shigeru Morinobu, Shigeto Yamamoto, Shigeto Yamawaki Departmnet of Psychiatry and Neurosciences, Hiroshima University, Hiroshima, Japan As psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in PTSD, it is hypothesized that impaired extinction may be involved in PTSD. Rats subjected to single prolonged stress (SPS) exhibit the enhanced negative feedback of the HPA axis, exaggerated startle response, and analgesia. Thus, SPS is a good model of PTSD. We examined whether extinction of fear memory was impaired in SPS rats, using the contextual fear conditioning. SPS rats exhibited the significant longer freezing during re-exposure to the context 2-4 days after the conditioning. Furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in SPS rats. We measured the levels of NMDA receptor subunits (NR1, NR2A, 2B, 2C), glycine transporter 1, and EAAC1, by real-time PCR. No significant changes were found in the hippocampus. Based on these findings, it is speculated that the increase in other types of glutamate transporters or NMDA receptor modification may play a role in impaired extinction in SPS rats.
Ichiro Masai Masai Initiative Research Unit, RIKEN, Wako, Japan
In human, there are hereditary retinal diseases such as retinitis pigmentosa. To understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. Here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal ERG and OKR response. In the tli mutant, photoreceptors initially differentiate but degenerate later. Electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that Tli is required for the formation of photoreceptive membranes. In the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. We found that the els gene encodes cGMP phosphodiesterase 6 ␣ -subunit (PDE6C), a component of cone-type PDE. Since genetic mutations of PDE6C have not been reported in human, the els mutant provides a good model for studying roles of cone-PDE6 in visual functions.
Shinichi Nakagawa 1 , Masatoshi Takeichi 2,3 , Fumi Kubo 1,3 1 Nakagawa Initiative Research Unit, RIKEN, Wako, Japan; 2 RIKEN CDB, Kobe, Japan; 3 Department of Biostudies, Kyoto University, Kyoto, Japan
The marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. We have previously shown that Wnt2b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. Interestingly, Wnt2b inhibits cellular differentiation in the absence of Notch activity, a well-known signaling receptor that inhibits neuronal differentiation. Wnt2b antagonizes proneural gene functions independent of the Notch signaling pathway, presumably through unidentified transcriptional repressors. We have isolated several candidate genes that are upregulated upon an activation of the Wnt signaling pathway, and some of them are expressed in the stem cell containing region. Physiological roles of those genes will be discussed.
Research funds: KAKENHI (16570184) SY1-6-06-3 Identification of cell lineage of retinal progenitor cells by cell surface markers Sumiko Watanabe, Hideto Koso, Shinya Satoh Department of Molecular and Developmental Biology, University of Tokyo, Tokyo, Japan I would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. We found c-kit and SSEA-1 to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and SSEA-1 during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/SSEA-1 double-positive cells showed stronger proliferation activities than ckit single-positive ones. Although the number of SSEA-1-positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by Notch signaling, suggesting that c-kit and SSEA-1 have intrinsically distinct characters. Prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to SCF, suggesting a role for c-kit in retinal development. The retinal photoreceptor cells play a primal and central role in the phototransduction system. They are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. We have been demonstrated that transcription factors, Otx2 and Crx play critical roles in retinal photoreceptor development. While Otx2 is a key molecule for retinal photoreceptor cell fate determination, Crx is essential for the terminal differentiation and maturation of photoreceptors. Meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. Our investigation of a role of aPKC, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that aPKC is required for the formation of outer & inner segments and ribbon synapse. In addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. We would like to present our recent analysis of photoreceptor development.
Research funds: KAKENHI (16790070, 16027257, 17024061)
Raj Ladher RIKEN Center for Developmental Biology, Kobe, Japan
The inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. The inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. Induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. In the chick, the inner ear is induced by localized FGF19 found in the mesoderm. We find that although FGF19 can induce the inner ear, it is unable to support differentiation of the inner ear. Differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member FGF3 and is actually inhibited by FGF19. For full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. These components develop from diverse origins but are intimately linked during development. We have been trying to understand how integration occurs and present one model by which this could occur.
Research funds: Center support grant, MEXT Leading Projects grant SY1-6-06-6 How is olfactory receptor-dependent axonal wiring conducted?
Shou Serizawa, Kazunari Miyamichi, Haruki Takeuchi, Yuya Yamagishi, Tokiko Tsubokawa, Hitoshi Sakano Department of biophysics and biochemistry, CREST JST, University of Tokyo, Tokyo, Japan In the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (OR) expressed, forming the olfactory sensory map. To study how the OR-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (OSNs) express a particular OR gene. We found that the expression of the immunoglobulin superfamily gene Kirrel2, encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. The expression level of Kirrel2 in each OSN is found to be correlated with the type of OR species expressed in the OSN. Moreover, Kirrel2 promoted fasciculation of OSN axon termini in the mosaic gain-of-function experiment. Here, we propose that the information of which type of OR is expressed in the OSN is converted to the expression level of Kirrel2 which determines the adhesiveness of axon termini, contributing to OR-dependent segregation of OSN axons.
In spite of its morphological similarity to the other species in the melanogaster species subgroup, Drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant Morinda citrifolia, known as the Tahitian Noni fruit. The ripe fruit of M. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. D. sechellia is attracted to these two fatty acids, while the other species are repelled by them. Using inter-species hybrid between D. melanogaster deficiency mutants and D. sechellia, Odorant binding protein 57e was identified as the gene responsible for this behavioral difference among the species. Obp57e forms a gene cluster with Obp57d, and these two genes are expressed in the same cells associated with the chemosensory organ. The history of dynamic Obp57d/e-cluster evolution was revealed by comparison of the genomic sequences of the Obp57d/e region obtained from 30 species phylogenetically located between D. melanogaster and D. pseudoobscula. SY1-6-14-4 An approach of dissociating complex traits into fine genetic elements using consomic strains of mouse Aki Takahashi 1,2 , Akinori Nishi 1,2 , Toshihiko Shiroishi 1,2 , Tsuyoshi Koide 1,2 1 SOKENDAI, Kanagawa, Japan; 2 National Institute of Genetics, Shizuoka, Japan
Much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. We have previously shown that laboratory strain C57BL/6 (B6) and wild-derived strain MSM/Ms have great differences in many behavioral traits. Consomic strains were established by natural mating between B6 and MSM, and those strains have the same genetic background as B6 except for one chromosome from MSM. By examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. One strain B6-17MSM, which have MSM chromosome 17, showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. To identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region.
Research funds: KAKENHI (16.12064) SY1-6-14-5 Cloning of the major quantitative trait locus underlying capsaicin resistance in mice
Capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. However, sensitivity to capsaicin differs among individuals. A genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. Capsaicin resistance linked on chromosome 2 (Capsq1) is the major locus affecting reduced taste sensation in KJR mice. Here we show that intracellular recycling of capsaicin receptor (TRPV1) was impaired in KJR neurons in contrast to that of C57BL/6J mice. By searching the candidate genes, EH domain-containing four (EHD4), a TRP-binding scaffold protein encoding gene was found. EHD4 binds to C-terminal of TRPV1. Three mutations were found in EHD4 of KJR, which remarkably diminished the binding, leading to changes in the intracellular distribution of TRPV1. This study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to TRPVs. SY1-6-14-6 Comprehensive behavioral analysis of genetically-engineered mice Tsuyoshi Miyakawa HMRO, Kyoto University, Kyoto, Japan
One of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. Final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. In our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. The battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. So far, we have assessed more than 45 different strains of mutant mice with the battery. Surprisingly, more than 90% of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. Representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. Also, a potential impact of our approach to "large-scale neuroscience" will be discussed.
Research funds: KAKENHI (16680015, 16653065, 17017021, 17025023) , JST BIRD
Hiroshi Takashima Department of Neurology and Geriatrics, Kagoshima University, Kagoshima, Japan Inherited neuropathies are clinically and genetically heterogeneous. At least 28 genes and 12 loci have been associated with Charcot-Marie-Tooth disease (CMT) and related inherited neuropathies. Most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of CMT gene discovery has accelerated. These recently discovered CMT causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. Their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. On the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of CMT have been identified. To clarify the specific features and molecular mechanisms, we reviewed recent progress in CMT research, especially CMT4F caused by PRX, and SCAN1 caused by TDP1. SY1-6-22-2 Gangliosides are important for the maintenance of the nodes of Ranvier Nobuhiro Yuki, Keiichiro Susuki Department of Neurology, Dokkyo University School of Medicine, Tochigi, Japan Gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. Some patients with Guillain-Barre syndrome have autoantibodies to gangliosides such as GM1, who show failure of peripheral motor nerve conduction. Sensitization of rabbits with GM1 can produce the disease model. In ventral roots from the paralyzed rabbits, IgG and complements deposited on the nodes of Ranvier, and sodium channel clusters were disrupted. In ganglioside-deficient mice with disrupted GM2/GD2 synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. Some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. The abnormality became prominent with age. These findings using different models showed that gangliosides are important for the maintenance of the node of Ranvier and saltatory conduction along the myelinated nerve fibers.
Hiroshi Ueda Division of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. This is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. In our recent paper (Nature Medicine, 2004) , we firstly demonstrated that lysophosphatidic acid (LPA) and its receptor (LPA1) activation initiate the neuropathic pain. In this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). I will discuss four issues, LPA-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying LPA-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific LPA production in terms of demyelination and allodynia.
Research funds: KAKENHI (17109015)
Toshihide Yamashita Department of Neurobiology, Graduate School of Medicine, Chiba University, Chiba, Japan Axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. Some of the axon growth inhibitory effects are associated with myelin. Three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. These proteins induce activation of Rho in some neruons. Inhibition of Rho or Rho-kinase promotes axon regeneration in vivo. These findings establish Rho and Rho-kinase as key players in inhibiting regeneration of the central nervous system. I will review recent findings regarding the signaling mechanism of axon growth inhibitors. Our experiments suggest that several new candidate proteins may be axon growth inhibitors. These proteins activate not only Rho/Rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. These findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system.
Sha Mi Biogen Idec, USA LINGO-1 is a CNS-specific protein expressed in both neurons and oligodendrocytes. In neurons, LINGO-1 mediates the inhibition of axonal growth as a component of the NgR1/p75/LINGO-1 and NgR1/Troy/LINGO-1 signaling complex. Inhibition of endogenous LINGO-1 by soluble LINGO-1 or dominant negative LINGO-1 can reverse the inhibition of neurite outgrowth by myelin components. Soluble LINGO-1 treatment significantly improves functional recovery of spinal cord injured rats as determined by BBB scores. Soluble LINGO-1 treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. In oligodendrocytes, LINGO-1 mediates the inhibition of differentiation and myelination. Loss of LINGO-1 function using dominant negative LINGO-1, LINGO-1 RNAi, or soluble LINGO-1 or LINGO-1 knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of LINGO-1 led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. The discovery of a significant role for LINGO-1 in neurons and oligodendrocyte biology are an invaluable step for understanding CNS axon regeneration and myelination.
Alex Reyes New York University, USA Neurons in the auditory cortex exhibit a wide range of firing patterns. To elucidate the cellular properties and circuitry that give rise to these responses, a 2D sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (ICN) modified to contain both feedback and feedforward circuits. A disc of neurons was stimulated and the resultant firing pattern and spread was documented. Simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. A computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. This waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. Using the ICN method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. We replicated many of the responses recorded in vivo. Morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons.
Research funds: NIH DC005787-01A1 SY1-7-15-2 Neuronal avalanches reveal neuronal wirings of layer 2/3 cell assemblies Jun-nosuke Teramae, Tomoki Fukai Brain Science Institute, RIKEN, Japan How cortical neurons process information crucially depends on how their local circuits are organized. Spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. They obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. However, the explicit network structure underlying the power-law statistics remains unclear. Here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. We demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. The resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. We investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback.
Research funds: KAKENHI (17700318) SY1-7-15-3 Spike-timing dependent and homeostatic plasticity from an optimality viewpoint Taro Toyoizumi 1 , Jean-Pascal Pfister 2 , Kazuyuki Aihara 1,3 , Wulfram Gerstner 2 1 Department of Complexity Science and Engineering, University of Tokyo, Japan; 2 School of Computer and Communication Science & BMI, EPFL, Japan; 3 Aihara Complexity Modelling Project, ERATO, JST, Japan Maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. Under the assumption of Poisson firing statistics, the synaptic update rule exhibits all the features of the Bienenstock-Cooper-Munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. The learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. An interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed.
Research funds: Grant-in-Aid for JSPS fellows 03J11691 and Sci. Res. on Priority Areas 17022012 from MEXT of Japan, and Swiss Natl. Sci. Found. 200020-108097/1 SY1-7-15-4 Timing computations in the auditory brain stem John Rinzel Center for Neural Science and Courant Institute of Mathematical Sciences, New York University, USA Sound localization involves precise temporal processing by neurons in the auditory brain stem. The first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (ITDs) in the sub-millisecond range. These cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated K+ current, I-KLT. This I-KLT contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. We will describe the temporal feature-selecting properties of MSO cells based on biophysical (HH-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory.
Neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. However, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. In response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. Onsets and spike numbers of these bursts are highly reproducible. We asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. Using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. Differences in spike patterns at the scale of milliseconds encode differences in the input as long as 200-300 ms. When single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. This suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. We propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds.
Accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. Although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. To address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. The results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. The degenerative process is not necessary for triggering the upregulation. It is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. The molecular basis underlying such neurogenesis will be discussed.
Keiichi Itoi 1 , Ikue Otaki 1 , Saya Suzuki 1 , Yasunobu Yasoshima 2 , Kazuto Kobayashi 2 1 Laboratory of Informational Biology, Graduate School of Informational Science, Tohoku University, Sendai, Japan; 2 Institute of Biomedical Science, Fukushima Medical University, Japan
In order to examine functional roles of the noradrenergic (NA) neurons in the locus coeruleus (LC) we developed a novel method to ablate specifically the NA neurons in the LC, and examined the behavioral and stress responses using the animal model. A transgenic mouse line was used in which human interleukin-2 receptor ␣ subunit (hIL-2R␣) was expressed under the control of dopamine -hydroxylase gene promoter. Anti-hIL-2R␣ antibody fused to Pseudomonas exotoxin was microinjected into bilateral LC of a transgenic mouse stereotaxically to destroy specifically the NA neurons. As behavioral paradigms, elevated plus maze and open field test were used. Plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. Thus, the effect of LC ablation how it affects the behavioral and stress responses will be elucidated. -8-16-5 Integrated circuits controlling the stress response James P. Herman Department of Psychiatry, University of Cincinnati, OH, USA
The hypothalamo-pituitary-adrenocortical (HPA) axis is a primary stress-response system in all vertebrates. The end-product of HPA activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. However, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of HPA activity a priority for the organism. This control is exerted in large part by limbic structures in the brain. Our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles HPA axis regulation. The amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on HPA activity. The role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. All of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (PVN) neurons controlling the HPA response, allowing convergence of information from multiple limbic sources prior to the PVN.
SY1-8-24-1 Molecular mechanism for the inverse incidence of Parkinson's disease and cancer: Synuclein as stimulator of tumour differentiation Makoto Hashimoto Department of Chemistry and Metabolism, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan Neurodegenerative disease and cancer are major age-associated disorders. However, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. In Parkinson's disease (PD) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of PD. Furthermore, epidemiological studies described that the incidence of cancer was reduced in PD patients. Recent study provides evidences of the inverse relationship of PD and some cancers at the molecular level. For example, loss of neuroprotection of DJ-1 is causative for familial PD, while increased expression of this molecule stimulates oncogenesis. In this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for PD, may induce differentiation of cancer cells. Thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields.
Kiyomitsu Oyanagi Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan Neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. However, it is well known that critical periods of some malignancies are in fetal period, which are (1) leukemia in patients exposed with atomic bomb during the IInd World War, and (2) brain tumors in rats with ethylnitrosourea administration. As to neurodegenerative diseases, (3) many genetic/familial diseases show clinical symptoms at the middle or old age. (4) Epidemiological study revealed that emigrants from Guam to the main land of USA show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. (5) Relating to Parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [Oyanagi et al., in press] . These findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood.
To understand the role of Synuclein, the major component of pathological inclusions, we examine the expression of Synuclein in the embryonic mouse cerebral cortex. We found that a-Synuclein and b-Synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. In another line of inquiry, we are interested in a zinc finger protein containing POZ domain, RP58, which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. When the RP58 gene is disrupted, apoptosis is enhanced, and a-Synuclein, but not b-Synclein, is upregulated in the mutant cortex, suggesting that a-Synuclein is involved in the cell death. Interestingly, in the mutant cortex the expression of S-phase marker, PCNA increased, suggesting that RP58 mutant mice are useful to analyze the relation among neurodegeration, Synuclein and cell cycle.
Minoru Saitoe, Junjiro Horiuchi, Daisuke Yamazaki Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan Age-related memory impairment (AMI) is a striking feature of ageassociated neuronal dysfunction. To identify gene mutations that affect AMI, we screened ∼100 Drosophila lines and found that heterozygous mutants for the PKA catalytic subunit (DC0/+) exhibit robust suppression of AMI without affecting memory at young ages. This result suggests a causal relationship between PKA and AMI. Of particular interest, IGF/PI3K/Akt signaling, which results in decreased GSK3 activity, has also been shown to ameliorate AMI. Both PKA and GSK3 phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. While IGF signaling suppresses activity of GSK3 at young ages, declining IGF levels during aging may increase GSK3 activity in aged animals. In support of this idea, we found suppression of AMI in flies fed GSK3 inhibitors. We hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by PKA and GSK3 causes neuronal dysfunction during normal aging.
Research funds: KAKENHI SY1-8-24-5 Molecular mechanism of cancer progression by gamma-synuclein Koji Okamoto Radiobiology Division, National Cancer Center Research Institute, Tokyo, Japan Synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣Synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as Alzheimer disease and Parkinson disease, whereas overexpression of ␥Synuclein is associated with progression of breast and ovarian cancer. However, the normal cellular function of Synucleins remains largely unknown. In order to get an insight into biological function of Synucleins, we focus on cancer progression induced by ␥Synuclein. We introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. Using such cells, the attempt to elucidate the biochemical function of ␥Synuclein is underway. The impact of Synuclein over-expression, especially on known tumor suppressor pathways such as the p53 pathway, will be discussed.
Research funds: KAKENHI
Ryuichi Sakai Growth Factor Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. Shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. In the neural systems, two unique Shc family molecules, ShcB and ShcC, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of NGF/TrkA signaling.
It was recently found that tyrosine phosphorylation of ShcC is frequently detected in majority of neuroblastoma cell lines. We showed that hyperphosphorylated ShcC detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (ALK) caused by the gene amplification. Identification of binding partners of ShcC and expression of mutant ShcC in several cancer cell lines revealed novel roles of ShcC as a regulator of differentiation and proliferation of neuroblastic tumors.
Research funds: KAKENHI SY1-8-24-7 Identification of estrogen receptor target genes and role of their gene products in cancer and nervous system Satoshi Inoue 1,2 1 Department of Geriatric Medicine, University of Tokyo Hospital, Tokyo, Japan; 2 Research Center for Genomic Medicine, Saitama Medical School, Saitama, Japan Estrogen has crucial roles in the cancer growth and in the neural function. Here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (GBSC) method. One of the first identified genes is the estrogen-responsive RING finger protein (Efp). Efp expression was observed in uterus, mammary gland and certain regions of the brain where ER is also expressed and positively regulated by estrogen. We revealed that Efp targets proteolysis of 14-3-3 sigma, a negative cell cycle regulator that causes G2 arrest and that Efp is an essential oncogenic factor in breast cancer growth. On the other hand, another gene identified by GBSC is NR2D, an NMDA receptor. This gene was regulated by estrogen in the hypothalamus, together with ER, PR and Efp. These estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms.
Keiji Tanaka 1 , Hossein Esteky 2 , Kiani Roozbeh 2 , Tadashi Sugihara 1 , Gang Wang 3 1 RIKEN Brain Science Institute, Wako, Saitama, Japan; 2 Institute for Studies in Theoretical Physics and Mathematics, Tehran, Iran; 3 Kagoshima University, Kagoshima, Japan Individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. Then, questions arise where and how view-general objects and object categories are represented. A possibility is the representation by a population of inferotemporal cells. To examine it, we recorded responses of 670 inferotemporal cells to 1100 object images in a fixation task. We also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. The results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to 60 • rotation.
Research funds: KAKENHI 17022047
Alexander Thiele 1 , Gene Stoner 2 , Louise S. Delicato 1 , Mark Roberts 1 1 University of Newcastle upon Tyne, UK; 2 The Salk Institute, Japan A variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. We have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. Using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area MT underlies the binding of an object's component features. Recent reports have focused on the role of synchrony in the mediation of attention. We will discuss the role of synchronized activity in primate V1 while attentional load was varied, and how it could be mediated by cholinergic mechanisms.
Research funds: HFSP, Wellcome Trust, BBSRC SY2-1-01-3 Context-dependent changes in noise correlation in MT William Newsome, Marlene R. Cohen Stanford University and Howard Hughes Medical Institute, USA Changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. We studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded MT neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. We created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. We found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. Specifically, noise correlations were higher in the cooperative than in the competitive context. This result suggests that MT neurons receive inputs of central origin whose strength changes with the task structure. The changes in correlation appear to reflect differences in how MT neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention.
Research funds: Howard Hughes Medical Institute SY2-1-01-4 Effects of task demands on color processing in area TE of the monkey Hidehiko Komatsu 1,2 , Kowa Koida 1 1 National Institute for Physiological Sciences, Okazaki, Japan; 2 SOKENDAI, Okazaki, Japan Color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. Lesion studies suggested that inferior temporal (IT) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area TE of IT cortex. To study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. We found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. In several extreme cases, large gain change almost completely eliminated the responses in one task. These results suggest that color signals are gated by the top-down signal representing task demands in area TE and color channels specific to different tasks are formed at this level of the visual cortex.
Yoichi Sugita Neuroscience Research Institute, AIST, Tsukuba, Japan Early visual experience is indispensable to shape the maturation of cortical circuits during development1. Monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception2-4. Here, I show exposure only to monochromatic illumination in infancy results in the disruption of color perception. Infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. After extensive training, they were able to perform color matching. But, their judgment of color similarity was quite different from that of normal animals. Furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. These results indicate that early visual experience is also indispensable for color perception.
Research funds: CREST SY2-2-02-1 Dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing Purkinje cell
Kazuyoshi Tsutsui 1 , Hirotaka Sakamoto 2 , Katsunori Sasahara 1 , Hanako Shikimi 1 , Kazuyoshi Ukena 1 , Mitsuhiro Kawata 2 1 Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, Japan; 2 Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto, Japan New findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. Such steroids synthesized de novo in the brain are called neurosteroids. Recently we have identified the Purkinje cell as a major site for neurosteroid formation in the brain. This is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. In mammals, the Purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. Subsequently, our recent studies on mammals using the Purkinje cell have demonstrated organizing actions of neurosteroids. Both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in Purkinje cells.
Research funds: KAKENHI (15207007 and 16086206 to KT) SY2-2-02-2 Roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-Studies with knockout mice and RNAi Sonoko Ogawa Kansei, Behavioral and Brain Sciences, University of Tsukuba, Tsukuba, Japan
The gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ERs), ligand dependent transcription factors. A series of studies using single and double knockout mice for ER-␣ and/or ER- genes have revealed that activation of ER-␣ and ER- differentially regulate a number of behaviors as well as neuroendocrine functions. Our studies have suggested a unique role of activation of ER- in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. In this talk, our findings from behavioral studies using ER-␣ and ER- knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. Our most recent studies on brain site-specific manipulation of ER gene expression with the use of small interference RNA combined with adeno-associated virus will then be presented.
Research funds: KAKENHI (17330151, 17051001) SY2-2-02-3 Sex steroid receptor function in sexual behavior Shigeaki Kato 1,2 , Takashi Sato 1 , Takahiro Matsumoto 1,2 1 IMCB, University of Tokyo, Tokyo, Japan; 2 ERATO, JST, Saitama, Japan Androgen actions are believed to mediate nuclear androgen receptor (AR)-mediated gene regulations. AR is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. We generated the floxed AR mice to avoid testicular feminization mutant (Tfm) abnormalities with infertility, and then crossed with female AR(−/+) heterozygoutes expressing Cre to generate AR(−/−) female mice. The AR(−/Y) KO males grew healthy with typical features of Tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (Kawano et al., 2003) . No sexual behaviors and reduced aggressive behaviors were seen in AR(−/Y) male mice (Sato et al., 2004) . Female AR KO mice were normal in sexual behavior but exhibited premature ovarian phenotype (Shiina et al., 2006) . Together with these results, the AR function will be discussed in terms of AR function as a transcription factor.
References Kawano, H., et al., 2003 . PNAS USA 100, 9416. Sato, T., et al., 2004 . PNAS, USA 101, 1673 . Shiina, H., et al., 2006 Research funds: PROBRAIN SY2-2-02-4 Annexin 1: A mediator of cell-cell communication in the neuroendocrine system Julia Buckingham 1 , Helen Christian 2 , John Morris 2 1 Imperial College London, UK; 2 Department of Human Anatomy and Genetics, University of Oxford, UK Annexin 1 (ANXA1) plays an important part in mediating the regulatory effects of glucocorticoids (GCs) on neuroendocrine function, particularly within the HPA axis. It is expressed by folliculostellate (FS) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. GCs act on cells expressing ANXA1 to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. This process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly ABCA1. The released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. The nature of the ANXA1 receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard.
Katsuhiko Nishimori 1 , Yuki Takayanagi 2 , Masahide Yoshida 1 , Yoshiyuki Kasahara 1 , Masaki Kawamata 1 1 Graduate School of Agricultural Science, Tohoku University, Sendai, Japan; 2 Department of Physiology, Jichi Medical University, Minamikawachi-machi, Japan
We examined the behaviors of mice lacking OXTR gene and discovered that OXTR null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. Those are implying impaired mother-infant relationship. OXTR null males also showed more aggressive and having social amnesia as well as the phenotype of OXT null mice. In addition, Oxtr null mice failed to maintain their body temperature after acute cold exposure. Their rectal temperature rapidly dropped in comparison of that of wildtype animals at 5 • C ambient temperature. Our studies demonstrate that OXTR plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism.
Research funds: Grant-in-Aid for Scientific Research (B) (14360046) SY2-2-08-1 Cortical mechanisms mediating visuomotor control of primate grasp Roger N. Lemon UCL Institute of Neurology, UK Primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. A network of parietal and frontal motor areas is thought to play a key role in this behaviour. Our work shows that: hand shape can be unambiguously determined from EMG activity of hand and digit muscles.
Information about grasp is represented by neuronal populations in the ventral premotor cortex (area F5); F5 activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (M1). Local field potential activity in F5 and M1 is also tuned to grasp, and there is strong beta coherence between F5 and M1, indicating reciprocal transmission of information. This is also seen in synaptic responses of M1 neurones to stimulation of F5 (and vice-versa). Single pulse stimulation in F5 strongly modulates corticospinal outputs from M1 through corticocortical pathways between these two areas. Paired-pulse TMS can probe the excitability of these pathways in humans. Facilitation of MEPs is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp.
Research funds: Wellcome Trust, BBSRC SY2-2-08-2 Where tactile signals are ordered in time Shigeru Kitazawa 1,2 1 Department of Neurophysiology, Juntendo University Graduate School of Medicine, Tokyo, Japan; 2 CREST, Japan Science and Technology Agency, Saitama, Japan How does the brain order successive events? It is generally accepted that the brain can resolve the order of two stimuli that are separated in time by 30 ms. This applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. However, crossing the arms caused misreporting (that is, inverting) of the temporal order. The reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., 1.5 s). When the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. We recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. The results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. Results from functional imaging support this idea.
SY2-2-08-3 Decision making and underlying neural mechanisms-Auditory-visual ambiguity solving and preference Shinsuke Shimojo 1,2 1 Biology/CNS, California Institute of Technology, Pasadena, CA, USA; 2 JST.ERATO Shimojo Implicit Brain Function Project, Atsugi, Japan
We explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). We've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. 122-channel MEG was employed, while the observer reported number of flashes. Partial Directed Coherence was applied to see if there was a causal influence by the auditory on the visual cortices. The results indicate a strong causal influence in A-V direction in Alpha (8) (9) (10) (11) (12) and ranges only in the illusion-reported trials, while stimulus parameters were identical. No such difference was found in V-A direction. For preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. Our fMRI study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. While such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision.
Research funds: JST.ERATO, HFSP SY2-2-08-4 Why look there? Insights from spatial neglect and the medial frontal cortex Masud Husain UCL Institute of Neurology, UK Why do we look where we do? Studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. Our eyes go to the most salient locations where local feature contrast is greatest. These findings have led to the concept of a salience map which directs attention and gaze bottomup. In humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. Recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. These include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. Gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. Our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. Parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do.
Research funds: Wellcome Trust (061140) SY2-2-08-5 Recognizing self actions through externalized eyes Atsushi Iriki 1,2 1 Symbolic Cognitive Development, RIKEN Brain Science Institute, Saitama, Japan; 2 Cognitive Neurobiology, Tokyo Medical and Dental University, Tokyo, Japan
We can recognize ourselves and our own actions through the mirror or video images. Thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. Human fMRI studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. Japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. The emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. Thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions.
Yoshiyuki Kubota Division of Cerebral Circuitry, NIPS, Okazaki, Japan GABAergic nonpyramidal cells in the neocortex are composed of several different subtypes. We found that most of GABAergic cell types, including FS basket and somatostatin Martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. Most postsynaptic spines also received an asymmetrical input, called double innervated (DI) spines. To better characterize the other asymmetrical input on the DI spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (VGLUTs): VGLUT1, existing mostly in cortical pyramidal cells, and VGLUT2, found in thalamocortical fibers. GABAergic inputs were rarely observed in spines innervated by VGLUT1-expressing terminals (n = 289), but were found in-10% of spines innervated also by VGLUT2-expressing terminals (n = 444). Symmetrical synapses on DI spines were positive for GABA, as shown by postembedding immunohistochemistry. These results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by GABAergic synapses from cortical nonpyramidal cells.
Research funds: KAKENHI SY2-3-03-2 GABAergic recruitment of excitation by cortical axo-axonic cells Gabor Tamas, Csaba Varga, Gabor Molnar, Szabolcs Olah, Pal Barzo, Janos Szabadics University of Szeged, Hungary
The axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (AACs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). Thus, AACs are considered as strategically placed inhibitory neurons controlling cortical information flow. We applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in AACs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. The excitatory action of AACs is based on a depolarized reversal potential for axonal relative to perisomatic GABAergic inputs as determined in paired perforated patch recordings. Powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼44-fold decrease in the potassium-chloride co-transporter 2 (KCC2) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling.
In my talk I will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. These dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. Previous studies have most often reported global linear or sublinear summation in these dendrites. Using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. Next I will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of NMDA-spike in local synaptic plasticity processes.
Yumiko Yoshimura Department of Visual Neuroscience, Research Institute Environmental Medicine, Nagoya University, Nagoya, Japan
Neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. We previously showed that layer 2/3 pyramidal cells in visual cortex share common excitatory inputs from layer 4 and from within layer 2/3, when they are directly connected. Here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. We recorded photostimulation-evoked synaptic currents from pairs of layer 2/3 cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. Fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. Adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. Therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs.
Research funds: KAKENHI (17023026,17500208) SY2-3-03-5 Local circuitry of cortical inhibitory neurons Edward Callaway, Takuma Mori, Xiangmin Xu The Salk Institute, USA We used laser scanning photostimulation to map local input to inhibitory neurons in layer 1 of rat visual cortex and layer 2/3 of mouse barrel cortex. Mouse studies used transgenic animals with GFP expressed in subsets of inhibitory neurons. In layer 1, axondescending cells receive excitatory input predominantly from layer 2/3 while neurogliaform cells receive stronger input from deeper layers. Layer 2/3 neurons also receive inputs that vary systematically by cell type. Two subtypes of Martinotti cells, distinguished by calretinin (CR) expression, also differ in morphology and intrinsic physiology. CR+ Martinotti cells receive excitatory input predominantly from layer 2/3, while the CR− Martinotti cells also receive strong excitation from layer 4. Irregular-spiking basket cells also receive strong excitatory input from layers 2/3 and 4, but they often have a gap at the top of layer 4, with little or no input. Fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers 2/3 and 4, and only weak input from deeper layers.
Research funds: NIH SY2-3-03-6 Physiological genomics of cortical microcircuits Sacha B. Nelson Brandeis University, Czech Republic Cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. Presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. In order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers (30-50) of fluorescently labeled, hand-sorted neurons. Subsets of pyramidal neurons and GABAergic interneurons were labeled genetically with GFP or anatomically with fluorescent microspheres. Labeled neurons were characterized electrophysiologically and sorted for expression analysis. The resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. Based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types.
Research funds: R03 EY015273 , McKnight Neuroscience of Brain Disorders Award SY2-3-09-1 Axon guidance mediated by localized Ca 2+ signals in the growth cone Hiroyuki Kamiguchi Laboratory for Neuronal Growth Mechanisms, RIKEN Brain Science Institute, Wako, Japan Axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (CAMs). Many guidance cues attract or repel the growth cone via asymmetric Ca 2+ signals. Its turning direction depends on the occurrence of Ca 2+ -induced Ca 2+ release (CICR) through the ryanodine receptor type 3 (RyR3). The activity of RyR3 is controlled by CAMs via cAMP and PKA. In this way, axon-guiding and CAM-derived signals are integrated by RyR3, that serves as a key regulator of axon guidance. Attractive Ca 2+ signals facilitate intracellular membrane transport to the leading front and subsequent VAMP2-mediated exocytosis on the side with elevated Ca 2+ . In contrast, repulsive Ca 2+ signals do not trigger such membrane dynamics. Growth cone attraction, but not repulsion, is prevented by inhibition of VAMP2-mediated exocytosis. The results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery.
SY2-3-09-2 Molecular mechanisms for signaling through plexin-A1
Hitoshi Kikutani, Atsushi Kumanogoh, Toshihiko Toyofuku Research Institute for Microbial Diseases, Osaka University, Suita, Japan Sema3A acts as a guidance cue for axons through a receptor complex comprising neuropilin-1 as the ligand-binding subunit and plexin-A1 as the signal-transducing subunit. The FERM domain-containing GEF, FARP2, associates directly with plexin-A1. Sema3A induces the dissociation of FARP2 from plexin-A1, resulting in activation of FARP2's Rac GEF activity, Rnd1 recruitment to plexin-A1 and down regulation of R-Ras. Simultaneously, the FERM domain of FARP2 sequesters PIPKI␥661 from talin, thereby inhibiting its kinase activity. These activities are necessary for Sema3A-mediated repulsion of outgrowing axons. Plexin-A1 also functions as a ligand binding receptor of a transmembrane semaphorin, Sema6D and contributes to cardiac morphogenesis. Sema6D exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. Plexin-A1 forms a receptor complex with Off-track in the ventricle or with VEGF receptor type 2 in the conotruncal segment, which are responsible for the effects of Sema6D on the respective regions.
Research funds: CREST SY2-3-09-3 Axonal transport elicited by axon guidance molecules Yoshio Goshima Department of Molecular Pharmacology & Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
For the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. We previously reported that Semaphorins3A (Sema3A), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (DRG) . To address if the individual molecules rides on the Sema3A-facilitated cargo, we used time-lapse imaging of several EGFP-fusion proteins expressed in DRG. Sema3A stimulated the transport of Neuropilin-1::EGFP, Plexin-A4::EGFP, and Fyn::EGFP, which are the components of Sema3A signaling, but not Neuropilin-2::EGFP. Interestingly, Sema3A accelerated the anterograde transport of TrkA::EGFP. These facts suggest that Sema3A selectively facilitates the transport of its own signaling components and that Sema3A may modulate NGF-sensitivity of neurites by accelerating the transport of TrkA.
Kozo Kaibuchi Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Japan
Neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. However, it remains largely unknown how neuronal polarity is established. We previously showed that collapsin response mediator protein-2 (CRMP-2) is enriched in growing axon, and play a crucial role in axon specification. CRMP-2 interacts with tubulin dimers to promote microtubule-assembly, and binds to Sra-1, an effector of Rac1 to regulate WAVE-dependent reorganization of actin filaments. CRMP-2 links kinesin-1 to tubulin dimmers and Sra-1, and participates in the kinesin-1-dependent transport of tubulin dimmers and the Sra-1/WAVE complex to growing axons. We also found that the PAR-6/PAR-3 complex and the Ras/PI3-kinase/Akt/GSK-3b pathway are involved in neuronal polarization. Akt appears to phosphorylate GSK-3b and inactivates its kinase activity downstream of Ras/PI3-kinase, thereby increasing non-phosphorylated active CRMP-2 which promotes axon growth. This time, I summarize and discuss functions of these polarity molecules in regulation of neuronal polarity.
Research funds: Grant-in-Aid for Creative Scientific Research SY2-3-09-5 Regulation of actin dynamics during neurite initiation and axon guidance Frank Gertler, Adam Kwiatkowski, Doug Rubinson, Erik Dent, Leslie Mebane MIT, USA Nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. Axons are guided to their targets by motile growth cones. Both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. The Ena/VASP protein family regulates cell migration and axon guidance. Ena/VASP proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. Results of recent work on phenotypes arising in mice lacking all three Ena/VASP proteins (Mena, VASP, EVL) will be presented. Such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. The mutants also contain little if any cortical axonal fiber tracts. Analysis of primary cells from the mutants indicates Ena/VASP function is required for neurite initiation. Mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions.
Kimitaka Anami Department of Psychiatry, National Center Hospital for Mental, Nervous and Muscular Disorders, NCNP, Tokyo, Japan Recent years, studies using EEG and fMRI in simultaneous manner has become flourished. This is because the feasibility that any EEG events is, in principle, able to be mapped on the MRI tomographic view has attracted many researchers. Applications of this methodology are to basic EEG researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. And applications of this methodology is not matured yet but still developing. In this presentation, we will introduce our study using this method to epilepsy and to other EEG events.
Masaya Misaki 1,2 , Takashi Abe 1,2,3 , Shigeyuki Kan 1,4 , Satoru Miyauchi 1,5 1 National Institute of Information and Communications Technology, Kobe, Japan; 2 Japan Society for the Promotion of Science, Tokyo, Japan; 3 Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshim, Japan; 4 Kyushu Institute of Technology, Kitakyushu, Japan; 5 CREST, Japan Science and Technology Agency, Tokyo, Japan
Recording fMRI and an EEG simultaneously is effective for studying spontaneous brain activities. We used this method to examine the relationship between an EEG rhythm and a BOLD signal. Some studies have hypothesized that the hemodynamic response for a change in power of certain EEG frequency bands, such as alpha waves, is canonical in shape. However, the appropriate response shape for a change in the rhythmic EEG has not yet been determined. We measured the EEG and fMRI simultaneously while subjects were in a resting or sleeping state. We applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic EEG waves and fMRI signals without a priori hypothesis of the response shape.
Research funds: CREST of JST and Grant-in-Aid for JSPS Fellows
Norihiro Sadato, Hiroshi Toyoda Department of Cerebral Research, National Institute for Physiological Sciences, Okazaki, Japan Previous studies have demonstrated a nonlinear relationship between Blood oxygenation level dependent (BOLD) response and stimulus parameters. However the origin of this nonlinearity still remains unclear. To investigate the origin for the nonlinearity of BOLD response, we underwent simultaneous measurement of fMRI and near infrared spectroscopy (NIRS) . Temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as BOLD signal were simultaneously measured during a visual stimulation with various durations. To quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. This model was applied to the NIRS measures as well as BOLD responses. The nonlinearity of the response in oxygen extraction fraction (OEF) was also estimated from NIRS measures. The non-linearity of BOLD was almost identical to OEF across the wide range of nonlinearity of the neuronal responses. And hence we conclude that the non-linearity of BOLD responses to the neural activity is mainly due to the nonlinear response of OEF.
The BOLD-fMRI signal is ambiguous regarding the underlying neurophysiology. In our work we attempt a) to better understand the neurophysiological basis of fMRI and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. In one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-Hb concentration (the inverse of BOLD) is related to neuronal inhibition. In another series of experiments, we combine EEG with fMRI in order to identify BOLD correlates of neuronal background rhythms such as alpha rhythm, Mu rhythm, etc. In a third series of experiments, we combine fMRI with the measurement of high-frequency oscillations in EEG. The latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fMRI
Haruhiko Akiyama Tokyo Institute of Psychiatry, Japan Activation of microglia is a part of host defense mechanisms in the brain. Microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. Brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. Nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. Neuroinflammation is a term that refers to such conditions. In Alzheimer's disease (AD), microglia play a central role for phagocytic removal of amyloid beta-protein (Abeta) from the brain. The process is enhanced by complement activation. However, these cellular and humoral responses to Abeta may be toxic to neurons in AD. Neuroinflammation could be a double-edged sword in the brain. In patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration.
Makoto Sawada Department of Brain Life Science, RIEM, Nagoya University, Japan Microglia, macrophage-like cells in the CNS, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the CNS degeneration as well as are one of important cells in the CNS cytokine network. They are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. Activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in AIDS dementia is completely neurotoxic. These suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. Recently we generated HIV-derived nef protein tranduced microglia. They are found to increase both the potential to produce O2-and MPO-like peroxidase activity resulting in the neurotoxicity. Therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. There is abundant evidence that extracellular ATP have an important role in pain signaling. The focus of attention now is on the possibility that ATP receptor of microglia might be involved in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain state is generally resistant to currently available treatments. We recently reported that the expression of P2X4 receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X4 receptors produce a reduction of the neuropathic pain behaviour (2003. Nature 424, 778-783) . More recently, we have reported that brain-derived neurotrophic factor (BDNF) released from microglia by the stimulation of P2X4 causes the depolarizing shift in reversal potential of anion in LI neurons of rats with nerve injury (2005 ( . Nature 438, 1017 ( -1021 . Understanding the key roles of these ATP receptors may lead to new strategies for the management of neuropathic pain.
Research funds: KAKENHI (15209051) SY2-5-05-4 PET imaging of microglia using peripheral benzodiazepine ligands Richard B. Banati University of Sydney, Australia Brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. The response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. Part of the structural and functional plasticity of microglia is the de novo expression of the 18 kDa transposor protein or "peripheral benzodiazepine binding site" (PBBS). The PBBS is bound by the isoquinoline PK11195, which labeled with carbon-11 can be used for positron emission tomography (PET). Using 11C-(R)-PK11195 PET in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional PBBS up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway.
SY2-5-10-1 Application of simulation of light propagation in tissue to NIRS imaging of brain function Eiji Okada Department of Electronics and Electrical Engineering, Keio University, Japan
In NIRS imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. The serious problem of NIRS imaging is ambiguity in light propagation in the head caused by the scattering of tissue. This poses results in poor spatial resolution and contrast in the image. The development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the NIRS imaging. In this study, simulation of light propagation in the head model for NIRS imaging is briefly reviewed. The heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (CSF), has a strong effect on the light propagation in the brain. The sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the CSF. The simulation of NIRS imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs.
Yoko Hoshi Integrated Neuroscience research Team, Tokyo Institute of Psychiatry, Tokyo, Japan Quantification of near-infrared spectroscopy (NIRS) data has been a central issue in the NIRS field. Over the past 25 years, many approaches to quantification have been tried, and in the case that Hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (TRS) and phase-resolved spectroscopy (PRS) has been established. When the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. The temporal profile of detected light intensity measured by TRS carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. Thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. Diffuse optical tomography (DOT) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. In this symposium, I will outline these approaches.
SY2-5-10-3 Brain functional imaging in cerebral ischemic disorders: Comparison of NIRS and fMRI Kaoru Sakatani Department of Neurological surgery, Nihon University School of Medicine, Tokyo, Japan
We compared the evoked cerebral blood oxygenation (CBO) responses measured by NIRS and activation maps of BOLD-fMRI in stroke patients with mild and severe (misery perfusion) cerebral ischemia (CI). In the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (PSMC) during contralateral motor tasks. The PSMC on the non-lesion side exhibits normal CBO response pattern. On the lesion side, the mild CI did not affect the CBO response pattern, but the severe CI caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. The mild CI caused only slight reduction of the activation volumes of BOLD imaging on the lesion side; however, the severe CI, caused markedly reduction of the activation volumes on the lesion side. Misery perfusion caused marked reductions of activation volumes of BOLD imaging associated with an increase of deoxyhemoglobin during activation. BOLD-fMRI should be performed, giving consideration to the baseline circulatory conditions.
Masato Fukuda, Toru Uehara, Masahiko Mikuni Department of Psychiatry and Human Behavior, Gunma University Graduate School of Medicine, Gunma, Japan Near-infrared spectroscopy (NIRS) has been increasingly employed in psychiatry researches such as personality (2005 . Neuropsychobiology 52, 45), aging (2004 . NeuroImage 22, 1715 , and psychiatric disorders ("Progress in Schizophrenia Research", Nova Science Publishers, 2006) . Frontal lobe reactivity was investigated using multichannel NIRS machines in unipolar depression, bipolar depression, and schizophrenia (2004. Biol. Psychiatry 55, 501; 2006. NeuroImage 29, 172) by monitoring changes of oxy-hemoglobin concentration ([oxy-Hb]) every 0.1s during a verbal fluency task. The unipolar depression was characterized by smaller [oxy-Hb] increase, the bipolar depression by comparable but delayed [oxy-Hb] increase, and the schizophrenia by reduced [oxy-Hb] increase during the task period followed by [oxy-Hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively.
Collaborators: Itsuro Ida, Akihiko Oshima, Makoto Ito, Tomohiro Suto, Masaki Kameyama, Yutaka Yamagishi, Toshimasa Sato, Masashi Suda SY2-5-10-5 Clinical application of NIRS to neurorehabilitation
Optical imaging using near-infrared spectroscopy (NIRS) is suitable for assessing cortical activation during human gait because of its flexibility and portability. In healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. In hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. A longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. Sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. Partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. Thus NIRS may contribute to establishing brain-based strategies for neurorehabilitation.
Research funds: Funds for comprehensive research of aging and health SY2-5-10-6 Measurement of language related brain activities during recovery from aphasia Eiju Watanabe 1 , Yumiko Muroi 2 , Chizuru Nakajima 2 1 Department of Neurosurgery, Jichi Medical University, Tochigi, Japan; 2 Department of Neurosurgery, Tokyo Metropolitan Police Hospital, Tokyo Mechanism which supports the recovery of language after aphasia is not well understood. It has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. Several studies suggest the compensation by these areas using fMRI and PET. We used optical topography (OT) to know the participation of these areas during the recovery from the aphasia. We measured 17 aphasics who showed recovery from the aphasia after apoplexy. Word generation task was used. In seven cases OT was measured more that twice. Seven cases showed the activity on the non-dominant frontal lobe. They all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. These findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase.
Tamami Fukushi Research Institute of Science and Technology for Society (RISTEX), Japan Science and Technology Agency (JST), Tokyo, Japan Recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. For example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fMRI) and near infrared spectroscopy (NIRS) show the possibility to read the mind of others, which may lead the criminal and commercial applications. Brain-machine interface (BMI) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. In advance to the expansion of "Applied Neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". In this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. We will also discuss the future perspective of the neuroethics in Japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public.
Judy Illes Stanford University, USA Akin to genetic testing in the 1990s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. Potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. We will discuss how these issues converge in 21st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future.
SY2-6-06-3 Neuroethics from primate's eyes Naotaka Fujii BSI, Laboratory for Symbolic Cognitive Development, Japan
Neurophysiologists working on monkeys have been trying to understanding how their brains are working. The aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. This was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. However, recent advancement of technology has changed the world. Findings in primates can be directly applied to human studies with or without researcher's intention. Technologies invented in primate physiology are now readily transferred into human without much discussion. Brain machine interface is one example. Now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. As an experienced primate neurophysiologist but with little ethical training, I will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics.
SY2-6-06-4 Neuroethics of nurturing the brain Takao K. Hensch Critical Period Mechanisms Group, RIKEN Brain Science Institute, Japan At no time in life is the brain so easily shaped by experience than in infancy and in early childhood. It is during these critical periods that neural circuits acquire language and other basic brain functions. Unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. Conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. This raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? To what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? How do we determine what is good or bad for the maturing brain?
Research funds: RIKEN BSI SY2-6-06-5 Neuroethics beyond laboratories and across cultures Daofen Chen National Institute of Neurological Disorders and Stroke, USA Recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. Potential uses of many of these recent advances go beyond their intended medical applications. A growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. While it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. Moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. We will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research.
SY2-6-06-6 Bridging neuroscience and public: Neuroethics in cultural contexts Osamu Sakura 1,2 1 Interfaculty Initiative in Information Studies, University of Tokyo, Tokyo, Japan; 2 Research Institute of Science and Technology for Society (RISTEX), JST, Japan
To bridge between neurosciences and public society-it should be one of the important aims of Neuroethics. For this purpose we need to draw the outline of neuroscience within the cultural context. The method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. Although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases.
Mitsuru Kawamura 1,2 , Akira Midorikawa 3 , Yoshiki Kaneoke 4 , Shinichi Koyama 1 , Masato Taira 5 , Argye Hillis 6 1 Showa University School of Medicine, Japan; 2 CREST, JST, Saitama, Japan; 3 National Institute of Neuroscience, NCNP, Tokyo, Japan; 4 National Institute for Physiological Sciences, Okazaki, Japan; 5 Nihon University, Tokyo, Japan; 6 Johns Hopkins University, Baltimore, USA This symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. Focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. The topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. We will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience.
Research funds: KAKENHI (17022035), CREST SY2-6-11-2 Impairment of surface perception in patients with ventral occipital damages Shinichi Koyama 1 , Mitsuru Kawamura 1 , Akira Midorikawa 2 , Yoshiki Kaneoke 3 , Masato Taira 4 , Argye Hillis 5 1 Showa University, Tokyo, Japan; 2 National Institute of Neuroscience, NCNP, Tokyo, Japan; 3 National Institute for Physiological Sciences, Okazaki, Japan; 4 Nihon University, Tokyo, Japan; 5 Johns Hopkins University, Baltimore, USA We examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. Patient 1 was a 61-year-old woman with bilateral damage in the fusiform and parahippocampal gyri. Although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (Gregory 1970) . In addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. Patient 2 was a 63-year-old men with bilateral damage in the lingual and fusiform gyri. His recognition of faces and objects became poorer when they were presented in inverted pictures. Based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed.
Research funds: Grant-in-Aid for JSPS Fellows SY2-6-11-3 How do pictorial cues influence 3D information processing in the parietal association cortex?
Masato Taira 1,2 1 ARISH, Nihon University, Tokyo, Japan; 2 Department of Applied System Neuroscience, Nihon University Graduate School of Medical Science, Tokyo, Japan Pictorial cues are one of the most influenced cues for 3D perception. Basically, it is thought that the parietal association cortex processes 3D visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. However, recent studies have suggested that there are many crosstalk of 3D information between these association areas. Our recent studies have shown that a group of neurons in the parietal cortex (area CIP) is involved in perception of 3D surface orientation and used both disparity and pictorial cues. Functional MRI data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in 3D perception. Furthermore, human psychophysical data gives us some insights of how the pictorial cues influence 3D information processing in the parietal association cortex.
Research funds: KAKENHI 17022038 SY2-6-11-4 Case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects Akira Midorikawa 1 1 National Center of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan It has been presented that severe type of Bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. Up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. In this report, I will talk about a patient with Bálint syndrome due to degenerative disease known as posterior cortical atrophy (PCA), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. The crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. In addition, discrepancy between the patients who could walk and not walk will be discussed.
Research funds: CREST, Japan Science and Technology Agency SY2-6-11-5 Neural mechanism underlying visual perception of motion as revealed by non-invasive human study Yoshiki Kaneoke Department of Integrative Physiology, National Institute for Physiological Sciences Magnetoencephalography (MEG) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. Thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. Our MEG studies have revealed several important findings in the human visual motion detection system. First, the response properties for the apparent motions indicate the importance of the human MT/V5+ for the perception and the existence of the parallel processing for the motion and light blinking. Second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. Third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. Based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception.
Research funds: KAKENHI (15500221) SY2-6-11-6 Neural correlates of visual attention Argye Hillis 1 , Mitsuru Kawamura 2 , Akira Midorikawa 3 , Yoshiki Kaneoke 4 , Shinichi Koyama 2 , Masato Taira 5 1 Johns Hopkins University, USA; 2 Showa University, Tokyo, Japan; 3 National Institute of Neuroscience, NCNP, Tokyo, Japan; 4 National Institute for Physiological Sciences, Okazaki, Japan; 5 Nihon University, Tokyo, Japan In this paper I report a series of studies of Unilateral Spatial Neglect (USN) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. Other data reveal that different types of USN are observed when there is hypoperfusion of temporal cortex versus parietal cortex. Still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. Finally, I will review new evidence that right USN is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of USN is very different from the distribution of types of USN after right hemisphere stroke.
Takeo Kubota, Takae Hirasawa, Kaoru Nagai Department of Epigenetic Medicine, University of Yamanashi Faculty of Medicine, Chuo, Yamanashi, Japan How are brains controlled molecularly? This is one of fundamental questions in neuroscience. Several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. Epigenetics is one of such systems to control expression of the genes not based on DNA sequence, but based on 'beyond the DNA sequence' (chromatin modifications and small RNAs). The failure of this system is known to result in neurodevelopmental diseases, such as an autistic Rett syndrome. It has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. These findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. Topics from update epigenetic researches in neuroscience will be discussed in this symposium. As one of epigenetic disorders, we introduce studies of Rett syndrome (RTT) and its model mouse. RTT is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. Mutations of the MeCP2 gene, encoding methyl-CpG binding protein 2, cause RTT. MeCP2 acts as a transcriptional silencer. Abnormal expression of some genes due to MeCP2 dysfunction is thought to be the first step of RTT pathophysiology. To understand how MeCP2 mutation makes RTT, we have two approaches that are morphological investigation of brain and discovery of MeCP2-downstream genes. Using mecp2-null mice (mecp2 −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. Premature synaptogenesis may be the initial neuronal changes of RTT. We also found that MeCP2 directly regulates expression of Insulin-like growth factor binding protein 3 (IGFBP3) gene in human and mouse brains. Pathological features of mecp2 −/y have the similarity of IGFBP3 transgenic mice, which show reduction of early postnatal brain growth. Over-expression of IGFBP3 due to lack of MeCP2 may lead to delayed brain maturation. Growing evidence suggests that alterations in the epigenetic status such as DNA methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. However, in contrast to mRNA profiling, there are few established methods for profiling the genomewide epigenetic status to date. We developed a method for profiling the genome-wide DNA methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. In this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between DNA methylation and mRNA expression patterns will be presented. Understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. The recent finding that Long Interspersed Nucleotide Elements-1 or L1 (LINE-1) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. Together with their mutated relatives, retroelements sequences constitute 45% of the mammalian genome with L1 elements alone representing 20%. The fact that L1 can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. This characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. However, the extent of the impact of L1 on the neuronal genome is unknown. The characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. SY2-7-07-6 Notch-induced acquisition of astrocyte differentiation potential of neural stem cells Kinichi Nakashima 1 , Jun Kohyma 1 , Tetsuya Taga 2 , Masakazu Namihira 1 1 Grad. Sch. Biol. Sci., NAIST, Ikoma, Japan; 2 Inst. Mol. Embryol. Genet., Kumamoto Univ., Kumamoto, Japan Neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. We have previously reported that GFAP-positive astrocyte differentiation is dependent on the transcriptional activity of STAT3. A CpG dinucleotide in the STAT3 recognition sequence within the GFAP gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling STAT3 to induce GFAP expression. Thus, it is conceivable that the epigenetic modification such as DNA methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. Here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of Notch-signaling. The activated Notch-signaling accelerates demethylation of STAT3 binding element in the GFAP gene promoter.
SY2-8-12-1 Neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke Masayasu Matsumoto Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, Japan
In order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. In the present symposium, I will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. (1) Which type of brain infarction will be a future target of this therapeutic approach? (2) Which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? (3) Which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? The present research status and future directions will be presented and fully discussed.
Research funds: KAKENHI SY2-8-12-2 Gene therapy for cerebral ischemia Setsuro Ibayashi, Hiroaki Ooboshi Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Cerebrovascular disease is the leading cause of the disabled people in Japan and Western countries. Gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. Cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. Several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. As for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. Our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. Approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. Thus, gene therapy has a potential for the future therapy against cerebral ischemia.
Isao Date Department of Neurological Surgery, Okayama University, Okayama, Japan Cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. In this presentation, the author will summarize our recent basic research and clinical application reported in the literature. It is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. In order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. We transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. We have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. As donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. The effect of neural protection increases when neurotrophic factor secreting genes such as GDNF were inserted into neural stem cells. Cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. We now know that (1) motor function may recover after minor injury to the primary motor cortex, (2) this recovery is, at least in part, associated with reorganization of cortical motor representation, (3) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and (4) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. Furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. Virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. Those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function.
Nobuyuki Nukina Laboratory for Structural Neuropathology, Riken Brain Science Institute, Japan A major hallmark of the polyglutamine (pQ) diseases is the formation of pQ inclusions. Recently, misfolding has come to be considered one of the primary factors for pQ protein aggregation, although, the nature of misfolding is not yet well known. The protein misfolding induced by pQ expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pQ. Expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. The surface of the mutant myoglobin with expanded pQ was partially unfolded and destabilized. We also investigated the early phase of fibrillization by small-angle X-ray scattering and electron microscopic studies, revealing that the expansion of pQ to 50 repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. This structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pQ diseases. Furthermore using cellular model system we also studied the aggregates interacting proteins (AIPs) by analyzing the purified polyglutamine inclusions and the lists of AIP including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of AIPs in the disease cascades.
SY3-1-01-3 Neuronal dysfunctions in dentatorubralpallidoluysian atrophy (DRPLA) Shoji Tsuji 1 , Toshiya Sato 2 , Mitsunori Yamada 3 1 Department of Neurology, The University of Tokyo, Tokyo, Japan; 2 Center for Bioresource-based Researches, Japan; 3 Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan
To investigate molecular mechanisms of neurodegeneration in DRPLA, a polyglutamine disease caused by expansions of CAG repeats of DRPLA gene, we have established transgenic mice harboring a single copy of the full-length human mutant DRPLA gene with 129 CAG repeats. The Q129 mice exhibited neurological phenotypes similar to juvenile type of DRPLA characterized by ataxia, myoclonus and epilepsy. Electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. Neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to DRPLA. Expression profiling analyses revealed down-regulated genes including cAMP responsive genes. These results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in DRPLA. Huntington's disease (HD) is caused by an expansion of a CAG repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. Using a transgenic mouse model of HD, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. Further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in HD, and understand how gene-environment interactions can modulate these pathogenic pathways. Our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for HD and related brain disorders.
SY3-1-06-1 Control of neural organization in the developing cerebral cortex Yasuto Tanabe Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan
In the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. Over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, Cajal-Retzius cells, cortical projection neurons, and cortical interneurons. The analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. In the first part of my talk, I would like to deal with the issue of ontogeny of Cajal-Retzius cells, and present the way Cajal-Retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. Then, I would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex.
Research funds: KAKENHI 17390086, 17023061 SY3-1-06-2 Mechanisms of the regulation of neuronal migration and corticogenesis Kazunori Nakajima 1,2 1 Dept. of Anat., Keio Univ. Sch. of Med., Tokyo, Japan; 2 Inst. of DNA Med., Jikei Univ. Sch. of Med., Tokyo, Japan
Mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. To determine whether the migration from the ventricular zone (VZ) to beneath the marginal zone (MZ) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the MZ. We analyzed VZ cells and cells from the intermediate zone (IMZ) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a Reelin-independent manner. These findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the MZ. In silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as Reelin signaling.
Kenji Shimamura Division of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan
Neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. To investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. We found that a morphogen-like activity of Sonic hedgehog (Shh) precisely defines positions of neurons with distinct properties, and that some GABAergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. We also provide evidence that Shh produced by the zona limitans intrathalamica (ZLI), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. Our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. Layers and nuclei are important functional units in the vertebrate CNS. Neurons in these structures have common physiological and anatomical features. Despite their importance, mechanisms for nucleogenesis are poorly understood. We focused on the lower rhombic lip (lRL)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (EYFP) gene, to study the process of nucleogenesis. After the unilateral transfer of EYFP to the lRL of embryonic day 12.5 mice, EYFP-labelled neurons migrate tangentially from the lRL in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. The former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. Before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. Perturbation experiments such as introduction of dominant negative constructs and siRNA suggested involvement of several molecules in the migration of these neurons. The brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. Here we show that Ca 2+ signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. An acute administration of ethanol reduced the frequency of transient Ca 2+ elevations in migrating neurons and cGMP levels, and increased cAMP levels. Experimental manipulations of these second messenger pathways, through stimulating Ca 2+ and cGMP signaling or inhibiting cAMP signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. Each second-messenger has multiple but distinct downstream targets, including CaMKII, calcineurin, PP1, Rho GTPase, MAPK and PI 3 K. These results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways.
SY3-2-02-1 Membranes, water and diffusion Denis J. Le Bihan SHFJ/CEA, France Among 1905 Einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. Molecular diffusion was explained by Einstein on the basis of the thermal random translational motion of molecules. In the mid 1980s it was shown that water diffusion in the brain could be imaged using MRI. A dramatic application of diffusion MRI has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. Also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. This feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. More recently, it was discovered that diffusion MRI could detect transient swelling of activated cortical cells. This represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. This approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes.
SY3-2-02-2 Diffusion tensor fiber tractography using a 3tesla MR system Yukio Miki Department of Diagnostic Imaging and Nuclear Medicine, Kyoto University, Kyoto, Japan Diffusion tensor imaging (DTI) is an MR imaging technique that is sensitive to orientation of mobility in water molecules. DTI reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. White matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. DTI of the brain can be reconstructed to display 3D macroscopic fiber tract architecture, in a process known as fiber tractography. With recent advances in actively shielded 3-T magnets and parallel imaging techniques, high-field MR imaging has become practical in clinical settings. We have demonstrated that depiction of most fiber tracts was improved on 3-T tractography compared to 1.5 T. We have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using 3-T MR was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. To test whether MR tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. We analyzed the data of 14 patients who presented to our institute with sensorimotor symptoms. There was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. Next, we applied the technique to patients with evolving symptoms after admission to hospital. We specifically assessed the change in the tract-infarct relationship over time. The data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on DWI diminished. Finally, we studied whether the use of tractography could help predict a patient's prognosis. To simplify the analysis, we specifically focused on patients with lenticulostriate artery (LSA) infarcts. We analyzed the correlation between the extent of CST involvement within the infarcts and the severity of motor deficits. The data indicated that the tractographic technique could be useful to predict a patient's outcome.
SY3-2-02-4 Anatomical and functional tractography: A combined approach with diffusion tractography and corticocortical evoked potential Riki Matsumoto Department of Neurology, Kyoto University Graduate School of Medicine, Japan Recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. The probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. However, the results depend merely on anisotropic movement of water molecules and need validation. For presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. Combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. Results of the combined approach will be presented. Parkinson disease (PD) is the second commonest neurodegenerative disorder after Alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. Pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. Although familial forms of PD can be encountered up to 15% of the patients, the remaining cases are sporadic. It has been postulated that nigral neurodegeneration in PD is induced by the interaction of genetic risk factors and environmental factors. Epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of PD; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. In addition, certain employments were reported to be associated with increased risk of PD; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. These studies suggest importance of environmental factors in the pathogenesis of PD. Recent progress in these areas will be discussed.
Masami Ishido National Institute for Environmental Studies, Tsukuba, Japan There are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. It has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. Epidemiologic studies also suggested that Parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. Thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. Oral administration of an endocrine disruptor (12-60 mg/kg) into male Wistar rats (from 5 days to 3 weeks of age) significantly caused hyperactivity at 4-5 weeks old. Immunohistochemical analyses of the brain tissues at 8 weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. TUNEL-positive cells were seen in the substantia nigra. Thus, environmental insults in early life may be of particular etiologic importance.
SY3-2-07-3 Non-thermal effects of mobile phones upon the rat brain Leif G. Salford, B. Persson, J. Eberhardt, G. Grafstrom, L. Malmgren, A. Brun Dept of Neurosurgery and the Rausing Laboratory, Lund University, Sweden
We have shown that RF electromagnetic fields can cause significant leakage of albumin through the BBB of exposed rats as compared to non-exposed animals. One remarkable observation is that SAR values around 1 mW/kg give rise to a more pronounced albumin leakage than higher SAR values -all at non-thermal levels. If the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other RF emitting sources could be managed by reduction of their emitted energy. The SAR value of around 1 mW/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about 150 -200 meters from a base station. Another remarkable observation in our studies is the fact that a significant (p < 0.002) neuronal damage is seen in rat brains 50 days after a 2 hour exposure to GSM at SAR values 200, 20 and 2 mW/kg. We have followed up this observation in a study where 96 animals were sacrificed 14 and 28 days respectively after an exposure for 2 hours to GSM mobile phone electromagnetic fields at SAR values 200, 20, 2, 0.2 and 0 (controls) mW/kg. Significant neuronal damage is seen after 28 days and albumin leakage after 14. Our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after 28 and 50 days.
SY3-2-07-4 The mitochondrial toxin 3-nitropropionic acid: An environmental toxin to study striatal degeneration in Huntington disease Emmanuel Brouillet Neuronal Death Laboratory, URA CEA-CNRS 2210, France Huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding Huntingtin. The mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in Huntington disease, are unknown. The behavioral and anatomical similarities found between Huntington disease and animal models of striatal degeneration using the environmental toxin 3-nitropropionic acid (3NP) support the hypothesis that mitochondrial defects could play a role in Huntington disease. We will discuss the mechanisms of 3NP toxicity and show that 3NP and mutated Huntingtin have certain mechanisms of toxicity in common. In particular, we show that mutated Huntingtin can alter the expression of mitochondrial complex II, the respiratory chain enzyme specifically inhibited by 3NP. In summary, the 3NP story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. We recently demonstrated that rats received intrecisternal injection of 6-OHDA or environmental chemicals, such as bisphenol A, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at 5 days of age showed behavioral hyperactivity at 4-5 weeks of age. Immunohistochemical studies revealed a deficit in the development of dopamine (DA) neurons. Adult rats received these chemicals showed degeneration in nigro-striatal DA neurons similarly to Parkinson's disease. In this study, we investigated the mechanism of 6-OHDA-induced neurotoxicity, using PC12 cells as an in vitro model system. We observed the generation of reactive oxygen species (ROS) and p-quinone via auto-oxidation of 6-OHDA. We also characterized the oxidation of cellular proteins by 6-OHDA and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. We will discuss about apoptotic cell death pathway including cytochrome C release and caspase activation induced by 6-OHDA, ROS and p-quinone.
SY3-2-07-6 Environmental factors in the pathogenesis of Alzheimer's disease Joanna L. Jankowsky California Institute of Technology, USA Epidemiological studies indicate that environmental factors significantly influence the risk of developing Alzheimer's dementia. Foremost among those factors are education, occupation, and leisure activities. Although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. Animal models for Alzheimer's disease have recently been used to explore the mechanism of this effect. Transgenic mice designed to recapitulate Alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. Both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. Intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. The efficacy of synaptic inhibition depends on the number of GABA A receptors expressed on the neuron surfaces. In the present study, we have elucidated the role of PRIP (PLC-related inactive protein) in trafficking of the receptors by analyzing PRIP knockout (KO) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of KO mice, suggesting the dysfunction of the ␥2 subunit-containing receptors. We then examined the mechanisms by which PRIP molecule regulates cellsurface expression of ␥2 subunit-containing receptors. Disruption of the direct interaction between PRIP and the  subunit of receptors by PRIP-binding peptide inhibited cell-surface expression of ␥2 subunit-containing receptors in GH3 and HEK293 cells. Constitutive internalization of the receptors was also modified by the peptide. Collectively, PRIP molecules are involved in trafficking of ␥2 subunit containing GABA A receptors to/from cell-surface membrane.
Research funds: KAKENHI (16109010) SY3-3-03-2 Involvement of BDNF in the induction of LTP at visual cortical inhibitory synapses Yukio Komatsu Dept. Visual Neurosci., Res. Inst. Environ. Med., Nagoya Univ., Nagoya, Japan High-frequency stimulation (HFS) induces long-term potentiation (LTP) at inhibitory synapses of layer 5 pyramidal cells in rat visual cortex. This LTP requires a postsynaptic Ca 2+ rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that LTP is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. In this study, we investigated whether BDNF could act as such retrograde messengers. LTP did not occur when HFS was applied in the presence of K252a at 200 nM, inhibiting Trk receptor tyrosine kinases selectively at that dose. HFS induced LTP when K252a application was started soon after HFS or when K252a was loaded into postsynaptic cells. LTP did not occur in the presence of TrkB-IgG or anti-BDNF antibodies. In cells loaded with BAPTA, the addition of BDNF to the medium enabled HFS to induce LTP without affecting basal synaptic transmission. These results suggest that BDNF released from postsynaptic cells activates presynaptic TrkB, enabling the induction of LTP.
Research funds: KAKENHI (17300101) SY3-3-03-3 Autocrine mGluR1 activation in cerebellar Purkinje cells regulates GABA-mediated synaptic inhibition Trevor Smart, Ian C. Duguid University College London, UK In the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-Purkinje cell (IN-PC) synapses. Endocannabinoids, via CB1 receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic NMDA receptors, induces a sustained increase in GABA release. We now report that dendritically released glutamate also acts as an autocrine messenger, activating mGluR1 on PCs to enhance synaptic inhibition via the release of endocannabinoids. This process was triggered by repetitive PC stimulation and blocked by uncoupling the mGluR1-Gq/11 transduction pathway as well as being initiated by direct mGluR1 activation during PC depolarisation. Glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mGluR1 activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. Our study suggests that autocrine mGluR1 activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission.
SY3-3-03-4 Regulatory mechanism of inhibitory synaptic transmission in the cerebellum Shin-ya Kawaguchi 1,2 , Tomoo Hirano 1,2 1 Dept. Biophys., Grad. Sch. Sci., Kyoto Univ., Kyoto, Japan; 2 CREST, JST, Kawaguchi, Japan At the GABAergic synapses between inhibitory interneurons and a Purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by GABA A receptors (rebound potentiation: RP). The signaling cascades regulating the induction of RP has been clarified. The balance of activities of protein kinases and phosphatases determines whether RP is induced or not. Here we show another molecular mechanism involved in the RP induction. Using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of GABA A receptormediated responses is brought about. RP induction was impaired by inhibition of Ca 2+ -activated protease calpain or by disturbance of association of GABA A receptor ␥2 subunit with GABARAP (GABA A receptor associated protein). Binding of GABARAP to microtubule was also involved in the regulation of RP. Our results suggest that structural alteration of GABARAP caused by calpain activity is critical for establishment of RP.
SY3-3-08-1 Contribution of Hebb's "Organization of Behavior" to the development of brain science Masataka Watanabe Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, Tokyo, Japan More than 50 years have passed since the publication of "Organization of behavior". This book has been one of the most influential books in neuroscience. Around the time of the 50th anniversary of this book, special issues and articles concerned with this book appeared in several journals. His idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. However, he also made some mistakes, for example he advocated frontal lobotomy. In this symposium, I will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and Hebb synapse.
SY3-3-08-2 Detection of cell assembly in neuroscience experiments and brain-machine interfaces Yoshio Sakurai 1,2 , Susumu Takahashi 2 1 Department of Psychology, Kyoto University, Kyoto, Japan; 2 CREST, Japan Science & Technology Agency, Japan
The reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. First in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. Second, we demonstrate a newly developed method, brain-machine interface (BMI), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. We introduce our recent BMI system with independent component analysis (ICA) and specific multi-electrodes and show some neuronal and behavioral data obtained by the BMI system. Hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. How relevant is it in protein synthesis-dependent late-phase plasticity (LP)? Synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific LP. Using live-imaging techniques, we measured entry of Vesl-1S-EGFP into dendritic spines (VE trapping) of rat hippocampal neurons in culture, and found that VE trapping activity serves as synaptic tag in many criteria. VE trapping conforms to the Hebb's rule in a sense that it required both presynaptic activity and postsynaptic NO-PKG pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. NO spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. Thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states.
Tomoki Fukai Laboratory for Neural Circuit Theory, RIKEN Brain Science Institute, Saitama, Japan
In the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. Such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by Hebbian learning. In this talk, I will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. I propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. In this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. However, the same experimental findings can lead us to a different type of cortical network models. In this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". I will discuss the characteristic properties of both types of models and the possible implications in cortical computations.
Research funds: KAKENHI (17022036) SY3-3-08-5 Impact of Hebbian hypothesis on neuroscience Keisuke Toyama Shimadzu Institute of Basic Technology, Seikacho, Hikaridai, Kyoto 619-0237, Japan Hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and Hebbian synapses to construct that cell assembly. The former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. Recently, these concepts refreshed the impact with new dressing of 'dynamics'. Cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic Hebbian synapses conceptualized as the spike-timing dependent plasticity (STDP). I would like to discuss about speaker's talks in this context. SY3-4-04-1 Tonic GABA A receptor mediated conductances: Properties, functions and plasticity Alexey Semyanov RIKEN Brain Science Institute (BSI), Japan
Communications mediated by non-synaptic receptors are important for information processing in the brain. High affinity extrasynaptic GABA A receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of GABA. This phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. Our findings have revealed functional significance of GABA A receptors mediated tonic conductance in the hippocampus. We have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. The magnitude of the conductance is regulated by efficiency of GABA uptake and membrane potential. GABA A receptor mediated tonic conductance undergoes adaptive plasticity. It is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. In mice lacking GAD65 the amount of the tonic inhibition is reduced in CA1 hippocampal interneurons, while unchanged in pyramidal cells.
SY3-4-04-2 Modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal CA1 region Yoshihiko Yamazaki 1 , Yasukazu Hozumi 2 , Kenya Kaneko 1 , Satoshi Fujii 1 , Hiroshi Kato 1 1 Dept. of Neurophysiol., Yamagata Univ. Sch. of Med., Yamagata, Japan; 2 Dept. of Anat. & Cell biol., Yamagata Univ. Sch. of Med., Yamagata, Japan Glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. We focused on interneuron/peri-interneuronal glial cell (PG) pairs in the hippocampal CA1 region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. Direct depolarization of PG suppressed the excitatory postsynaptic currents in an adjacent interneuron. This suppression was inhibited by adenosine A1 receptor antagonist. Moreover, PG activation modulated the firing pattern of the interneuron. Since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via PG would have marked effects on the information processing of neurons in the CA1 region.
Research funds: KAKENHI (15082201) SY3-4-04-3 Carbachol-induced beta oscillations in rat hippocampal slices Kiyohisa Natsume, Jun Arai Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Fukuoka, Japan Rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. The input involves the generation of hippocampal rhythm, theta, gamma rhythm. To mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. Carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. In the present paper, we introduce the beta oscillations. The application of 30 M carbachol induces beta oscillations which occur intermittently with the interval of 20-30 s. During the intervals, gamma oscillations are induced. The mean frequency of the oscillations is 16.9 ± 0.9 Hz (mean ± S.E.M.). The oscillations are induced via muscarinic M1, 3, 4 receptors. The frequencies of them are significantly decreased by the application of bicuculline, a GABA A antagonist. They are sensitive to bicuculline, while theta oscillations are not. It is indicated that the character of beta oscillations are different from those of theta oscillations. The neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. To examine ongoing network interactions among these distinct cortices during neocortical slow oscillations (1-3 Hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. We have found that (1) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, (2) no bimodal up-down transition was present in hippocampal neurons. Hippocampal granule cells were directly driven by entorhinal up-state activity, while CA3 and CA1 neurons discharged during both up and down states, (3) gamma and fast (ripple) oscillations were observed in hippocampal CA1 area irrespective of up-down transition. These observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. The cholinergic neurons in the mesopontine reticular formation (MPRF) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the MPRF elicits REM sleep and hippocampal theta wave. In this study, we recorded neuronal activity in the MPRF and pontine and hippocampal EEG during REM sleep and investigated time-relationship between them. Our results are summarized as follows: (1) most of the MPRF neurons were active during REM sleep; (2) the MPRF activity increased over ten seconds before transition from NREM to REM sleep, i.e. from non-theta to theta period;
(3) the theta wave was instantaneously accelerated concomitant with activation of the MPRF neurons. These results suggest that cholinergic neuron in the MPRF is important in generation and maintenance of REM sleep and theta wave. Because hippocampal theta waves are involved in memory consolidation during REM sleep, our findings might help to clarify this mechanism.
Research funds: KAKENHI (17605001) SY3-5-09-1 Clock mechanisms of the SCN involving in the entrainment to the morning and evening light Sato Honma, Natsuko Inagaki, Nobuko Tokumaru, Ken-ichi Honma Dept. Physiology, Hokkaido Univ. Grad. Sch. Med., Sapporo, Japan
The circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. The mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. By using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. Rats and mice were housed under light-dark (LD) 18:6 h and LD 6:18 h. Clock gene expression patterns in the entire SCN were examined by in situ hybridization on the first day of constant darkness. The phase relations of Per1 and Per2 rhythms suggest that light-on resets Per1 rhythms in both light conditions, while Per2 rhythm also relates to the light-off. In cultured SCN of transgenic mice expressing luciferase under the control of Per1 promoter, we observed two bioluminescent peaks a day only in the anterior SCN from the mice kept in LD 18:6. The finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior SCN. The suprachiasmatic nucleus (SCN) is the center of the mammalian circadian clock. Tissue transplantation of the SCN restores the behavioral circadian rhythm in SCN-lesioned mice in spite of the impaired neural connection with the host brain. We have investigated whether grafted SCN regulates the circadian oscillator in peripheral organs using the SCN-transplanted mice that have a limited time information transmission paths. As a result, the grafted SCN restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. Many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, Per1 and Per2 showed low amplitude of oscillation. The findings suggest that diffusible signal molecules released from the transplanted SCN entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes.
SY3-5-09-3 Genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes Norio Ishida Clock Cell Biology Group, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan Recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. Adrenal gland is important for circadian regulation for mammalian peripheral clocks. To identify circadian expressed genes regulated by adrenal glands pathways, we performed DNA microarray analysis using hepatic RNA from adrenalectomized (ADX) and sham-operated mice. We identified 169 genes that fluctuated between day and night in the livers, 100 lost circadian rhythmicity in ADX mice. These included the genes for key enzymes of liver metabolic functions such as glucokinase, HMG-CoA reductase, and glucose-6-phosphatase. The present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as CLOCK, and the other 100 genes depend on adrenal glands pathway such as glucocorticoids.
Hitoshi Okamura Kobe University Graduate School of Medicine, Japan Light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. In the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (SCN)-sympathetic nervous system. Moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. The abolishment after SCN-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. The surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and CSF. The light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment.
SY3-5-09-5 Neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus Shigenobu Shibata, Naomi Hayasaka, Takashi Kudo, Tsuyoshi Yaita Department of Pharmacology, School of Science and Engineering, Waseda University, Tokyo, Japan
The clock genes are expressed not only in the suprachiasmatic nucleus (SCN) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. In the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. Although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the SCN, we do not know the resetting signals on peripheral clock function. Communication between the SCN and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. This symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function.
SY3-6-10-1 A to Z of gene transfer with adenoviral vector-Application to neuronal birth date-specific gene transfer
Using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. When adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (E)10.5 to E14.5, the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. This technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. This adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. I will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk.
Research funds: CREST SY3-6-10-2 Live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors Kaori Kashiwagi, Naoaki Saito Lab. Mol. Pharmacol. Biosig. Res. Ctr, Kobe Univ, Kobe, Japan
Live imaging analysis has revealed that each protein kinase C (PKC) subtype shows spatio-temporally distinct targeting in response to various stimuli. We demonstrated that the trans-synaptic stimulation induced translocation of ␥PKC-GFP in cerebellar slices from bitransgenic mice (NSE-tTA/TetOp-␥PKC-GFP) which express ␥PKC-GFP in time and region-specific manner. This translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of Purkinje cells. In order to gain further insight in to the molecular mechanisms of PKC translocation, we introduced viral vectors to primary cultured Purkinje cells. The propagative ␥PKC-GFP translocation was also observed in cultured Purkinje cells derived from NSE-tTA mice. The molecular mechanisms of PKC translocation in Purkinje cells were analyzed by live imaging with various kinds of viral vectors. The combination of TG mice and viral vectors is useful to understand the physiological role of PKC in the specific neuronal cells.
Research funds: KAKENHI (17024040) SY3-6-10-3 Visualization and manipulation of the signaling systems in the CNS using Sindbis viral vectors Sho Kakizawa Dept. of Pharmacol., Grad. Sch. of Med., The University of Tokyo, Tokyo, Japan Virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. Because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. Sindbis virus is an enveloped plus-strand RNA virus belonging to the Alphavirus genus of the Togaviridae family. The sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. These facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. In this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol 1,4,5-trisphosphate, in the cerebellar Purkinje cells.
Research funds: Grant-in-Aid for Scientific Research on Priority Areas-Molecular Brain Science-from the Ministry of Education, Culture, Sports, Science and Technology of Japan SY3-6-10-4 Rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer Kazuhisa Kohda, Wataru Kakegawa, Kyoichi Emi, Michisuke Yuzaki Dept. of Physiol., Keio Univ. Sch. of Med., Tokyo, Japan Even after the completion of genome project in major species, functions of many molecules remain uncharacterized. A transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. Here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦2 (GluR␦2) in the cerebellum. By introducing a Sindbis virus carrying a wild-type GluR␦2 into GluR␦2-null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-Purkinje cell synapses. By examining whether a mutant GluR␦2 lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. Alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important.
Research funds: KAKENHI 16650071 SY3-6-10-5 Gene transfer into in vivo cerebellar Purkinje cells by HIV-derived lentiviral vectors Hirokazu Hirai Advanced Science Research Center, Kanazawa University, Ishikawa, Japan Cerebellar Purkinje cells are key elements regulating motor coordination and motor learning. Gene transfer into Purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. Although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for Purkinje cells, while the latter causes substantial damage to the infected cells. To achieve effective gene transfer into Purkinje cells, we used human immunodeficiency virus (HIV)-derived lentiviral vectors. Purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. Gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. These results suggest that HIV-derived lentiviral vectors are useful for the study of gene function in Purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect Purkinje cells.
Research funds: JST/PRESTO, KAKENHI (17300100) SY3-7-11-1 Physiological basis for stereotaxic surgery in basal ganglia
Atsushi Nambu Division of System Neurophysiology, National Institute for Physiological Sciences, and School of Life Science, The Graduate University for Advanced Studies, Okazaki, Japan Stereotaxic surgery in movement disorders such as Parkinson's disease dramatically improves the symptoms of such diseases. I assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. I will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-STN-GPi), direct (cortico-striato-GPi) and indirect (cortico-striato-GPe-STN-GPi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing.
Research funds: KAKENHI (17022042) SY3-7-11-2 Deep brain stimulation of subthalamic nucleus on Parkinson's disease Fusako Yokochi Department of Neurology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan Parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. Consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. Many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. Stereotaxic surgery was started for treating severe tremor and rigidity in the mid-20th century. Stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (DBS), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. In particular, DBS of the subthalamic nucleus (STN) is an effective treatment for the symptoms. Much basic research on the function of STN has been reported, but STN function is still unclear. Clinical outcomes including the side effects of STN DBS, the neural activities recorded from STN and the localization of clinical effects are reported in this paper. SY3-7-11-3 Firing patterns of basal ganglia neurons and effects of deep brain stimulation in Parkinson's disease Takao Hashimoto Center for Neurological Diseases, Aizawa Hospital, Matsumoto, Japan High-frequency electrical stimulation of the subthalamic nucleus (STN), internal segment of the globus pallidus (GPi) and thalamus can improve motor signs in patients with Parkinson's disease, however, its mechanism of action remains unclear. A leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the GPi and substantia nigra, which in turn suppress thalamocortical output. On the other hand, altered firing patterns in the basal ganglia have been reported in MPTP-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the GPi and STN, and synchronization of GPi, or GPi and striatal neurons. Synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level.
Kaoru Takakusaki Department of Physiology, Asahikawa Medical College, Asahikawa, Japan Locomotion is composed of volitional and automatic processes. Particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. However, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. Because each process is seriously impaired in Parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. The basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. On the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. In this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control.
Research funds: Grant-in-Aid for Scientific research (C) and Priority Area (Area No.454) SY3-7-11-5 Characteristics of neuronal activity within the globus pallidus interna (GPi) in patients with dystonia Yoichi Katayama 1,2 1 Department of Neurological Surgery, Nihon University School of Medicine, Tokyo, Japan; 2 Division of Applied System Neuroscience, Nihon University Graduate School of Medical Science, Tokyo, Japan Dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic GPistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of GPi neurons. Little is yet known, however, regarding characteristics of neuronal activity within the GPi underlying dystonia. We analyzed activity of GPi-neurons in patients with dystonia or Parkinson's disease, which were recorded during surgery for chronic GPi-stimulation. As compared to GPi neurons in patients with Parkinson's disease, GPi neurons in patients with dystonia were distinctive with following three characteristics; firing rate (49.4 ± 31.7 Hz, n = 27) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. These findings suggest that dystonia may be related to unstable movement-related sensory processing within the GPi. It has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. The purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (WMS). The genomic mutation of WMS occurs in the gene encoding plasma membrane Ca 2+ -ATPase isoform 2 that is located on the 6th chromosome. Recent immunohistochemical and electrophysiological investigations on WMS have shown that (1) D2-type dopamine receptors are downregulated presynaptically in the striatum, and (2) a large number of Purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. In this symposium, the possible correlation between these data and dystonic phenotypes will be discussed.
Kei-ichiro Maeda Reproductive Science, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan GnRH has been well established to regulate reproduction through two modes of secretion: surge and pulse. The surge mode is female-specific and induces LH surge and then ovulation through positive feedback action of estrogen. The pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. Environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of GnRH release. Pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. The following four papers aim to unveil the physiological mechanism underlying two modes of GnRH secretion in various experimental models.
SY3-8-05-2 Metastin: A neuropeptide playing a central role in the regulation of ovulatory cycle Hiroko Tsukamura, Kei-ichiro Maeda Graduate School of Bioagricultural Sciences, Nagoya University, Japan Estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. Gonadotropinreleasing hormone (GnRH)/luteinizing hormone (LH) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. The central mechanism of positive feedback action of estrogen on GnRH/LH secretion, however, is not fully understood yet. Metastin was first isolated as a natural ligand for a G-proteincoupled receptor, GPR54 (2001. Nature 411, 613) . Recent studies reported that a genetic alteration leading to homozygous loss of function of GPR54 impairs pubertal development in mice and human. We have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating GnRH/LH surges to control estrous cyclicity in the female rat (2005. Endocrinology 146, 4431) . The present paper focuses on the role of metastin in regulating GnRH/LH surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen.
Suzanne Moenter, Catherine Christian University of Virginia, USA A surge in gonadotropin-releasing hormone (GnRH) secretion is the CNS signal that triggers the luteinizing hormone (LH) surge, which causes ovulation. The GnRH surge depends on a switch in estradiol (E) feedback from negative to positive and in rodents on time of day, occurring in the PM. Treating ovariectomized (OVX) mice with a constant release E capsule (OVX+E) elicits daily PM LH surges; there is no diurnal change in OVX controls. Likewise, extracellular recordings of firing activity of GFP-identified GnRH neurons showed no diurnal changes in cells from OVX mice. In contrast, E increased firing in the PM compared to AM. GABAergic neurons form a major input to GnRH neurons, and activation of the GABAA receptor can be excitatory in these cells. Whole-cell patch-clamp recordings of synaptic activation of GABAA receptors on GnRH neurons revealed E-dependent decreases in transmission during the AM (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. Together these data indicate one mechanism by which E induces the GnRH surge is by altering GABA transmission to GnRH neurons.
Yoshitaka Oka Grad. Sch. Sci., Univ. of Tokyo, Tokyo, Japan
The gonadotropin-releasing hormone (GnRH) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory GnRH neurons. Here, I introduce our recent studies on the physiological properties of neuroendocrine preoptic (POA) GnRH neurons in comparison with the neuromodulatory terminal nerve (TN) GnRH neurons. To study the both types of GnRH neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. The POA-GnRH neurons had quite different basic electrophysiological membrane properties from those of TN-GnRH neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of TN-GnRH neurons. Now that we have various experimental approaches (electrochemical measurement of GnRH release, Ca 2+ imaging after single-cell electroporation, single-cell RT-PCR, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of GnRH neurons.
Research funds: KAKENHI 15370032 SY3-8-05-5 Metabolic regulation of puberty onset using the prepubertal rat model Helen I'Anson Biology Department and Neuroscience, Washington and Lee University, USA We hypothesize that glucose availability determines the timing of puberty onset in rats. Abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. Such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. We hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. When glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. Conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. Blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. In summary, central glucose availability may be an important signal timing puberty onset.
Research funds: Jeffress Memorial Trust and Washington and Lee University SY3-8-12-1 Molecular mechanisms of thyroid hormone action in developing brain Toshiharu Iwasaki, Noriyuki Koibuchi Department of Integrative Physiology, Gunma University Graduate School of Medicine, Maebashi, Japan Thyroid hormone (TH) plays an important role in the developing brain. The action is mainly exerted by controlling gene expression through binding to its specific receptor, TH receptors (TRs). TRs are ligand-regulated transcription factors that are expressed in many organs, including brain. TRs bind to target DNA sequences known as TH-response element (TRE). Coactivators and corepressors are involved in the TR-mediated gene regulation through histone modification. We characterized the coactivator and the corepressor that were expressed in embryo brain. Although precise mechanism of the TH action for brain development has not been fully clarified, these cofactors may be involved in these actions. TH affects brain development only during a limited period, which is referred as the critical period of TH action. Recently, it has been speculated that environmental chemicals may cause the abnormal brain development. Possible mechanism of action of such chemicals on TR system will be discussed.
Research funds: KAKENHI 17510039, 17390060 SY3-8-12-2 Thyroid hormone and organic anion transporters in brain Hiroyuki Kusuhara, Yuichi Sugiyama Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan Organic anion transporting polypeptides (OATPs/SLCO) currently consist of 15 isoforms in human and rodents. They are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. Some members accept a variety of structurally unrelated compounds as substrate. Oatp1c1 is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for T4 and reverse T3, but the transport activity of T3 is quite low. It is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where Oatp1a4, the transport activities of T4 and T3 are similar, is also expressed. The uptake of T4 and T3 by the brain determined using in situ brain perfusion technique was saturable and inhibited by OATPs substrates and inhibitors. These two transporters may play a role in regulation of brain levels of T4 and T3.
Research funds: The Advanced and Innovational Research program in Life Sciences from the Ministry of Education, Culture, Science and Technology SY3-8-12-3 Alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals Takayuki Negishi 1 , Masaki Takahashi 1 , Yasuhiro Yoshikawa 2 , Tomoko Tashiro 1 1 Department of Chemistry and Biological Science, Aoyama Gakuin University, Kanagawa, Japan; 2 Department of Biomedical Science, The University of Tokyo, Tokyo, Japan
There is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (PCB) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. In this presentation, based on comprehensive DNA microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to 4-hydroxy-2,2 ,3 ,4 ,5pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to 4-hydroxy-2,2 ,3,4 ,5,5 ,6-heptachlorobiphenyl (thyroid hormone-like). Among genes whose mRNA expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. Hydroxylated metabolites of PCBs (OH-PCBs) have chemical structures similar to thyroid hormones (THs). We reported that low doses of two types of OH-PCB inhibited TH-dependent extension of Purkinje cell dendrites (2005. Dev. Brain Res. 154, 259) . Koibuchi et al. (2004) clarified that they interfere with TH-dependent gene expressions in reporter gene assays. Further, Takasuga et al. (2004) detected some PCB and OH-PCB congeners in human CSF, of which 4OH-CB187 is the highest concentration. To determine its effects on developing neurons, we examined 4OH-CB187 in rodent cerebellar culture cells. Interestingly, 4OH-CB187 promoted dendritic development of Purkinje cells in the absence of TH and increased significantly their survival numbers. These results indicate that OH-PCB congeners may disrupt normal brain development by different mechanisms depending on their chemical structures.
We have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (PTU), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this PTU rat can be a possible candidate of animal model for autism. In PTU rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to Purkinje cells were shown. These neurons transiently expressed serotonin-IR, therefore we treated SSRI or 5-HTP to examine the relevance of serotoninergic function. The treatment of 5-HTP but not SSRI recovered the delay of cell migration. These effects of serotonin manipulation in PTU rats on the behavioral impairment and the development of CNS will be discussed. It is hoped that embryonic stem (ES) cells will be used in transplantation therapy for neurological diseases. However, because grafts of neural stem cells derived from ES cells may contain residual undifferentiated cells, there would be a risk for teratomas. To reduce this risk, we applied to ES cells herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV) treatment. Stable mouse and cynomolgus monkey ES cell lines expressing HSV-tk were obtained. GCV sensitivity was higher in undifferentiated ES cells than in ES cell-derived neural stem cells. ES cell-derived neurons were resistant to GCV treatment. Nude mice with transplants of undifferentiated ES cells expressing HSV-tk formed teratomas, but the tumor growth was suppressed after the GCV treatment. Suicide gene delivery might increase the safety of the use of ES cells in cell replacement therapy. Enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. The physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase ABC. In transected mice, dopaminergic axons did not extend across the lesion. Chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type IV collagen deposits were developed in the lesion center. In chondroitinase ABC-treated mice, numerous dopaminergic axons were regenerated across the lesion. In these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. These results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (Kawano et al., 2005) .
Reference Kawano et al., 2005. J. Neurosci. Res. 80, 191-202. Research funds: KAKENHI 16500234 OS2A-8-03 Neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles Yoshimasa Koyama 1 , Kazumi Takahashi 2 , Yukihiko Kayama 2 1 Cluster of Science and Technology, Fukushima University, Fukushima, Japan; 2 Department of Physiology, Fukushima Medical University, Fukushima, Japan
The present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic (5HT) neurons in the dorsal raphe (DR) during sleep waking cycles. Using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. Spontaneous firing of the 5HT neurons in DR were excited by glutamate and orexin A or B. They were inhibited by noradrenaline. An ␣1 receptor agonist (phenylephrine or methoxamine) increased the firing rate during SWS or PS, but had no effect when applied during W. In PS-off type DR neurons, cessation of firing during PS was recovered by bicuculline, however in the DR neurons that did not stop firing during PS, bicuculline had almost no effect.
OS2A-8-04 Correlation between regional grey matter volume and proficiency increase in second language: A VBM study Arihito Nauchi 1,2 , Kazuyoshi Hirano 2,3 , Yukimasa Muraishi 2,3 , Kuniyoshi L. Sakai 1,2 1 Department of Basic Science, University of Tokyo, Tokyo, Japan; 2 CREST, JST, Japan; 3 Secondary Education School, University of Tokyo, Japan
Although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. In the present study using voxel-based morphometry (VBM), a whole-brain unbiased technique for detecting regional differences in MR images, we examined the relationship between the proficiency increase in second language (L2) and grey matter volume among students aged 13-17, who received special classroom training in the use of English verbs. In specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of English sentences. These results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in L2.
OS2A-8-05 Grammar center activation in honorification judgment of Japanese sentences Kanako Momo 1,2 , Kuniyoshi L. Sakai 1,2 1 Department of Basic Science, University of Tokyo, Tokyo, Japan; 2 CREST, JST, Japan One linguistic theory proposes that Japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. To investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fMRI under several types of normal/anomalous judgment for Japanese sentences including honorification. When activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (IFG) as well as the bilateral cerebellum for all tested participants. Moreover, the lower performance group showed greater activation in the left F3op/F3t and the bilateral cerebellum. These results suggest that syntactic process is required for Japanese honorification and that activation in these regions shows modulation according to performance level, even in native language.
Research funds: CREST, JST OS2A-8-06 Top-down modulation for melody-related activity in the right auditory areas: An MEG study Takuya Yasui 1,2,3 , Kimitaka Kaga 2 , Kuniyoshi L. Sakai 1,3 1 Dept. of Basic Science, Univ. of Tokyo, Tokyo, Japan; 2 Dept. of Otolaryngology, Univ. of Tokyo School of Medicine, Japan; 3 CREST, JST, Japan
We previously reported right-hemisphere dominance for melody error-induced fields (M140) (Neurosci. Res. 52, S61). In a subsequent study, we confirmed that M140 was independent from mismatch negativity usually induced by oddballs. In the present study, we examined whether M140 was induced by deviation from a memorized melody. We used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. Subjects learned these songs and judged whether there were one or two deviations in notes. There was no significant difference in dipole amplitudes between M140 elicited by the original songs and that by the modified ones. However, while M140 without the deviation showed no significant effect of lateralization, M140 with the deviation resulted in significant enhancement in the right hemisphere. These results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas.
Research funds: CREST, JST OS2A-8-07 Cortical plasticity in adulthood for learning phonics rules for English orthography and phonology Makiko Muto 1,2 , Kuniyoshi L. Sakai 1,2 1 Department of Basic Science, University of Tokyo, Tokyo, Japan; 2 CREST, JST, Tokyo, Japan
Although matching English orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. In the present fMRI study, we tested an English matching task during the course of phonics training in 16 sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. Comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. These results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology.
Research funds: CREST, JST OS2A-8-08 Hierarchical syntactic processing in the left frontal region: An MEG study Kazuki Iijima 1,2 , Naoki Fukui 3 , Kuniyoshi L. Sakai 1,2 1 Dept. of Basic Science, Univ. of Tokyo, Komaba, Japan; 2 CREST, JST; 3 Dept. of Linguistics, Sophia Univ., Yotsuya, Japan Previous ERP studies have shown word-related activation based on semantic association or context. However, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. In the present MEG study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. Sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. The first peak of the activity for a verb presentation was observed at the left frontal region as early as 130 ms after the onset. In the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. These results are consistent with the current linguistic theory (the Minimalist Program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level.
Research funds: CREST, JST OS2A-8-09 Individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition Koji Jimura, Seiki Konishi, Tomoki Asari, Junichi Chikazoe, Yasushi Miyashita Dept. Physiol. Univ. Tokyo Sch. Med., Japan Previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (MPFC) and the superior temporal sulcus (STS) during performance of theory of mind tasks. The present fMRI study explored individual difference of the MPFC and STS activity by employing false belief paradigms. The task consists of two sessions, study and test. During the study session, subject studied a brief story in which two characters have false beliefs. Then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. Consistent with previous studies, significant activity was observed in the MPFC and the STS during representing the false belief. The individual differences of the MPFC and the STS activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. These results suggest that the two indices represent distinct neural mechanisms participating in social cognition.
Research funds: Grant-in-Aid from MEXT (14002005), JSPS Research Fellowship (1611149) OS2A-8-10 Brain activity of happy facial recognition in mother-daughter relationship Jun Shinozaki 1 , Nobukatsu Sawamoto 1 , Toshiya Murai 2 , Takashi Hanakawa 1 , Hidenao Fukuyama 1 1 HBRC, Kyoto Univ. Grad. Sch. of Med. Kyoto, Japan; 2 Neuropsychiatry, Kyoto Univ. Grad. Sch. of Med. Kyoto, Japan
Relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. Eleven healthy females participated in this fMRI experiment. The subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. When happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. It has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. The activity in the striatum and anterior insula might be related to positive affection and empathy, respectively.
OS2A-8-11 Anatomical connections among functionally identified brain regions for sentence processing Yukari Yamamoto 1,2 , Atsushi Maki 1,2 , Kuniyoshi L. Sakai 2,3 1 Advanced Research Laboratory, Hitachi, Ltd., Tokyo, Japan; 2 CREST, Japan Science and Technology Agency, Saitama, Japan; 3 Dept. of Basic Science, Univ. of Tokyo, Tokyo, Japan
We have functionally identified the left dorsal inferior frontal gyrus (IFG), the left lateral premotor cortex, and the triangular/orbital part of the left IFG (F3t/F3O) as regions associated with sentence and discourse level processing. In the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. F3t and F3O of the left IFG were chosen as seed areas for fiber tracking. Fiber bundles that went through two spherical regions were extracted from the tracking data. The central coordinates of these regions were (−54, 27, 21) , (−39, 3, 42) , and (−51, 27, −6) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). Direct connections among these regions were consistently observed among the subjects. This result suggests a critical network among multiple regions that are associated with sentence processing.
OS2A-8-12 Effect of the incongruity controlled by semantic distance on visually evoked magnetic fields Nobuyoshi Harada 1 , Sunao Iwaki 1 , Mitsuo Tonoike 2 1 AIST, Osaka, Japan; 2 Chiba University, Chiba, Japan Visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. The semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a Japanese thesaurus. The words for mammalians were grouped into five semantic categories in the thesaurus. The heads of the animals were changed with those from another semantic category (Deviant, d = 4), and with those from an inner semantic category (Middle, d = 2), while others were not changed (Normal, d = 0). Peak amplitudes of waveforms of the root mean square values on the components of 170 ms (F (2/38) = 4.92, p = 0.013) and 220 ms (F (2/38) = 5.23, p = 0.0098) were significantly decreased with increments of the semantic distance in left occipital sensors. The gradient of the decreasing line of the amplitudes of the 170 and 220 ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. We call this capability, the structural sensitivity for prototype (SSP).
Ryohei Yasuda Duke University Medical Center, USA Calcium signaling in dendritic spines is important for many forms of synaptic plasticity. However, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. Identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. To visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. This technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including Ras GTPase proteins in response to physiologically relevant stimuli with single spine resolution.
Research funds: Burroughs Wellcome Fund, Dana Foundation OS2P-2-02 Mechanisms of P2Y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar GABAergic synapses Yumie Ono 1 , Xiaoming Zhu 1 , Takashi Tominaga 2 , Fumihito Saitow 3 , Shiro Konishi 1,2 1 Waseda-Olympus Bioscience Research Institute, Singapore, Singapore; 2 Department of Neurophysiology, Tokushima Bunri University, Kagawa, Japan; 3 Department of Pharmacology, Nippon Medical School, Tokyo, Japan Postsynaptic P2Y receptor activation by ATP enhances IPSCs at cerebellar interneuron-Purkinje cell (PC) synapses. To investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (N) and single channel conductance (I) of GABA A receptors in PCs using whole-cell recordings of evoked IPSCs in PCs of rat cerebellar slices before and 5-15 min after application of ATP. The ATP-induced enhancement of the IPSC amplitude was associated with a significant increase in the single channel conductance, but not the number, of GABA A receptors in PCs: I and N after ATP treatment were 160 ± 9.9% and 94 ± 6.1% of the controls, respectively. Pretreatment with the protein kinase A inhibitor H-89, but not the calmodulin kinase II inhibitor KN-62, completely abolished the ATP-induced IPSC enhancement.
OS2P-2-03 Activin induces long-lasting NMDA receptor activation via scaffolding PDZ protein ARIP1 Isao Inoue, Akira Kurisaki, Hiromu Sugino Institute for Enzyme Research, Tokushima University, Tokushima, Japan Calcium entry into the postsynaptic neuron through NMDA type glutamate receptors (NMDARs) triggers the induction of long-term potentiation (LTP). The Ca 2+ permeability of NMDAR is regulated by phosphorylation of its tyrosine residues. We report here that activin, a member of the transforming growth factor-b (TGF-b) superfamily, and one of proteins synthesised after LTP, promotes phosphorylation of NMDARs and increases the Ca 2+ influx through those receptors in primary cultured rat hippocampal neurons. This signal transduction occurs in a functional complex of activin receptors, NMDARs, and Src family tyrosine kinase, Fyn formed on a multimer of postsynaptic scaffolding PDZ protein, activin receptor interacting protein 1 (ARIP1). Activin-induced NMDAR activation persists more than 24 h, which is complimentary to the transient activation of NMDARs by brain derived neurotropic factor (BDNF). Our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms.
OS2P-2-04 Roles of CaM kinase I in the hippocampal longterm potentiation Kohji Fukunaga, Takashi Komori, Shigeki Moriguchi Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan CaM kinase I (CaMKI) family members are highly expressed in the adult rat hippocampus and CaMKI-alpha is predominantly localized in the cytosol. CaMKI activation requires phosphorylation of Thr177 by CaMKK as an upstream kinase. We here documented a marked increase in CaMKI-alpha-Thr177 phosphorylation following LTP induction in rat hippocampal CA1 region. Like CaMKII activation following LTP (Fukunaga et al., 1993) , the increased CaMKI-Thr177 phosphorylation remained elevated at least for 60 min after LTP induction. The increased CaMKI-Thr177 phosphorylation was closely associated with prolonged increases in phosphorylation of CREB and myosin light chains in the CA1 region. This is in contrast with transient increases in CaMKIV and ERK phosphorylation. Treatment with CaMKK inhibitor, STO-609 significantly inhibited both CREB and MLC phosphorylation with concomitant reduction of LTP in the CA1 region. Taken together, CaMKI likely mediates the late phase of CREB phosphorylation and an increased MLC phosphorylation in the hippocampal LTP. To investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce BAP and EPSP at the alveus and the proximal dendrite, respectively. The resulting coincidence of magnitude of BAP and EPSP at the distal dendrite was enhanced when the BAP was delivered at a timing (5 ms) to induce LTP. Furthermore, the magnitude of BAP at the distal dendrite was attenuated by the input from the proximal dendrite at a timing (20 ms) to induce LTD. These results suggest that the magnitude of BAP delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and BAP. This may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain.
Research funds: KAKENHI (17021037) OS2P-2-06 Mouse brains deficient in neuronal PDGF receptor- develop normally but are vulnerable to injury Yoko Ishii, Takeshi Oya, Lianshun Zheng, Masakiyo Sasahara Department of Pathology, Faculty of Medicine, University of Toyama, Japan
The platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) are widely expressed in the mammalian CNS. Here, we developed novel mutant mice in which PDGFR- subunit gene was genetically deleted in the neurons of CNS to elucidate the role of PDGFR-. Our mutant mice reached adulthood without apparent anatomical defects. The cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. Furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because NMDA-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered NMDA receptor expression. This is the first known report to address the postnatal function of PDGFR- expressed in CNS neurons, using genetically engineered mutant. It was clearly demonstrated that PDGFR- expressed in neuron protects CNS neurons from cryogenic injury and NMDA-induced excitotoxicity. Early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (GCs) to dynamically migrate from the dentate hilus and form the GC layer. To investigate the mechanism that regulates newborn GC migration, we developed a new slice coculture system. The hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (P6) rats that had received a single BrdU injection at P5 were substituted with the corresponding region of entorhino-hippocampal slices from P6 rats. After five days in vitro, newborn GCs, detected by BrdU and Prox1, migrated out of the hilar graft and reached the host GC layer. Chronic application of picrotoxin, a GABA A receptor antagonist, facilitated the migration of newborn GCs into the GC layer. These results indicate that GABA A receptors regulate the migration of newborn GCs in early postnatal days.
OS2P-3-02 cdk5 is required for neuroblast migration in the adult mouse brain Yuki Hirota 1,2,6 , Toshio Ohshima 3 , Takuji Iwasato 4 , Ashok B. Kulkarni 5 , Hideyuki Okano 2,6 , Kazunobu Sawamoto 1,2,6 1 Bridgestone Lab. Keio Univ., Tokyo, Japan; 2 Dept. Physiol., Keio Univ., Tokyo, Japan; 3 Dev. Neurobiol. Riken, Tokyo, Japan; 4 Behavioral Gen. Riken, Tokyo, Japan; 5 NIH, Bethesda, USA; 6 SORST, JST, Saitama, Japan Neuroblasts generated in the subventricular zone (SVZ) of the lateral ventricles migrate into the olfactory bulb (OB) through the pathway called rostral migratory stream (RMS). Molecular mechanisms regulating the directional long-distance migration remain largely unknown. Here we studied adult function of cyclin-dependent kinase 5 (cdk5) that has been revealed to play a role in neuronal migration in the embryonic brain. Crossing the floxed-cdk5 mice to Emx1cre mice resulted in decreased size of OB and abnormal distribution of neuroblasts. SVZ explants from these mice cultured in Matrigel showed decreased migration distance. Leading process of neuroblasts infected with Cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the SVZ compared to control cells. These results indicate that Cdk5 has a cell autonomous function in neuroblast migration in the adult brain.
OS2P-3-03 Colocaliztion of neuron markers and glial markers in GABAergic neuron progenitors as revealed by singlecell microarray analysis Shigeyuki Esumi 1 , Wu Sheng-Xi 2 , Yuchio Yanagawa 3,4 , Kunihiko Obata 5 , Nobuaki Tamamaki 1 1 Kumamoto Univ., Kumamoto, Japan; 2 Fourth Military Medical Univ., Xi'an, People's Republic of China; 3 Gunma Univ., Maebashi, Japan; 4 SOKENDAI, Hayama, Japan; 5 RIKEN, Wako, Japan GABAergic neurons and oligodendroglia share many characters in the murine forebrain. Both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. In addition, it is reported that they share several glial markers, such as NG2, PLP, and CNP at their prematured stages. In order to investigate its nature, we have established a single-cell microarray analysis method. Single GFP-positive GABAergic neuron progenitors were corrected from the subventricular zone of the GAD67-GFP knock-in mouse neocortex at E18-P0 by dissociation and picking. Complemental DNA from the single cells was amplified by universal PCR amplification and converted into biotin-labeled cRNA using T7 RNA polymerase. After these procedures, cRNA sufficient for a microarray analysis was obtained. As the result we found, MBP and S100- expression in the GABAergic neuron progenitors.
OS2P-3-04 Role of -catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone Kazuhide Adachi 1,2,3 , Masanori Sakaguchi 3 , Toru Yamashita 2,3,6 , Yuko Fujita 3 , Yukiko Gotoh 4 , Arturo Alvarez-Buylla 5 , Takeshi Kawase 1 , Hideyuki Okano 3 , Kazunobu Sawamoto 2,3 1 Neurosurgery, Keio Univ. Sch. Med., Tokyo, Japan; 2 Bridgestone Lab. Dev. Regenerative Neurobiol., Keio Univ. Sch. Med., Tokyo, Japan; 3 Physiol., Keio Univ. Sch. Med., Tokyo, Japan; 4 Inst. Mol. Cell Biosciences, Univ. Tokyo, Tokyo, Japan; 5 Neurosurgery, UCSF, San Francisco, USA; 6 Neurol, Okayama Univ, Med, Dentistry and Pharmaceutical Sci, Okayama, Japan
The subventricular zone (SVZ) continuously produces olfactory bulb neurons in the adult rodent brain. Neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. Here, we studied the role of -catenin signaling in the adult mouse SVZ. -Catenin accumulated in the nucleus of only the transitamplifying cells in the SVZ. Activated -catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. These results suggest that -catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse SVZ.
The ciliary marginal zone (CMZ) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. Wnt2b is expressed in CMZ, and has been shown to control the differentiation of the retinal progenitor cells.
We have isolated a novel BMP antagonist, Chick Tsukushi (C-TSK), which belongs to the Small Leucine-Rich Proteoglycan family. In the eye, the expression of C-TSK is observed in the CMZ which is similar with that of Wnt2b. To examine the molecular interactions between C-TSK and Wnt2b, we co-electroporated them into the optic vesicle at stage 9-10 chick embryo and observed the proliferation of the retinal progenitor cells. We found that C-TSK inhibited the Wnt2b activity that sustains prolonged proliferation of retinal progenitor cells. Our result suggests that C-TSK controls the proliferation of retinal progenitor cells interacting with Wnt2b. To reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (HDAC) 9 in developing cortical neurons. An expression vector of GFP-tagged HDAC9 was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. HDAC9 was primarily localized in nuclear until 1 week in vitro, but was translocated to cytoplasm in the later stages. Such translocation was found in a similar time course after birth in vivo. To examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. As a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. However, TTX addition to the culture medium produced the inverse translocation. These results suggest that activity-dependent intracellular localization of HDAC9 contributes to neuronal differentiation in cortical development.
Research funds: KAKENHI (16700286)
Dept. of Physiology, Fujita Health Univ. Sch. of Med., Japan
We described electrical synapses in alpha retinal ganglion cells (␣-GCs). Precise temporal synchronization of spikes is generated from ␣-GCs (Hidaka et al., 2004) . The fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. Junction conductance (maximum 2.45 nS) was measured. In high-voltage electron microscopy (Hitachi1250M, NIPS, 2005 , gap junctions (average size 0.86 m long) were present in contacts. In confocal laser-scanning imaging, connexin36 localization at contacts counted gap junctions (seven sites in a pair on average). Assuming that the density of connexons would be 5180/m 2 and a single channel conductance is 15 pS, the conductance of each junction would be 45 nS. The presence of seven junctions between a pair will lead to estimate a total junction of 315 nS. The measured conductance could allow to estimate a fraction of open channels as 0.8%. The open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. The visual system separates different types of information into parallel, anatomically distinct processing streams. Despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. To identify molecular correlates of functionally distinct streams, we fabricated a custom cDNA microarray of higher mammal ferrets. We successfully identified molecules whose unique distribution and developmental profiles define the LGN itself, its constituent layers, or identify cells comprising one of the physiological streams in the LGN. Using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret LGN.
Research funds: KAKENHI (17023014), COE Research
Akira Muto, Herwig Baier Department of Physiology, University of California San Francisco (UCSF), USA The visual system operates over a broad range of luminances. This is accomplished by adjustment of photosensitivity, called light adaptation. To study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. In this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. In a screen of almost 2000 genomes, we identified five mutants all of which showed substantially delayed recovery of the OKR. Positional cloning of one of the loci revealed a mutation in the DNA-binding domain of glucocorticoid receptor (GR). GR is known for its role in the stress response, but its function in the visual system is unexplored. We propose that GR is regulating genes essential for light adaptation in the retina.
OS2P-4-04 Multisite recording of the signal propagation pattern in the visual cortex Makoto Osanai, Yusuke Takeno, Satoshi Tanaka, Tetsuya Yagi Graduate School of Engineering, Osaka University, Suita, Japan
Recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. But the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. Therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a 60 channel multielectrode array and a calcium imaging system. In the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer 4 stimuli and propagated horizontally in the layer 2/3. In the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer 4 stimuli. Signal propagation was restricted within several tens m around the stimulus electrode by AP5 + CNQX administration and was completely blocked by TTX administration. Administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. We concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system.
OS2P-4-05 Presence of two phases in the sensitive period of orientation plasticity Shigeru Tanaka 1 , Toshiki Tani 1 , Kazunori O'hashi 1,2 , Jerome Ribot 1 1 Brain Science Institute, RIKEN, Saitama, Japan; 2 Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kita-kyushu, Japan
Recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. In this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. When kittens were returned to the normal visual environment by removing the goggles after 2 weeks of goggle rearing starting around the age of 3 weeks, the over-representation of the exposed orientation was preserved. On the contrary, when the goggle rearing started around the age of 5 weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. These findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase.
OS2P-4-06 Residual visuomotor processing in the animal model of blindsight: Comparison with normal, near-threshold vision Masatoshi Yoshida 1,2,3 , Tadashi Isa 1,2,3 1 Dept. Dev. Physiol., Nat'l Inst. Physiol. Sci., Okazaki, Japan; 2 Sch. Life Sci., Grad. Univ. Adv. Stud., Hayama, Japan; 3 CREST, JST, Kawaguchi, Japan
In two macaque monkeys with unilateral V1 lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. The luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield (80-90% correct). In the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. These results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. We propose that the residual visuomotor processing of monkeys with V1 lesion is unlike normal, near-threshold vision.
Research funds: KAKENHI 13854029, KAKENHI 16700343 and CREST, JST OS2P-4-07 Comparison of the angle representation in macaque visual areas V1 and V2
Minami Ito 1,2 , Hidehiko Komatsu 1,2 1 National Institute for Physiological Sciences, Okazaki, Japan; 2 The Graduate University for Advanced Studies, Hayama, Japan Previously, we have reported that fairly large number of area V2 neurons has angle selectivity. Here, we studied the angle selectivity of area V1, which is the major source of inputs to area V2. We conducted single-unit recordings from the superficial layer of area V1, while animals performed a fixation task. For comparison, we used a similar stimulus set. The stimuli were much larger than the size of the classical receptive fields. Area V1 neurons responded mainly to sharp angles (30 • ), straight lines (180 • ) or right corners (90 • ), but not to intermediate angles (60 • or 120 • angle width). This contrasted with area V2, where neurons showed a variety of the optimal angle width including intermediate angles. We also observed several V1 neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. We suggest that area V1 neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area V2.
OS2P-4-08 Firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing Xin Jin, Ying-Ying Zhang, Xue Liu, Hai-Qing Gong, Pei-Ji Liang Department of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China Population activities of retinal ganglion cells (RGCs) were recorded using a multi-electrode recording system. Single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. However, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. Differing from single direction-selective ganglion cell (DSGC), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. These results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information.
Yasuto Tanaka, Satoru Miyauchi, Masaya Misaki Brain Information Group, NICT, Kobe, Japan Visual long-range interaction was reported to be limited in space. Here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. Two horizontally collinear Gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. Detection threshold of GS probe reduced with cue-probe separations up to 10 • . The facilitation was highly tuned to the symmetrical locus. Furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. The reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. The anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision.
OS2P-4-10 Integrity of visuospatial attention in a split brain patient Noudoost Behrad, Seyed Reza Afraz, Maryam Vaziri, Hossein Esteky School of Cognitive Sciences (SCS), Iran Transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. We investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (MD). We asked MD to track one bouncing ball among four identical distracters while fixating at the center of the screen. Target crossed the vertical midline in half of the trials. Her performance in crossed conditions was significantly above chance level. Also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. In this alignment task MD was able to compare location of the two bilaterally presented stimuli well above chance level. Our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum.
Pei Sun, Justin L. Gardner, Mauro Costagli, Kenichi Ueno, R. Allen Waggoner, Keiji Tanaka, Kang Cheng Laboratory for Cognitive Brain Mapping, RIKEN Brain Science Institute, Wako-shi, Japan
Although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fMRI, tuning to particular stimulus orientations has not been directly demonstrated using this technique. In an effort towards revealing orientation selectivity and its spatial arrangement in human V1, we have conducted an fMRI study with a novel stimulation paradigm and a differential mapping method. We found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. Our results provide the first demonstration that orientation selectivity in humans can be directly studied using fMRI.
OS2P-4-12 Probing the spatial scale of classifier performance with high spatial resolution fMRI Justin L. Gardner 1,2 , Pei Sun 2 , Keiji Tanaka 2 , David J. Heeger 1 , Kang Cheng 2 1 Department of Psychology and Center for Neural Science, New York University, USA; 2 Laboratory for Cognitive Brain Mapping, RIKEN Brain Science Institute, Japan
Recently, classifier analysis with conventional resolution fMRI has been used to decode the orientation of a grating stimulus from the fMRI responses of early visual cortex. It has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. We have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fMRI (0.75 mm × 0.75 mm × 3 mm voxels) in human V1. We found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on T2*-weighted venograms. We suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels.
Research funds: NRSA fellowship from the NIH (1F32EY016260-01) OS2P-4-13 Relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area TE) Hisashi Tanigawa, Kathleen S. Rockland, Manabu Tanifuji RIKEN Brain Science Institute, Wako, Japan
We have studied the relationship between horizontal connections and functional structure in TE using a combination of optical imaging, unit recording, and anatomical tracing. Intrinsic signal imaging was performed in exposed TE, under anesthesia, during presentations of visual object stimuli. This resulted in multiple optical spots evoked by each stimulus. In some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. Then, an anterograde tracer was injected into one of the spots. Both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. However, these regions and the injection site were not always connected by horizontal axons. Some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. These results suggest that horizontal axons can connect regions with different stimulus preferences in TE, in contrast to like-to-like connectivity, as understood in early visual cortices. We recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations (18-350 ms; ISI = 1 s) were presented. Presentation of visual stimuli at all of the tested durations resulted in prolonged responses. The brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. There was a significant difference in average firing rate of late phase (350-550 ms) of response to optimal stimulus across presentation durations. But no such differences were found for the first phase (70-270 ms). In addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. But the suboptimal stimulus (∼50% of max response) evoked similar response magnitudes in the first and second phase. These results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate.
OS2P-4-15 The perceptual learning effect in myopes by the lateral masking procedure Keiko Mizobe 1 , Kazuto Terai 2 , Osamu Hieda 3 , Shigeru Kinoshita 2 1 Dept. of Ophthal., Kyoto Second Red Cross Hospital, Kyoto, Japan; 2 Dept. of Ophthal., Kyoto Pref. Univ. of Med., Kyoto, Japan; 3 Baptist Eye Clinic Hospital, Kyoto, Japan Purpose: The study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. We asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, NEUROVISION. Method: Nine low myopes were studied. Non-corrected digital visual acuities (VA) ranged from 0.4 to 1.2. The logMAR average was 0.31. Eight sessions of NEUROVISION treatment were performed to each individual. The estimation was done by comparison of VA before and after the treatment. Results: Four eyes showed more than one octave improvement of VA. The logMAR average of the four was 0.03, improved from 0.50. The residual five eyes showed less or no improvement. The change of logMAR average was from 0.15 to 0.08. Conclusion: Some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology.
OS2P-5-01 The hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (PCP) genes Hironori Wada 1 , Hideomi Tanaka 1,2 , Satomi Nakayama 1 , Miki Iwasaki 1,2 , Hitoshi Okamoto 1,2 1 Laboratory for Developmental Gene Regulation, BSI, RIKEN, Japan; 2 CREST, JST, Japan Many neurons migrate tangentially through one cell layer at a specific depth within the brain. In the developing zebrafish hindbrain, the facial (nVII) motor neurons originate in rhombomere (r) 4 and migrate tangentially to r6 near the pial surface of the hindbrain. In this study, we demonstrate that expression of the planar cell polarity (PCP) genes celsr2 and frizzled3a in neuroepithelial cells maintain the nVII motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. Mosaic analyses show that expression of the frizzled3a gene in the surrounding neuroepithelial cells prevented the entry of the nVII motor neurons in the neuroepithelial layer. The demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex.
OS2P-5-02 Disrupted-in-schizophrenia 1 (DISC1) regulates the transport of the NUDEL/LIS1 complex to axons via direct interaction with Kinesin-1 Shinichiro Taya, Kozo Kaibuchi Department of Cell Pharmacology, Nagoya University, Nagoya, Japan DISC1 is a candidate gene for susceptibility to schizophrenia. In a Scottish family, the chromosome translocation interrupts the coding sequence of DISC1. DISC1 is reported to interact with NUDEL, which forms a complex with LIS1. Although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of DISC1 remain largely unknown. Here we report that DISC1 interacted with Kinesin-1, a motor protein of anterograde axonal transport. Kinesin-1 interacted with the NUDEL/LIS1 complex through DISC1, and these molecules accumulated at the distal part of axons. The knockdown of DISC1 by RNAi of DISC1 induced the delocalization of NUDEL and LIS1 from the axons and reduced axonal growth. The knockdown of Kinesin-1 induced the delocalization of DISC1 from the axons. Taken together, these results indicate that DISC1 links Kinesin-1 to the NUDEL/LIS1 complex and regulates its transport as a cargo receptor for axon elongation.
Research funds: MEXT OS2P-5-03 Role of a novel collapsin response mediator protein-2 interacting molecule, synaptotagmin-like protein in hippocampal neuron Nariko Arimura, Saeko Kawabata, Atsushi Hattori, Kozo Kaibuchi Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan During the development, neurons recognize the extracellular signals and extend the axons to proper directions. Certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. However, the mechanism of controlled recruitment of receptors remains unsolved. Here, we report that synaptotagmin-like protein 1 (Slp1) can mediate the vesicle transport. Slp1 is known to associate with Rab27. We found that Slp1 associates with collapsin response mediator protein-2 (CRMP-2), which is a key regulator of axon formation. Slp1 could form the trimeric complex with Rab27B and CRMP-2, and also associate with Kinesin-1 through CRMP-2. Slp1 is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. These findings suggest that Slp1 functions as a mediator of recruitment of certain receptor depending on CRMP-2 and Kinesin-1.
OS2P-5-04 Absolute quantification of mdr1a, mrp1, mrp4 and bcrp proteins at the mouse brain blood barrier by LC-MS/MS Junichi Kamiie 1,2 , Yuki Katsukura 1,2 , Sumio Ohtsuki 1,2 , Xiao-Kun Cai 1,2 , Tetsuya Terasaki 1,2 1 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan; 2 SORST, JST, Japan
The ABC transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. However, contribution of each transporter to the BBB function is not clarified. The purpose of this study was to clarify the protein amounts of mdr1a, mrp1, mrp4, and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using LC-MS/MS. By this method, the standard curve showed linearity between 10 and 1000 fmol, and amino acid sequence of the detected fragment was confirmed by MS/MS spectrum. In the brain capillaries, the protein amounts of mdr1a, mrp4, bcrp were 3.9, 2.7 and 4.8 fmol/g, respectively, while it of mrp1 was under detection limit of standard curve. This quantitative profile suggests that mrp4 and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr1a.
OS2P-5-05 Dominant expression of claudin-5 in highly purified brain capillary endothelial cells Sumio Ohtsuki 1,2 , Hirofumi Yamaguchi 1 , Saori Sato 1 , Tmoko Asashima 1,2 , Tetsuya Terasaki 1,2 1 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan; 2 SORST, JST, Japan
Claudins are major constituents of tight junctions (TJs). The purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (BCECs), which form the blood-brain barrier. Mouse BCECs were highly purified using endothelial surface antigen (PECAM-1) and magnetic cell sorting. mRNA expression of caludin-1-23 was measured by real-time RT-PCR. Claudin-5 showed the highest mRNA expression in the purified mouse BCECs. mRNA levels of claudin-1 and -12 were 0.0027% and 0.20% of that of claudin-5. Claudin-5 mRNA was concentrated in the purified BCECs, while claudin-1 and -12 mRNA in the purified BCECs were lower than that in the whole brain. Rat claudin-5 mRNA was also concentrated in rat brain capillary fraction, but claudin-12 mRNA did not. These results suggest that claudin-5 is a dominant TJs protein in BCECs, and expression of claudin-1 and -12, which was reported as TJ protein in BCECs, are not restricted in BCECs.
OS2P-5-06 Effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier F. Ahmad 1 , A. Pauzi M. Yusof 1 , P.D. Mourad 2 , M. Bainbridge 3 , S. Ab Ghani 1 1 Universiti Sains Malaysia; 2 University of Washington, Seattle, USA; 3 Brody School of Medicine, East Carolina University, NC, USA H 2 O 2 is the main peroxides produced in mammalian cells that consume O 2 . The main source of H 2 O 2 in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. Therefore, we look into the effects of H 2 O 2 towards the gap junction communication in astrocytes and permeability of blood brain barrier. In this study, by using a H 2 O 2 microsensor, we investigated the level of H 2 O 2 in the brain that altered the permeability of bbb. The microsensor was implanted in the rat's brain and operated amperometrically. We measured H 2 O 2 level from the current generated by the electron transfer at the electrode. We observed a change in permeability when external H 2 O 2 was injected into the brain. Fatality occurs when the injected H 2 O 2 exceeds 250 M. These finding showed that the altered paracellular permeability in the presence of H 2 O 2 is caused by a series of events that happen one after another.
Research funds: Short Term Grants 304PKIMIA633134 and 304PFAR-MASI670008 OS2P-6-01 Somato-ovarian sympathetic reflex discharges in anesthetized rats Sae Uchida, Fusako Kagitani, Harumi Hotta Dept. Auton. Nerv. Syst., Tokyo Metropol. Inst. Gerontol., Tokyo, Japan Ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (T9-11) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. In central nervous system (CNS) intact rats, stimulation of the T9-11 spinal afferent nerve produced early and late A-reflex discharges, and a late C-reflex discharge. After spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an A-reflex with the same latency as the early A-reflex in CNS-intact rats and an early C-reflex discharge with the similar latency as the late A-reflex in CNS-intact rats. On the other hand, stimulation of the tibial afferent nerve produced late A-reflex and C-reflex discharges in CNS intact rats, those were not observed after spinalization. It was concluded that ovarian sympathetic Aand C-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in CNS-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin.
OS2P-6-02 Responses of renal sympathetic nerve activity and sodium excretion to 3 days sodium loading in rats Misa Yoshimoto, Nozomi Iinuma, Rie Itokawa, Eri Hayashi, Kenju Miki Integrative Physiol. Grad. Sch. Humanities and Sci. Nara-Women's Univ., Nara, Japan
In the present study, a month recording of renal sympathetic nerve activity (RSNA) in freely moving rats was made to explore the long-term regulation of RSNA and sodium excretion. Wistar male rats were instrumented chronically with electrodes for the measurements of RSNA and electrocardiogram. After the 7 days recovery period, RSNA, heart rate and sodium balance were measured over three weeks. Animals were allowed to drink four different concentration of sodium chloride solutions (0, 50, 154, 308 meq./L NaCl) over 3 days. The sodium loading with 308 meq./L NaCl suppressed RSNA significantly and then it gradually recovered while either 0 meq./L NaCl or 154 meq./L NaCl loading had no effects on RSNA. Sodium excretion changed significantly in proportion to the each sodium loading levels. These results indicated that the changes in RSNA were not always correlated with the changes in sodium excretion in rats.
OS2P-6-03 Cross correlation analysis of respiratoryrelated optical imaging signals Yoshitaka Oku 1 , Haruko Masumiya 1 , Yasumasa Okada 2 1 Dept. Physiol., Hyogo Col. Med., Nishinomiya, Japan; 2 Dept. Med. Keio Univ. Tsukigase Rehab. Ctr. Shizuoka, Japan
We aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals.
Optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. Cross correlation between integrated C4 ventral root (C4VR) activity and each pixel was calculated after cycle-triggered averaging and detrending. The maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (LAGmax) were displayed as 3D pseudocolor maps.
In all preparations, two respiratory-related regions were consistently identified: (1) a continuous column extending from the para-facial region to the pre-Bötzinger complex, and (2) a region corresponding to the ventral horn. Pixels where LAGmax were negative (meaning that the activity preceded the C4VR activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate pH was lowered.
OS2P-6-04 Slow afterhyperpolarization determines the firing pattern of action potentials in rat GnRH neurons Masakatsu Kato, Yasuo Sakuma Department of Physiology, Nippon Medical School, Tokyo, Japan Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. GnRH neurons must be able to continuously fire in response to depolarizing stimuli. For this type of firing, GnRH neurons may have a certain intrinsic property. To address this issue, we investigated the Ca 2+ -activated voltage-independent K + currents underlying afterhyperpolarization. Dispersed GnRH neurons from adult GnRH-EGFP transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. The GnRH neurons showed a slow afterhyperpolarization current (I sAHP ). In contrast to previous reports, the I sAHP observed in rat GnRH neurons was potently blocked by an SK channel blocker apamin. In current clamp condition, GnRH neurons evoked a train of action potentials to depolarizing current pulse. Apamin increased the susceptibility to spike failure. The results indicate that rat GnRH neurons exhibit an apaminsensitive I sAHP , which regulates the firing pattern.
Research funds: KAKENHI 16590180, 16086210
OS2P-6-05 The effect of music to sex hormones of elderly person Hajime Fukui 1 , Kumiko Toyoshima 2 , Kiyoto Kuda 1 , Katsuhiko Iguchi 3 1 Nara Univ. of Edu., Nara, Japan; 2 Grad. School of Human Sciences, Osaka Univ. Japan; 3 Nara City Medical Clinic, Japan
It has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. Also, it is pointed out that the decline of T and EST accelerates depression. Therefore the treatment such as Hormone Replacement Therapy (HRT) has been tried to cure depression and Alzheimer's disease. However, It has been pointed out that HRT has serious side effects. On the other hand, there are reports that music influences on a steroid hormone. In addition, it is known that music has certain therapeutic gain toward AD and dementia. In this study, from a point of view of the prevention of AD and dementia, we examined the effect of music to sex hormones of normal elderly person. Four males and 36 females participated music session and T and EST were evaluated. As a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. From above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. Tuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. This study identifies a specific protein marker in CSF, which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% (n = 5) of confirmed and 90% (n = 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa protein band was analyzed by LC-MS/MS analysis. In the present study we have identified two mycobacterial proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B) and one host derived protein as the components of the TBM specific 30-kDa protein. Involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and MPP + -induced rat models of Parkinsonǐs disease (PD). HPLC-EC, patch clamp, fluorimetry, immunoblot and RT-PCR were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mRNA of mitochondria-linked signaling in brain. We report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in MPP + -, but an extrinsic mode of cell death in rotenone-induced model. Drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme Q 10 , Vitamin D 3 and melatonin act via interfering the signaling events in neurons. Loss of complex-I and -IV activities and changes in some of the protein subunits in PD postmortem brains were confirmed in PD and control cybrids. Results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death.
OS2P-7-02 Involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl Tsugunobu Andoh 1 , Boon Chock 2 , Dennis L. Murphy 3 , Chuang C. Chiueh 4 1 Dept. Applied Pharmacol., Univ. Toayama, Toyama, Japan; 2 Lab. Bioch., NHLBI, NIH, MD, USA; 3 Lab. Clin. Sci., NIMH, NIH, MD, USA; 4 Cent, Brain Diseases and Aging, Taipei Med. Univ., Taipei, Taiwan
The present study investigated whether the induction of thioredoxin (Trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of MAO-B. After confirming (−)-deprenyl protects against MPP + -induced cytotoxicity in human SH-SY5Y cells, we observed further that (−)-deprenyl induced Trx for protection against oxidative injury caused by MPP+. The induction of Trx was blocked by PKA inhibitor through a PKA-sensitive phosphoactivation of MAP kinase Erk1/2 and the transcription factor c-Myc. (−)-Deprenyl-induced Trx and associated neuroprotection were concomitantly blocked by the antisense against Trx mRNA in human SH-SY5Y cells. Consistently, Trx increased the expression of MnSOD and Bcl-2 supporting cell survival. In conclusion, (−)-deprenyl augments the gene induction of Trx leading to elevated expression of antioxidative MnSOD and antiapoptotic Bcl-2 proteins for protecting against MPP + -induced neurotoxicity.
OS2P-7-03 PGD 2 induces neuronal apoptosis via 15d-12,14 -PGJ 2 Tatsurou Yagami 1 , Noboru Okamura 2 , Toshiyuki Sakaeda 2 1 Facul. Health Care Sci., Himeji Dokkyo Univ., Himeji, Japan; 2 Kobe Univ. Grad. Sch. Med., Japan Prostaglandin D 2 (PGD 2 ) is abundant in the brain, but its neuropathologic role has been unclear. Here, we found that PGD 2 induced neuronal apoptosis in rat cortical cultures. However, a PGD 2 receptor blocker did not suppress neurotoxicity of PGD 2 . Little PGD 2 receptor was detected, suggesting an involvement of PGD 2 metabolites in the apoptosis. Among PGD 2 metabolites, 15-deoxy-12,14 -prostaglandin J 2 (15d-12,14 -PGJ 2 ) caused neuronal apoptosis most potently and rapidly. Although 15d-12,14 -PGJ 2 is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (PPAR␥), PPAR␥ activators did not kill neurons, suggesting that 15d-12,14 -PGJ 2 induces apoptosis independently of PPAR␥ activation. We found specific binding sites of [ 3 H]15d-12,14 -PGJ 2 (JBS) in plasma membranes. There was a close correlation between the neurotoxicity of various eicosanoids and their affinity for JBS. In conclusion, we demonstrated that PGD 2 induced apoptosis via 15d-12,14 -PGJ 2 in rat cortical neurons, and suggested that JBS in the plasma membrane was involved in the 15d-12,14 -PGJ 2 -induced apoptosis.
Yoshiki Iwamoto, Daisuke Umetsu, Shigeru Ozaki, Naohito Terui Department of Physiology, University of Tsukuba, Tsukuba, Japan
Stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. We reported last year that bilateral sternocleidomastoid muscles (SCM) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. In the present study, we analyzed the relationship between SCM activity and vehicle acceleration. EMGs of the right and left SCM of drivers were recorded during rapid forward acceleration. The time course of the rectified, smoothed EMGs did not match that of vehicle acceleration. For a given acceleration, EMG was larger when acceleration was increasing than when it was decreasing. We compared EMGs and a linear sum of acceleration and its time derivative, jerk. With optimal weights for the two variables and a proper time lag, the linear sum reproduced the EMG profile. The optimal weight and lag varied across subjects and vehicles. We suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. Grasping is a highly developed movement in primate including human. In contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. Here, we show the first direct evidence suggesting the significant contribution of spinal neurons. We trained Japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (C6 to T1). Majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. Spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. We suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement.
Research funds: KAKENHI 17022043 OS2P-7-06 Compartmentalization of the cerebellar nuclei: Aldolase C expression and the olivonuclear projection pattern
Izumi Sugihara, Yoshikazu Shinoda Dept. Systems Neurophysiol., Tokyo Med. & Dental Univ., Tokyo, Japan
The cerebellar cortex is compartmentalized into more than 20 longitudinal stripes by the aldolase C (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. However, no equivalent compartmentalization has been known in the cerebellar nuclei. We mapped aldolase C labeling of terminals of Purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. The cerebellar nuclei were divided into the caudoventral aldolase C-positive and rostrodorsal negative parts, indicating Purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. Olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. These results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase C expression is tightly related with the functional organization of the cerebellum. We examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (PPTN) in monkeys during visually guided saccade tasks. About half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. On the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. For visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. We further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of PPTN. In the reinforcement learning theory, the midbrain dopamine (DA) neurons send reward prediction error signal to the striatum. The cholinergic pedunculopontine tegmental nucleus (PPTN) is one of the strongest excitatory input sources to DA neurons. We hypothesized that PPTN may play an important role for relaying necessary components of reward prediction error signals to DA neurons. During recording of PPTN neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. For more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. From another population, we recorded reward related activity. In conclusion, PPTN neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error.
Research funds: KAKENHI (17022027) OS2P-7-09 Timing activity in supplementary eye field during a saccadic eye movement task Shogo Ohmae 1 , Xiaofeng Lu 1,2 , Yusuke Uchida 1 , Toshimitsu Takahashi 1,2 , Shigeru Kitazawa 1,2 1 Dept. of Neurophysiol., Juntendo Univ. Grad. Sch. of Med., Tokyo, Japan; 2 CREST, JST, Tokyo, Japan
To act properly in our daily life, the ability to detect and predict timing of events is always required. How do we deal with timing in the brain? To address this question, we trained two Japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of 16 targets following a gosignal given at a random timing between 500 and 800 ms after the appearance of the target. We recorded neuronal activity from the supplementary eye field (SEF) during the task. We found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. These cells were classified into two types: (1) those that showed buildup activity during the delay period until the go-signal, and (2) those that displayed changed activity after the go-signal in relation to the length of the delay period. The results suggest that SEF is involved in timing the onset of the go-signal during the saccadic eye movement task. In reaching, a spatial visuomotor transformation should occur in our brain. We can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. We trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. We recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (PMv) and the primary motor cortex (MI) during reaction time. It was revealed that a majority of the MI neurons and a part of the PMv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the PMv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. The results indicate that the PMv contributes to gain control of reaching during visuomotor transformation. Local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. Time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. This result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. The human sensorimotor processing may be hierarchical and similar to an artificial neural computer. We have shown that the trigeminal oral nucleus (Vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (JC) or jaw-opening (JO) motor nucleus in the cat. It is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. Thus, we conducted this study to reveal whether there are Vor neurons that receive cortical projections and project to the JC or JO nucleus in the rat. We injected a retrograde tracer, Fluorogold (FG), in the Vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (SI). Thus, we injected an anterograde tracer, biotinylated dextranamine (BDA), in the rostral SI, and also FG in the JC or JO nucleus in the same animals. We found a considerable number of FG-labeled Vor neurons made contact with BDA-labeled axon terminals. These results suggest that SI neurons control jawreflexes through Vor neurons.
Tsunehiko Kohashi, Yoichi Oda Grad. Sch. Science, Nagoya Univ., Nagoya, Japan
The Mauthner (M) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. To investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of Mcell activity on the behavior. The rostral portion of the zebrafish, 30-200 h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. Thirty hpf embryos, in which M-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (>9 ms) than larvae (>80 hpf, 3 ms). M-cell activity monitored with confocal Ca 2+ imaging during the tail flip (>80 hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without M-cell firing appeared in some cases (<25%) after 100 hpf. Thus, the development of the escape behavior coincided with that of M-cell circuit. Junctophilins (JPs) expressed in the ER/SR interacts with plasma membrane thereby constructing junctional membrane complexes (JMC). We here report that lacking neural JPs subtypes exhibit an irregular hindlimb reflex and impaired memory. To define neural mechanism of memory deficit in JP-DKO mice, we performed whole-cell patch clamp recording of hippocampal neurons. In wild mice, an obvious afterhyperpolarization (AHP) was observed and its AHP was totally blocked by apamin. By contrast, AHP was absent in the JP-DKO mice and was insensitive to apamin treatment. The ER Ca 2+ release through ryanodine receptors, triggered by glutamate receptor-mediated Ca 2+ influx, is essential for the activation of SK channels toward AHP generation in the hippocampal neurons. Therefore, JP-mediated JMC formation likely plays an essential role in neural excitability underlying neural plasticity and memory.
OS2P-8-02 Distribution of voltage-gated calcium channel ␣ 2 ␦-4 mRNA in mouse central nervous system
Takeshi Houtani, Satoru Sakuma, Masahiko Kase, Tetsuo Sugimoto Department of Anatomy and Brain Science, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
The ␣ 2 ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ 1 subunits. These auxiliary subunits are composed of four genetically different molecules, ␣ 2 ␦-1 to ␣ 2 ␦-4. The distributions of ␣ 2 ␦-1, -2, -3 mRNA have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ 2 ␦-4 remains to be determined. We cloned ␣ 2 ␦-4 cDNA fragment from mouse brain by RT-PCR and examined the distribution of ␣ 2 ␦-4 mRNA-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled cRNA probe. While the ␣ 2 ␦-4 mRNA was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mRNA expression. Our results suggest that ␣ 2 ␦-4 subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. The mushroom bodies of insect brains are essential in associative olfactory learning. Here we show that the Drosophila larval mushroom body calyx, the dendritic region, is organized in about 34 glomeruli, which we have mapped. Individual glomeruli receive specific innervation from second order olfactory neurons. By contrast, they contain dendrites from hundreds of mushroom body neurons (Kenyon cells), which show low specificity for individual glomeruli. Glomeruli therefore potentially transmit specific sensory inputs to a large fraction of Kenyon cells. Quantitative analysis of dendritic termini of single larval-born Kenyon cells suggests that they arborize in about 6 glomeruli in an apparently random manner.
This pattern of connectivity is consistent with a model in which Kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors.
Withdrawn OS2P-8-05 Hypothalamic defense reaction involves Purkinje cells in the flocculus folium p via orexin and GABA In anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in Purkinje cells located in folium p of the flocculus. Iontophoretic application of an orexin antagonist (SB334867) depressed the excitation, while bicuculline depressed the inhibition. H 1 or H 2 histamine antagonist had no effect. Labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. Stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. These observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact Purkinje cells directly and also indirectly via other GABAergic neurons.
OS3A-5-01 Activity-dependent development of corticispinal synapse in mouse slice co-culture Takae Ohno, Masaki Sakurai Dept. Physiol., Teikyo Univ. Sch. Med., Tokyo, Japan
We showed NMDA-dependent synapse elimination of corticospinal (CS) tract in vitro in rat. In order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of CS synapses in C57 BL/6 mice. By recording field EPSP (fEPSP) along 80 m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fEPSPs were recorded diffusely throughout the spinal gray matter at 7-8 DIV, then the amplitudes of fEPSPs in the ventral side began to decrease at 9-10 DIV, and dominated in the dorsal area at 14 DIV. CS axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at 7-9 DIV but the axons terminals in the ventral area were eliminated until 14 DIV. This synapse elimination from the ventral side was blocked by APV application from 6 DIV, indicating that this process is also NMDA-dependent. In slice coculture study, we showed that corticospinal (CS) axons grow rapidly and reach the ventral spinal gray until 7 DIV. The number of those ventral axons is reduced before 14 DIV. To study the behavior of the CS axons at the single axonal level, we transfected a small number of cortical neurons with EYFP expression vector pCAG-EYFP by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage CO2 incubator. Some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at 6 DIV. Some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during 7-11 DIV. Some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. These observations provide direct evidence that there are early CS axons that once reach the ventral spinal gray and then retract to stay dorsally. We identified CLICK-III/CaMKI␥ as a novel brain-enriched isoform of the CaMK-I family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of CLICK-III into lipid rafts fractions. In situ hybridization revealed the abundant presence of CLICK-III transcript throughout the central nervous system in mouse embryos. To test the role of CLICK-III during early neuritogenesis, a shRNA vector specific for CLICK-III was delivered into dissociated cortical culture. We found that knock-down of CLICK-III resulted in significant decrease in the number and total length of dendrites. Results from introduction of CLICK-III into a CLICK-III-null context confirmed this finding. Surprisingly, lipid modifications of CLICK-III seemed to contribute to fully elicit such an effect. We thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a CaMK can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis.
OS3A-5-06 Transcription factor control of dendrite arbor ultrastructure Adrian Moore, Reiko Amikura, Shiho Nakao, Andrew Liu, Emi Kinameri RIKEN Brain Science Institute, Japan
The different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. The Drosophila larva dendritic arbourization (da) neurons consist of four classes (I-IV) with increasing levels of arbor complexity. These diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. Here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. We have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. Using this method we illustrate that the dendrites of class I neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class IV neurons, with a complex arbor, contain a low density meshwork of microtubules. We are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code.
OS3A-5-07 Segmental and Hox related cues are involved in the establishment of the somatotopy Yasunori Murakami IGBMC, Strasbourg, France
In the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. Little is known about the mechanism underlying the development of the somatotopic organization. By fate mapping of specific rhombomeres (r), we found that principal sensory (PrV) neurons derived from r3 receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. By conditional inactivation, we found that early expression of Hoxa2 in r2 is required for pathfinding and positioning of trigeminal nerve afferents. At later stages, Hoxa2 expression in PrV neurons provides instructive cues for topographic arborization of maxillary axons. Moreover, while PrV neurons appeared normally specified, loss of Hoxa2 function resulted in selective loss of Eph4 expression, and altered axonal projections from PrV to the ventral posterior medial (VPM) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. Thus, Hoxa2 dependent cues are required to determine the territory for whisker representation in r3 and the assembly of a somatosensory circuit.
OS3A-5-08 The second wave of corticospinal innervation after synapse elimination of the first wave Tsutomu Kamiyama, Masaki Sakurai Dept. Physiol, Teikyo Univ. Sch. Med., Tokyo, Japan
In the previous study we showed that the rat corticospinal (CS) terminals and synapses were widely distributed at P7 and those in the venrtolateral (VL) area were eliminated from P8 to P10 and that the number of terminals in the dorsomedial (DM) and VL area began to increase again from P12 and further increased thereafter. In the present study we further studied the subsequent developmental time course of CS terminal distribution. CS axons were anterogradely labeled by injection of biotin dextrane (BDA) into the sensorimotor cortex. The number of the terminals began to increase from P12, reaching peak around the third postnatal week. Labeling of single or a few by microinjection axons revealed that at P14 some additional CS axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. However, the number of axons did not increase in the brainstem and the upper cervical cord. These suggest that the second wave of innervation is explained mainly by branching of CS axons just before and after entering the spinal gray matter.
OS3A-5-09 Proteomics of the growth cone: I. Protein profiling of the growth cone The growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. In higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. Proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. We obtained the whole growth cone (GCP) obtained from neonatal rat forebrain and the membrane subfraction of the GCP (GCM), and then those fractions were analyzed using proteomics. We have identified several hundreds of the distinct proteins of these fractions. Here, we show the profiling of GCP and GCM, and will discuss the overview of these protein profiles in relation to the growth cone functions. Axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. However, the interplay between these two mechanisms remains largely unknown.
To study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (TC) axon branching by using organotypic cocultures. During the second week in vitro, yellow fluorescent protein-labeled TC axons formed branches primarily in the target layer. Spontaneous firing was found to increase when branches were formed abundantly. Pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. Moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. These findings suggest that synaptic activity modifies layer-specific TC axon branching by regulating the remodeling process with molecular cues expressed in the target layer.
Research funds: KAKENHI (17023030), KAKENHI (15300107) OS3A-6-01 The application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain
Naoki Tani, Yoichi Saitoh, Haruhiko K., Satoru Oshino, Masayuki Hirata, Amami Katoh, Toshiki Yoshimine 1 Department of Physiology, University of Osaka, Osaka, Japan
Repetitive transcranial magnetic stimulation (rTMS) has been applied to control intractable deafferentation pain (DP). But nobody has investigated which cortical area is the most effective target for pain relief. Therefore, we stimulated M1, S1, SMA, premotor accurately with a navigation-guided rTMS and compared their effects of pain relief. At the same time, rTMS (1, 5, 10 Hz, 500 stimulations) was compared in 20 DP patients. The pain relief was evaluated with visual analogue scale. High frequency (5, 10 Hz) rTMS of M1 was the only effective stimulation for treating intractable pain in 10 of 20 patients (50%). The pain relief continued for 3 h significantly. We would like to discuss the mechanism of pain relief with high frequency rTMS of M1.
OS3A-6-02 Involvement of ATP on nociceptive modulation in rat model of masseter muscle pain Yasuo Sugiura, Noriyuki Ozaki, Masamichi Shinoda Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan
We determined the role of P2X 3 R on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (MM) . The pain in the MM was assessed by the pressure pain threshold (PPT) defined as the amount of pressure required to induce head flinching. The MM injection of ␣,-meATP (P2X 1,3,2/3 Rspecific agonist) significantly enhanced the behavioral response to the pressure. This enhanced response was completely blocked by the co-application of ␣,-meATP with PPADS (P2X 1,2,3,5,1/5,4/5 R-specific antagonist). Excessive muscular contraction of MM produced by the electrical stimulation significantly decreased the PPT indicating mechanical hyperalgesia of the MM. Administration of PPADS to the exerted MM produced a complete recovery of decreased PPT. P2X 3 Rpositive neurons innervating the exerted MM increased in trigeminal ganglia. Our results suggest that P2X 3 R plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of MM. The present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. Here we found sciatic nerve ligation (SNL) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (PAG). Using the fluoro-gold (FG) microinjection into the PAG, numerous FG-labeled cells were detected in the hypothalamus. In the arcuate nucleus (ARC) of the hypothalamus, the immunoreactivity (IR) for an excitatory neuronal maker, fosB was increased, whereas the -endorphin (-EP)-IR was decreased 7 days after SNL. Furthermore, the subpopulations of -EP-positive cells were co-labeled with fosB in the ARC. The present data suggest that the hypothalamus can be received by SNL-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the PAG. This phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway.
OS3A-6-04 The Cantor-like patterns in rat hippocampal CA1 pyramidal neurons Tsuda and Kuroda proposed a mathematical model for the Cantor coding in the hippocampal CA1. This prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since Marr's theory of simple memory in the hippocampus. However, our mathematical model is too abstract to describe physiological feature of neurons. Then, we have tried to find Cantor-like patterns experimentally from the CA1 pyramidal neurons. Temporally associated and non-associated electrical stimulations were delivered to Schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. In our results, Cantor-like patterns were observed in hippocampal CA1 pyramidal neurons. Young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. We prevented zebra finches from hearing their own vocalizations by exposure to loud noise after 35 days of age, before which they had been reared with song tutors from birth. When the noise stopped at 102-200 days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. The similarity to the tutor syllables steadily increased until the time of song crystallization (30 days later). These findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d90 of age) and that zebra finches can develop songs using the memory well after the normal period of song development. The temporal order of syllables resembled the tutor model only in birds released from the noise before 80 days of age. Thus, different schedules and processes may govern the learning of syllable phonology and syntax. In addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (TGL) was identified in rats (Takemura, 2005) . A tracer study revealed that there is an interconnection between the dorsal part of the TGL and the lateral nucleus of the amygdala, suggesting a functional implementation of TGL neurogenesis in amygdala-dependent emotional memory processing. To investigate this possibility, we performed a TGL region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. The TGL-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the TGL neurogenesis for aversive memory reduction. We (Tsukada and Pan, 2005) systematically examine the functional difference between spatio-temporal learning rule (STLR) proposed by Tsukada (1996) and Hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence.
In this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the CA1 hippocampal network. Tsuda and Kuroda proposed a mathematical model for the Cantor coding in the hippocampal CA1. They also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal CA3. This prediction includes an attractor dynamics expected in the associative network, which was proposed by Marr and others. The time series of events, which could be output from CA3, may be encoded in CA1 in an efficient way. The proposed Cantor coding is effective, because the topology of time series is naturally measured on the Cantor set since each element of Cantor set represents a single time series. However, our mathematical model is too abstract to describe physiological feature of neurons. Then, we have tried to make more realistic model of CA1, using 2-compartment model of neuron, and we found the Cantor coding of information of time series in the model CA1. It is known that neurons can propagate action potentials with high temporal precision. However, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. Here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. We found that closely neighbouring pyramidal neurons in the CA1 region of the hippocampus synchronize with sub-millisecond precision. The optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. Moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. The synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. Therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. The alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. We evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-ERD) evoked in motor area using synthetic aperture magnetometry in 15 patients with chronic ischemia due to diverse vascular occlusive diseases (n = 12) and moyamoya disease (n = 3). Contrary to the normal motor activation, ipsilateral beta-ERD was dominant during grasping task of affected hand in 8 patients. This abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. And it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. Ipsilateral beta-ERD may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia.
OS3A-7-02 Hypothermia protects against cerebral ischemia by suppressing ␦PKC activation Takayoshi Shimohata 1,2 , Heng Zhao 2 , Gary Steinberg 2 1 Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan; 2 Department of Neurosurgery, Stanford University, Stanford, USA Hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦PKC is reported to induce apoptosis upon activation. Its activity is modulated by phosphorylation, translocation and proteolytic cleavage. We investigated effects of hypothermia on ␦PKC activation using a rat permanent distal MCA occlusion model. Mild hypothermia (30 • C) reduced infarct size by 84%. Western blots indicated that ␦PKC cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < 0.05). p-␦PKC (T505) dephosphorylated after stroke; this effect is blocked by hypothermia. Full-length and cleaved form ␦PKC as well as p-␦PKC (S643) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦PKC activator suppressed the protective effect of hypothermia. Taken together, hypothermia blocks ␦PKC activation after focal ischemia. This effect might contribute to hypothermic neuroprotection. Calcium responses in situ following ischemia remain unclear. We sought to determine, in rats, the calcium changes following transient forebrain ischemia. In anesthetized adult rats, 4-vessle occlusion was induced. Fluo-3/AM was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached Nipkow-disk scanner (CSU-21, Yokogawa, Japan) equipped with 10× objective lens] was inserted into the brain. 4-vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. Fluorescence intensity of fluo-3 increased up to 115%, and persistently increased up to 130% during 20-min reperfusion, indicating the long-lasting Ca 2+ increase in the CA1 region. In contrast, in the frontal cortex, 10-min ischemia increased fluorescence intensity during ischemia but not reperfusion. In the CA1 region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in Ca 2+ in situ.
Research funds: KAKENHI #16390407, #16047212
OS3A-7-04 Reevalution of classical view on resident microglia: Neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults
Hiroaki Matsumoto 1 , H. Watanabe 1 , Y. Kumon 1 , T. Ohnishi 1 , Chi Ii 2 , Y. Imai 2 , J. Tanaka 2 1 Dept. Neurosurgery, Ehime University, Japan; 2 Dept. Molecular and Cellular Physiology, Ehime University, Japan
Resident quiescent microglia (MG) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (NO). In the present study, however, we found that the majority of resident MG died through apoptosis within 36 h after the onset of ischemic and traumatic brain insults. We further noticed that traditional MG markers isolectin B4 and CD11b recognized with OX42 antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than Iba1+ MG or macrophages. Accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible NO synthase. Iba1+ amoeboid-shaped MG started to accumulate 3 days after the insults. The data prompted us to reevaluate the roles and the fate of resident MG in the brain.
OS3A-7-05 Insulin regulates the hepatic clearance of amyloid  peptide Tetsuya Terasaki 1,2 , Chihiro Tamaki 1 , Sumio Ohtsuki 1,2 1 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan; 2 SORST, JST, Japan
The liver is the major organ that eliminates amyloid -peptide (A) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein 1 (LRP-1) is a molecule responsible for the hepatic clearance. Since epidemiologic investigations suggest the high incidence of Alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of A . Insulin infusion into the rat portal vein increased LRP-1 expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. Insulin treatment also increased the hepatic uptake of A(1-40), which reached 1.6-fold greater uptake than non-treated control after 10 min treatment. Increase of the hepatic uptake of A(1-40) by insulin was concentration dependent (EC 50 = 230 pM), and was completely suppressed by RAP (2 M), an LRP inhibitor. These results suggest that insulin induces translocation of LRP-1 to the plasma membrane of hepatocytes, leading to increase of A hepatic clearance from the circulation.
Research funds: SORST, JST OS3A-7-06 MR images of intra-arterially administered microglia surrounding -amyloid deposit in the rat brain The therapeutic use of microglial cells has recently received some attention for the treatment of Alzheimer disease (AD), but few noninvasive techniques exist for monitoring cells. Here we present a magnetic resonance imaging (MRI) technology to track micrgolia cells injected intra-arterially in a rat model of AD. We labeled microglia expressing GFP with Resovist using the HVJ-E vector. We administered labeled microglia into the carotid artery of the rats. MRI revealed clear signal changes attributable to Resovist-containing microglia in A-injected areas. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for AD. Extravasation of protease-activated receptor (PAR) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders, which causes pathophysiological changes in neurons and glial cells. To elucidate the mechanism of thrombin-induced activation of astroglial cells, we used 1321N1 astrocytomas that show a characteristic retraction of bipolar protrusions after activation of PARs with thrombin. The thrombin-induced morphological change of 1321N1 cells was inhibited by an inhibitor of IP 3 receptors, 2-aminoethoxydiphenyl borate (2-APB) or an endoplasmic reticulum Ca 2+ -ATPase inhibitor, cyclopiazonic acid (CPA). In parallel, thrombin-induced mobilization of Ca 2+ was inhibited by 2-APB and CPA. Moreover, removal of external Ca 2+ accelerated the reversal of thrombin effects. These results suggest that refilling of Ca 2+ store by Ca 2+ entry play an important role in the cytoskeletal dynamics of astroglial cells. To clarify the occurrence range of neurofibrillary tangles (NFT), we reexamined an autopsied Alzheimer patient with the onset at age 38 and a 25-year-clinical course. The brain showed severe atrophy (630 g). Microscopic examination disclosed that all telencephalic neocortices had NFT of more than 100 and SP of more than 10. All limbic cortices and nuclei had NFT of more than 100 and SP of more than 10. Although there was no SP, various numbers of NFT were observed in the following structures: claustrum 85, caudate 9, globus pallidus 6, hypothalamus 22, Meynert's nucleus 4, thalamus 74, substantia nigra 6, central gray 12, locus ceruleus 6, Purkinje cells 0, posterior root ganglion 0, adrenal medulla 3. This study revealed that there exist NFT-rich neurons and free neurons. The latter includes Purkinje cells and posterior root ganglion cells.
Considering the pathogenesis of NFT, it must be valuable to clarify qualitative/quntitative differences between NFT-rich neurons and free neurons.
OS3A-7-09 Transcriptional regulation of androgen receptor in aging mouse brain
Androgen receptor (AR) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. In aging mice brain, AR mRNA expression was measured by RT-PCR, AR promoter methylation by Southern hybridization, and proteins binding to promoter by EMSA. AR mRNA level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. Female mice exhibited higher methylation of AR promoter than males. Methylation was increased by testosterone, but decreased by estradiol. Furthermore, DNaseI accessibility to AR promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. Incubation of brain nuclear extract with 32Plabeled AR promoter yielded three specific complexes. The intensity of these complexes varied with age and sex. These findings show that AR mRNA expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. Such regulation of AR expression might influence androgen action and consequently brain function during aging. Reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. It has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (EAATs) contribute to glutamate removal. However, the role of presynaptic EAATs remains unclear. Using mouse retinal slices, we examined the contribution of EAATs at the rod to rod bipolar cell (RBC) synapse. The kinetics of the RBC current evoked by electrical stimulation of rods was slowed by pharmacological blockade of EAATs. Recordings of the evoked RBC currents from EAAT subtype-deficient mice and the EAAT-coupled anion current revealed that functional EAATs are localized to rod terminals but not to postsynaptic or glial cells. Model simulations suggest that rod EAATs are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. TRPV4 is a thermosensitive TRP channel, and activated by body temperature. We found functional-TRPV4 was expressed in soma, dendrites and synapses in the neurons. Since TRPV4 was firstly cloned as an osmotically activated channel, we hypothesized TRPV4 might be involved in volume regulation of the spines. Therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed TRPV4 expression related to the volume increase of spines. Next, we compared the resting membrane potential (RMP) between wild type and TRPV4-deficient neurons at 37 • C, and found RMP in wild type was more depolarized by approximately 5 mV than RMP in TRPV4-deficient neurons. We also performed current-injection experiments in both neurons, and found that TRPV4-deficient neurons required much bigger currents to get their firing. Thus, we conclude that TRPV4 is involved in regulation of both neural activity and spine motility in hippocampus.
OS3P-2-04 A system for rapid uncaging in defined patterns and its application
Hiroshi Kojima Department of Intelligent Information Systems, Tamagawa University, Tokyo, Japan
Neurons integrate many sysnaptic signals at dendrite. Understanding these information processes is a central topics in experimental and computational neuroscience. The use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. However, most experiments were carried out either at spatial locations or in a very simple scanning patterns. We developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. Our system is capable of patterned photorelease of caged neurotransmitters at 100 locations per 200 ms with submicron resolution. Ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. Simultaneously, imaging of neurons are obtained by 2-photon microscopy and electrophysiological experiments can be done. We briefly report the present system for rapid uncaging and its application to neurophysiological research.
OS3P-2-05 D1-like receptors selectively block P/Q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain A number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. Their functions, however, remain largely unknown. We tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by RNAi and to evaluate its effect on the synaptic transmission in the brain slices. For this purpose, we selected the nodose ganglion (NG), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. In anesthetized young Wistar rats, synthetic siRNA against the genes coding adenosine A 1 receptors (adora1) was introduced to the NG by electroporation. One to five days after siRNA delivery, the expression level of adora1 in the NG decreased by >90% of that in the non-treated NG, being not accompanied by a change in mRNA level for A 2A receptors. This technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. NMDA-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. Activation of neurons by NMDA results in intracellular generation of reactive oxygen species (ROS) and reorganization of cell metabolism. Exposure of rodent and human lymphocytes with NMDA results in ROS increase within the cells which is suppressed by NMDA antagonists. Moreover we have demonstrated by rt-PCR technique and by using anti-NMDA-antibodies the expression of NMDAreceptors on lymphocyte membranes. In addition, we shown that NMDA receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase-3 activity and interferon-␥ synthesis. In the presentation, properties of NMDA-receptors and their functional role in immunnocompetent system are discussed. Small molecule G-protein Arf1 in combination with phospholipase D (PLD) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to Golgi apparatus. However, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. To examine possible involvement of Arf and subsequent PLD in regulation of receptor-induced responses in neurons, we recorded K + -current response to dopamine (DA) in the ganglion cells of Aplysia under conventional two-electrode voltage clamp. Intracellular application of Arf1 blockers such as brefeldin A, Exo1, and Arf1 N-terminal peptide, markedly suppressed the DA-induced response. Furthermore, intracellular application of ␣-synuclein, a specific blocker of PLD, significantly depressed the K + -current response to DA. These results suggest that Arf1 and subsequent PLD may regulate the K + -current response induced by DA.
OS3P-3-05 p250GAP, a brain-enriched RhoGAP, is involved in the NMDAR-mediated signaling Takanobu Nakazawa 1 , Toshihiko Kuriu 2 , Ayako M. Watabe 3 , Toshiya Manabe 3 , Shigeo Okabe 2 , Tadashi Yamamoto 1 1 Div. of Oncology, Inst. Med. Sci., Univ. of Tokyo, Tokyo, Japan; 2 Dept. of Cell Biol., Tokyo Medical and Dental Univ., Tokyo, Japan; 3 Div. of Neuronal Network, Inst. Med. Sci., Univ. of Tokyo, Tokyo, Japan NMDAR regulates structural plasticity by modulating actin organization within spines. However, the signaling pathways that link NMDAR activity to the postsynaptic actin cytoskeleton are poorly understood. We identified a brain-enriched RhoGAP, p250GAP, which interacts with the NR2B subunit of NMDAR. Within neurons, p250GAP was highly concentrated in the postsynaptic density and co-localized with NR2B and an actin-binding protein, cortactin. p250GAP promoted GTP hydrolysis of Cdc42 and RhoA in vitro and in vivo. NMDAR stimulation led to de-phosphorylation and redistribution of p250GAP. When over-expressed in dissociated neuron, p250GAP suppressed the activities of Rho GTPases, which resulted in spine elongation. Taken together, the results suggest that p250GAP is likely to be involved in NMDAR activity-dependent actin re-organization in spines.
OS3P-3-06 Non-static method to directly quantify the transfer of firing correlation from one neural population to another: Fokker-Planck method
Hideyuki Cateau RIKEN Brain Science Institute, Saitama, Japan
Firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. Therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. To characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. Many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. Unlike this method, the Fokker-Planck (FP) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. Despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. Here, we provide a first non-static FP analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time.
OS3P-4-01 Spatial frequency tuning of disparity-selective neurons in macaque V4
Hironori Kumano 1 , Seiji Tanabe 2 , Ichiro Fujita 2 1 Grad. Sch. of Engineering Science, Osaka Univ., Osaka, Japan; 2 Grad. Sch. of Frontier Biosciences, Osaka Univ., Osaka, Japan
To examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area V4 of awake, fixating monkeys. For each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional (2-D) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (RDSs). Neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated RDSs. In a subset of V4 neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using 2-D filtered noise stereograms. The disparity tuning of most V4 neurons was consistent across a range of spatial frequencies to which they were sensitive. We suggest that V4 neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth.
OS3P-4-02 Predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area V4
Hiroshi Shiozaki, Seiji Tanabe, Ichiro Fujita Lab. Cognitive Neurosci., Grad. Sch. Frontier Biosciences, Osaka Univ., Japan
Many neurons in visual area V4 of macaque monkeys are selective for binocular disparity. Most disparity-selective neurons in V4 are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. However, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. We recorded single unit activity from V4 while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. The monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. Trial-to-trial fluctuation of visual responses of V4 neurons was correlated with the monkey's subsequent behavioral choice. Given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of V4 neurons. The results suggest that V4 neurons are involved in mediating stereoacuity.
OS3P-4-03 The role of disparity energy and binocular matching processes in stereopsis Takahiro Doi, Seiji Tanabe, Ichiro Fujita Lab. Cognitive Neurosci., Osaka Univ., Japan The early visual system computes disparity energy of stereo images. Some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. We addressed how the energy and correspondence computations underlie stereopsis. We asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. At each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. At small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. At large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. The results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. We introduce a novel receptive field (RF) analysis, LSRC, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. The visual stimuli are standard wide-field 2-D ternary dynamic random noise, generally refreshed every 26-40 ms. Unlike the conventional reverse correlation which computes a spike-triggered average (STA) of the stimuli themselves, LSRC computes the STA of the spectra of localized regions of the stimuli. Both simulations and recordings from cat V1/V2 neurons demonstrate that LSRC is capable of revealing details of complex cell RFs, cross-orientation suppression, variations of orientation tuning within RFs that might lead to shape selectivites. Since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, LSRC is highly suitable for multi-neuron, multi-area studies spanning retina, V1, and especially areas beyond.
Research funds: MEXT(13041033), JSPS(13308048), COE21
OS3P-4-06 Analysis of center-surround organization of V1 neurons as a high-order receptive field
Hiroki Tanaka, Izumi Ohzawa Graduate School of Frontier Biosciences, Osaka, Japan
Responses of area 17 (V1) neurons are influenced by stimuli not only in their classical receptive field (RF) center, but also in its surround. Such a center-surround organization may be considered as a unified higher-order RF. We have sought to obtain detailed structures of such a RF by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. Of 52 cells analyzed, 80% showed spatial frequency tuning curves that were well fitted with Gaussian. By taking the inverse Fourier transform of these curves, spatial center-surround RF was obtained as Gabor functions with spatial phases between ±90 degrees. Highly asymmetric structures were observed for cells with strong surround suppression. Estimated sizes of center and surround were well correlated with those from size tuning curves. Moreover, there was no space-time tilt in the center-surround RF. The results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli.
OS3P-4-07 Spatial organization of receptive fields of complex cells in the early visual cortex Kota Sasaki 1 , Izumi Ohzawa 1,2 1 Grad. School of Eng. Sci., Osaka Univ., Japan; 2 Grad. School of Frontier Biosci., Osaka Univ., Japan
Little is known about the quantitative internal structure of the receptive fields (RF) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. Therefore, we have analyzed the relationship between the spatial 2nd-order interaction kernels and the RF envelopes of complex cells. Extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. Threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with 51 × 51 dots was presented over an area 2 to 4 times larger than the RF of a complex cell. For each dot location, a 2nd-order kernel and its envelope (by Hilbert transform) were calculated. The RF envelope of the neuron was determined by summing the envelopes of 2nd-order kernels at all locations. 2nd-order kernels had roughly comparable extent as the RF, and contained 3.3 subregions on average (N = 53). Among 35 complex cells, whose RF envelopes were elongated, 16 cells exhibited the horizontal elongation.
Research funds: MEXT(13041033), JSPS(13308048), COE21
OS3P-4-08 Orientation tuning of neuron in cat lateral geniculate nucleus
Tomoyuki Naito 1 , Osamu Sadakane 1 , Masahiro Okamoto 2 , Hironobu Osaki 3 , Hiromichi Sato 1 1 Grad. Sch. Med., Osaka Univ., Osaka, Japan; 2 Grad. Sch. Front. Biosci., Osaka Univ., Osaka, Japan; 3 Med. Sch., Osaka Univ., Osaka, Japan
We examined the orientation selectivity of LGN neurons of anesthetized cats and found that although about 19% LGN neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (SF), and that 97% of LGN neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (CRF) and SF higher than the optimal for CRF response. Two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal SF condition. However, high SF grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. Our results suggested that elliptic CRF center produces orientation-biased response of LGN neurons. Furthermore, surround suppression of LGN neurons tuned to particular stimulus orientations enhances orientation selectivity of LGN neurons.
OS3P-4-09 Temporal dynamics of suppressive receptive field surround in cat V1
Satoshi Shimegi, Hiroyuki Kida, Ayako Ishikawa, Hiroshi Sakamoto, Hiromichi Sato Graduate School of Medicine, Osaka University, Toyonaka, Japan
In the primary visual cortex (V1), a neuronal response to stimulation of the classical receptive field (CRF) is suppressively modulated by the stimulus presented at the receptive field surround (SRF). Using stationary flashes (500 ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in V1 cells of anesthetized cats. Stimulus slightly larger than the CRF caused suppression in early response (<100 ms) but not in middle (100-200 ms) and late responses (200-500 ms). As stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. Radius of surround suppressive field progressively expanded in temporal sequence from 2.5 deg (early response) to 5 deg (middle response) and 7.5 deg (late response). Thus, modulation of early response seems to reflect whether stimulus is larger than CRF size or not, and late response to reflect how wide area is stimulated.
Research funds: KAKENHI (15500259) OS3P-4-10 Spatial-frequency dependent surround suppression in cat V1
Ayako Ishikawa 1 , Satoshi Shimegi 2 , Hiroyuki Kida 3 , Hiromichi Sato 2 1 Grad. Sch. Front. Biosci., Osaka Univ., Osaka, Japan; 2 Grad. Sch. Med., Osaka Univ., Japan; 3 Grad. Sch. Eng. Sci., Osaka Univ., Japan
We examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (SF) tuning of neurons in cat V1. We used a stationary flash (duration, 500 ms) of a circular sinusoidal grating patch with optimal orientation and SF as CRF stimulus, and that of an annulus (50 ms) with optimal orientation but varying SF as SRF stimulus. First, we stimulated CRF and SRF simultaneously (Stimulus-onset-asynchrony (SOA) = 0) and analyzed time course of surround suppression. SF tuning of the surround suppression changed along time course of response, and effective SF of surround suppression shifted from the SF lower than that optimal for CRF response (C-SF) to that near C-SF. Next, changing SOA, we examined surround suppression on different temporal phases of CRF response. SOA-dependency of surround suppression changed according to the temporal phase of response. These results suggest that multiple mechanisms with different SF-and temporal characteristics are involved in the surround suppression.
OS3P-4-11 Contrast-dependency of spatial summation property in cat V1 and LGN Masahiro Okamoto 1 , Tomoyuki Naito 2 , Osamu Sadakane 2 , Hiromichi Sato 2 1 Grad. Sch. Front. Biosci., Osaka Univ., Japan; 2 Grad. Sch. Med., Osaka Univ., Toyonaka, Japan
We examined contrast-dependent change in a receptive field (RF) size and strength of surround suppression of neurons in the primary visual cortex (V1) and the lateral geniculate nucleus (LGN) of anesthetized cats. RF structure was modeled by spatial interactions of excitatory and inhibitory Gaussians. Both in V1 and LGN, Ratio of Gaussians (ROG) model captured size-tuning curves of responses better than Difference of Gaussians (DOG) model. Under the high contrast stimulus condition, the peak of size tuning curve shrank by 0.76 and 0.68 times in V1 and LGN, respectively. In LGN, surround suppression was strengthened under high contrast stimulus condition, but in V1, the strength of surround suppression did not affected by stimulus contrast on average. We conclude that 1) ROG model describes the surround suppression better than DOG model both in V1 and LGN, 2) under high contrast stimulus condition, there is a reduction of RF size with a shrinking of excitatory Gaussian, which is confirmed with ROG model.
Hiroyuki Kida 1 , Satoshi Shimegi 2 , Ayako Ishikawa 3 , Hiroshi Sakamoto 3 , Hiromichi Sato 2 1 Grad. Sch. Eng. Sci., Osaka Univ., Japan; 2 Grad. Sch. Med. Sci., Osaka Univ., Japan; 3 Grad. Sch. Front. Biosci., Osaka Univ., Japan
In the primary visual cortex (V1), neuronal responses to stimulation of the classical receptive field (CRF) were suppressed by the presence of stimuli at surround receptive field (SRF). We examined whether the suppression varied according to spatial configuration of SRF stimuli in V1 neurons of anesthetized cat. The CRF stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. SRF was divided into 8 flanks (45 • step), and stationary stimulated with an annulus, oppositely-faced flanks (2-fk) or a flank (1-fk) stimulus. The durations of stimulus presentation were 500 ms for CRF and 50 ms for SRF stimulation. Localized SRF stimulation with either 2-fk or 1-fk exerted significant suppression on CRF responses. According to the analysis of spatiotemporal change in SRF effects, there was no particularly suppressive SRF area for 1-fk stimulation throughout the CRF response. However, 2-fk stimulation of end position to CRF had strong and long-lasting suppression on responses during 80-200 ms after onset.
OS3P-4-13 Temporal-frequency dependency of receptive field size and surround suppression in LGN and V1
Osamu Sadakane 1 , Tomoyuki Naito 1 , Hironobu Osaki 2 , Masahiro Okamoto 3 , Hiromichi Sato 1 1 Grad. Sch. Med., Osaka Univ., Japan; 2 Med. Sch., Osaka Univ., Japan; 3 Grad. Sch. Front. Biosci., Osaka Univ., Osaka, Japan Spatial summation property of neurons in the primary visual cortex (V1) varies depending on stimulus parameters (e.g., stimulus contrast). In this study, we examined how temporal frequency (TF) of grating stimulus affects size-tuning properties of cat V1 neurons. Our results showed that, when the TF was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that V1 neurons change their spatial property according to TF in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. We also tested the effect of changing stimulus size on TF tuning curve. Consistent with above-mentioned results, large grating made the peak and the high cut-off of TF-tuning curve higher than those for small grating. In the lateral geniculate nucleus (LGN), we obtained basically similar results to those of V1 neurons, suggesting that the subcortical TF tuning property contributes to that in V1.
Ryo Sasaki, Takanori Uka Department of Physiology (I), Juntendo University, Tokyo, Japan A few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (Schoups et al. 2001; Yang and Maunsell 2004) . The change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. Here we hypothesized that the read out of information from sensitive neurons was modified by learning. To test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (MT) neurons during learning of a depth discrimination task. Two monkeys were trained to report the depth of moving dots (NEAR or FAR), and we recorded from isolated MT neurons during the course of training. The monkeys showed improvement in discrimination thresholds across 70 daily sessions. In contrast, the sensitivity of MT neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. These results suggest that plasticity due to perceptual learning occurs within the neural pathway following area MT. We developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using 4.7 T MRI. The scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. The microelectrode tip was precisely localized up to a resolution of 50 m under particular geometrical scan condition. We then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. The microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution MRI. The accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. A transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. The time course of this signal was ahead of the BOLD signal and characterized by a steep onset. Diffusion-MRI thus represents a new exciting mechanism for fMRI. In order to increase its efficiency we aimed at defining a diffusion response function (DRF) as a counterpart of the hemodynamic response function (HRF). An volume of interest was defined using SPM with a boxcar function. Gamma-variate functions were used to model the steep onset. The parameters of the DRF were estimated by fitting the time-course with the DRF convolved with a boxcar. Although the magnitude of the signal change (around 1%) was smaller than that of BOLD (>2%), the temporal profile showed a constant precedence of the diffusion signal by 2.4s.
OS3P-5-03 New insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals
Laboratory for Human Brain Dynamics, Brain Science Institute (BSI), RIKEN, Wako-Shi, Japan
Tomographic analysis of Magnetoencephalography (MEG) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [Moradi, et al., NeuroImage; Ioannides et al., Cerebral Cortex] . This unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [Ioannides et al., Cerebral Cortex;Ioannides et al., NeuroImage] and pathological [Ioannides et al., J. Neurosc.] brain function. Novel ways of analyzing MEG data provide direct measures of regional brain activity over much longer timescales. These new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from MEG with hemodynamic measures of brain activity.
OS3P-5-04 Spatial registration of stand-alone fNIRS data to MNI space Ippeita Dan, Archana Singh, Masako Okamoto National Food Research Institute, Japan
The registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. However, this is difficult to achieve in functional near-infrared spectroscopy (fNIRS) because fNIRS data is primary obtained from the head surface and lacks structural information of the measured brain. Therefore, we present a method for probabilistic registration of fNIRS data to the standard Montreal Neurological Institute (MNI) template through international 10-20 system without using the subject's magnetic resonance image (MRI). The standard deviation in probabilistic registration thus performed for given head surface points is approximately within 1 cm. This means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fNIRS analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' MRIs are not available. Stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. It allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. We have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. Three groups of subjects [Alzheimer disease (AD), Obstructive Hydrocephalus (OH) and Normal controls] were examined. Mass fractal dimension is high for AD (1.33), approaching unity (∼1.02) for OH, and in between for control (1.14). Statistical analysis was performed and significant differences were observed for these groups (p < 0.01). The observations are accounted by a flow dynamics heterogeneity model. The implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. Thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. In this study, we revealed that mitogen-activated protein kinase (MAPK) pathways contribute to thrombin-induced brain injury in two experimental models. Firstly, we employed organotypic cortico-striatal slice cultures. Application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. The cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (ERK) but not p38 MAPK, while the striatal shrinkage was prevented by both of them. Secondly, thrombin was injected into rat striatum. Thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of ERK and p38 MAPK. These results suggest that MAPK pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. Positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in Parkinson's disease (PD). Eight PD patients performed mental and motor tasks: (1) resting, (2) staring at a standing human object, (3) thinking of standing, (4) standing with eyes open, (5) standing with eyes closed. Regional CBF data analyzed by SPM2 were compared with normal counterparts. The cerebellar vermis was more activated during imagination of standing in the PD group than in healthy group. As seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in PD patients, but the between-group comparison generated greater activations in the vermis and prevuneus in PD. The cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in PD.
OS3P-6-03 Potentiation of NMDA receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat Yu Kono 1,2 , Eiji Shigetomi 2 , Kiyoharu Inoue 1 , Fusao Kato 2 1 Dept. Neurol., Jikei Univ., Sch. Med., Tokyo, Japan; 2 Lab. Neurophysiol., Jikei Univ., Sch. Med., Tokyo, Japan
To elucidate the mechanism underlying the selective vulnerability of motoneurons (MNs) to metabolic failures (MFs), we compared the membrane current responses of MNs and non-MNs to MFs. Experiments were performed on neurons in the hypoglossal nucleus (XII) and dorsal motor nucleus of the vagus nerve (DMX) in the young rat brainstem in the presence of TTX. MFs were induced by NaCN or oxygen deprivation. In XII neurons, MFs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto XII neurons. Furthermore, NMDA receptor-mediated current evoked by exogenous NMDA was increased by NaCN. In DMX neurons, MFs evoked outward currents without affecting synaptic inputs. These pre-and postsynaptic responses to MFs in MNs might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis.
OS3P-6-04 Effects of MDMA on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei
Yuichi Suzuki, Megumi Higuchi, Takayuki Nakagawa, Shuji Kaneko Dept. Mol. Pharmacol., Grad. Sch. Pharmaceu. Sci., Kyoto Univ., Kyoto, Japan 3,4-Methylenedioxymethamphetamine (MDMA) is a recreational drug of abused which has been shown to increase serotonin (5-HT) release and cause degeneration of 5-HTergic nerve terminals via 5-HT transporter, although the mechanisms are unclear. In this study, we developed rat organotypic mesencephalic slice culture including the 5-HTergic raphe nuclei, and examined the effects of MDMA and methamphetamine (METH) on 5-HT release and 5-HTergic neurotoxicity. Immunohistochemical studies for tryptophan hydroxylase revealed abundant 5-HTergic neurons around the raphe nuclei. Treatment with a 5-HTergic neurotoxin 5,7-dihydroxytryptamine dramatically reduced the tissue contents of 5-HT and its metabolite, which was blocked by a selective 5-HT reuptake inhibitor. MDMA and METH (0.1-1000 M) increased 5-HT release, and reduced the tissue contents of 5-HT and its metabolite at higher doses. The mesencephalic slice culture including the 5-HTergic raphe nuclei may be useful to examine the mechanisms underlying 5-HTergic neurotoxic effect of MDMA in vitro.
OS3P-6-05 Studies on drug Dependence (Rept. 421): Involvement of platelet-derived growth factor (PDGF) receptor in the morphine-induced rewarding effect
Masami Suzuki, Minoru Narita, Michiko Narita, Tomoko Takeuchi, Yasuyuki Nagumo, Keiichi Niikura, Tsutomu Suzuki Dept. of Toxicol., Hoshi Univ. Sch. Pharm. Pharmaceut. Sci., Tokyo, Japan
The present study was undertaken to investigate the involvement of platelet-derived growth factor (PDGF) receptor in the morphineinduced rewarding effect in rodents. Extensive coexpression of tyrosine hydroxylase with PDGF receptor was apparently observed in the rat ventral tegmental area (VTA). The levels of dopamine and its major metabolites in the nucleus accumbens (N.Acc.) were markedly increased by the microinjection of PDGF into the rat VTA. The morphine-induced rewarding effect was suppressed by intra-VTA microinjection of PDGF receptor Fc chimera. The increased level of dialysate dopamine produced by morphine in the rat N.Acc. was significantly decreased by intra-VTA injection of PDGF receptor Fc chimera. These findings suggest that the stimulation of -opioid receptors in the VTA by morphine leads to the activation of PDGF receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents.
OS3P-6-06 Prostaglandin D 2 is a strong mediator of neuroinflammation in genetic demyelinating mouse model Prostaglandin (PG) D 2 , an inflammatory mediator, mainly produced by hematopoietic PGD synthase (HPGDS). Microglial activation and gliosis are commonly observed during the neuroinflammation. In twitcher (GALCt wi/twi ), a genetic demyelinating mouse model, we found that HPGDS expression was upregulated in activated microglia accompanied by the DP1 receptor induction in hypertrophic astrocytes. Using primary culture of glial cells, we demonstrated that activated microglia produced large amount of PGD 2 by HPGDS and that astrocytes expressed both DP1 and DP2 receptors and were activated by PGD 2 . We found that gliosis and demyelination were well suppressed in HPGDS-or DP1-null twitcher and twitcher treated with an HPGDS-inhibitor. These results suggest that PGD 2 is a key molecule of neuroinflammation involved in the demyelination.
Research funds: 09670806, 12558078 OS3P-7-01 On a sodium channel distribution enabling high frequency signal processing Go Ashida 1,2 , Kousuke Abe 2 , Kazuo Funabiki 1 1 Grad. Sch. Medicine, Kyoto Univ., Kyoto, Japan; 2 Grad. Sch. Informatics, Kyoto Univ., Kyoto, Japan Some auditory neurons, such as the owl's Nucleus Laminaris (NL) cells, can sense very high frequency signals (up to 8 kHz). From the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. First, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of Ranvier. Next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. Sodium conductance in the soma amplifies low frequency signal components below 1kHz, while that in the node does up to 10 kHz. Last, as a typical example, we discuss the capability of high frequency signal processing in the owl's NL neuron. Some biological evidences indicate that sodium channels in the NL neuron are distributed mainly in the nodes but less in the soma. By using an NL neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. Interaural time difference (ITD) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:NL). However, little is known about the cellular mechanisms of coincidence detection. Here, we report the results of in vivo intracellular recordings from the barn owl's NL. We used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. The inner sharp electrode was protected by the outer one during penetration of the cerebellum. We isolated 140 NL cells from 16 owls and achieved intracellular recordings in 38 of them, as judged by a sudden DC potential drop and the resting membrane potential (mean Rp = 58 ± 17 mV). NL neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (Sound Analogue PSPs:SAP). The amplitude of SAPs varied as a function of ITD. Spike rates changed in linear proportion to the amplitude of SAP. We evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. In total, 13 vision impaired (7 blind and 6 low vision) and 15 sighted persons participated. The stimuli were pure tone pulses. For each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. Localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. The discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. The discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. It was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.'
OS3P-8-01 Autocrine BMP signaling in astroglia sensitizes the glial scarring Masahisa Yamada 1 , Runa Araya 1 , Naoto Kitamura 1 , Yuji Mishinsa 2 1 Yamada Unit, RIKEN BSI, Saitama, Japan; 2 NIHS, NIEH, NC, USA Bone Morphogenetic Proteins (BMPs) affect growth of glial cells however, contribution of BMPs during glial scar formation is unknown. To study the role of BMP signaling in vivo, we disrupted Bmpr1a, one of the type I receptors for BMPs, in a telencephalic neuronal stem cell-specific manner. We found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. Although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. Disruption of astroglial Bmpr1a expression by Cre-adenovirus recapitulates the same phenomena. BMPs were upregulated in reactive astrocytes in after brain injury. Knocking down of Bmpr1a by small interfering RNA in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine BMP signaling in astrocytes. This result opens up the understanding of novel mechanisms underlying the autocrine BMP signaling on glial scarring after CNS injury. The present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. Here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. A robust increase in GFAP-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. In contrast, Iba1-positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with BrdU-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. Our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress.
Withdrawn OS3P-8-04 Fucosylation prevents overshooting of the migration by the vagus motor neuron precursors Shigeharu Kinoshita 1,2 , Hideomi Tanaka 1,2 , Sachiko Tsuruoka 3 , Hironori Wada 1,2 , Hitoshi Okamoto 1,2 1 RIKEN BSI, Wako, Japan; 2 JST CREST, Kawaguchi, Japan; 3 RIKEN RRC, Wako, Japan
The vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. Aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (SIDS). Therefore, control of precursor cell migration into the right position may be crucially important.
The zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmX and mmX). The dmX precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. In the towhead mutant embryos, ectopic neurons are distributed between bilateral dmX where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. Positional cloning and mRNA rescue analysis identified TOWHEAD as a GDP-mannose 4,6 dehydratase (GMDS), a key enzyme for de novo synthesis of a GDP-fucose. As a result, the mutant embryos showed exclusive reduction of fucosylated glycans. Our findings represent that fucosylation is responsible for maturation of these neurons. In development of the Drosophila visual center, photoreceptor cells extend their axons (R axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, Hedgehog (Hh). This mechanism helps to establish an orderly arrangement of connections between the R axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding R axons. It is found that the bHLH-PAS transcription factor Single-minded (Sim) is induced by Hh in the lamina neurons and is required for the association of lamina neurons with R axons. In sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with R axons. As a result, lamina neurons are set aside from R axons. The data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks.
Research funds: KAKENHI (17024013)
OS3P-8-06 Initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of PSD95-homolog Dlg
Hiroshi Kohsaka, Etsuko Takasu, Akinao Nose Department of Physics, University of Tokyo, Tokyo, Japan
Trans-synaptic interaction via cell adhesion molecules (CAM) is essential in constructing synapse structures. Although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. Here we used live-imaging and genetic analysis to show that a Drosophila CAM Fasciculin2 (Fas2) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. By visualizing GFP-tagged Fas2 genetically expressed on a muscle, we found Fas2 accumulated at postsynaptic site just after the contact between growth cones and its target muscle. Genetic and deletion analysis implied that trans-synaptic interaction with presynaptic Fas2 is crucial for the postsynaptic localization of Fas2. In addition, postsynaptic localization of a scaffolding protein Dlg, PSD95-homolog, and glutamate receptors was impaired in fas2 mutants. These results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses.
Gaudilliere Brice Harvard Medical School, USA Postsynaptic differentiation of dendrites is an essential step in synapse formation. We report here a requirement for the transcription factor myocyte enhancer factor 2A (MEF2A) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. A transcriptional repressor form of MEF2A that is sumoylated at Lys403 promoted dendritic claw differentiation. Activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of MEF2A at Ser408 and thereby promoted a switch from sumoylation to acetylation at Lys403, leading to inhibition of dendritic claw differentiation. Our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain.
Research funds: NS4102, AG11085
PS1A-A002 Characterization of mRNA species that are associated with postsynaptic density fraction by gene chip microarray analysis We previously reported the partial identification by random sequencing of mRNA species that are associated with the postsynaptic density (PSD) fraction (Tian et al., 1999) . We report here further characterization by gene chip analysis of the PSD fraction-associated mRNAs, which were prepared in the presence of RNase inhibitor. We confirmed that a large number of mRNA species are associated with the PSD fraction and found that mRNAs encoding various postsynaptic proteins were highly concentrated in the PSD fraction. We identified some mRNA species that were highly concentrated in the PSD fraction. We also constructed a cDNA library using the PSD fraction-associated mRNAs as templates, and identified 1152 randomly selected clones by sequencing. Our data suggested that the PSD fraction-associated mRNAs are a very useful resource, in which as yet uncharacterized genes are concentrated.
Tian et al., 1999. Mol. Brain Res., 72, 147-157. Research funds: KAKENHI (17500252) PS1A-A003 The distribution of SNAP-25 protein is regulated in isofom-specific manner Makoto Itakura, Saori Yamamori, Kouta Takano, Masami Takahashi Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Japan
Two isoforms of SNAP-25 derived from Exon 5 splicing are expressed in brain. We generated two specific antibodies for SNAP-25a and b, and studied the distribution in rodent brain. There was a sticking difference in expression of SNAP-25a and b during early postnatal period. SNAP-25b was low at the birth and increased remarkably thereafter. By the contrast, SNAP-25a increased transiently and attained a maximum level around seven day after birth. Furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of SNAP-25b but not SNAP-25a was recovered in raft-enriched fractions of Triton X-100-treated LP1 membrane after sucrose density gradient centrifugation. Immunohistochemistry demonstrated that SNAP-25b was widely distributed throughout brain, whereas, SNAP-25a was restricted to some particular regions of brain. These results indicate that expression and distribution of SNAP-25 protein are regulated differently in isoformspecific manners, and SNAP-25a and SNAP-25b play different functional roles in brain.
PS1A-A004 ERC(ELKS/Rab6IP2/CAST) regulates syaptic short-term plasticity by recruiting bMunc13-2 to the active zone CaMKII in the postsynaptic sites is localized as a PSD-anchored or a cytoplasmic form. CaMKII in the two sites is interchangeable by its translocation. Translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. We have previously suggested that postsynaptic CaMKII is also localized in lipid raft microdomain (2001. Mol. Brain Res. 89, 20-28) . In this report, we proved the lipid raft localization of CaMKII by detergent-treatment and successive sucrose floatation assay of SPM or Cos7 cells expressing CaMKII, and by cholesterol depletion from membrane using MbCD. We also investigated the mechanism and properties of CaMKII targeting to lipid raft. CaMKII targeted to lipid raft microdomain possibly through protein-protein interaction. Our data suggest that lipid raft microdomain is a major site of CaMKII distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. Glial glutamate transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, the decay kinetics of climbing fiber (CF)mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(-/-) mice are not significantly different from those in wildtype mice. Here we attempted to clarify the roles of glial glutamate transporters in CF-PC synapses using GLAST(-/-) and GLT-1(-/-) mice, and a novel antagonist of glial glutamate transporters, (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate. Our results indicate that glial glutamate transporters can retain the fast decay kinetics of CF-EPSCs in the normal range if a small proportion (approximately 20%) of functional transporters, GLAST and/or GLT-1, is preserved. Glutamate is well known as an essential neurotransmitter in nervous system. How glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. We found that the loss-of-function mutations in VGLUT (vesicular glutamate transporter) encoded by eat-4 gene led to abnormal sensory behaviors including thermotaxis in C. elegans. Thermotaxis defect of eat-4 mutant was caused by malfunction of both thermosensory neuron AFD and its downstream interneuron RIA, suggesting that thermal signals from AFD or RIA to their downstream neurons are transmitted by glutamate through EAT-4 VGLUT. A mutation in AVR-14 glutamate receptor also led to abnormal thermotaxis. We are trying to investigate whether AVR-14 functions in the downstream neurons of AFD or RIA, and to identify other glutamate receptors involved in thermotaxis. Through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level.
PS1A-A008 Biochemical characterisation of the vesicular glutamate transporter 1
Stephan Schenck, Shigeo Takamori Department of Neurology and Neurological Science, 21st Century COE Program, Tokyo Medical & Dental University, Tokyo, Japan Vesicular glutamate transporters (VGLUTs) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. The three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. While the third isoform constitutes only a minor fraction, VGLUT1 and VGLUT2 are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. So far, no clear difference in the function of VGLUT1 and VGLUT2 has been found. To further characterise the specific properties of the transporters we make use of the VGLUT1-KO mouse which gives us the opportunity to investigate a brain devoid of VGLUTl. We focus on synaptic vesicle fractions from KO-mice to study the VGLUT-associated transport biochemically. In recent years, three isoforms of vesicular glutamate transporters (VGLUTs) have been molecularly identified in mammals. Histological investigations have revealed that the distribution of three VGLUT isoforms in the CNS is largely complementary with limited overlap, suggesting that differential expression of VGLUT isoforms may contribute to functional diversity in glutamatergic synapses. However, functional differences among the isoforms remained poorly understood. To get insights into their isoform-specific property, we searched for interacting protein(s) to the C-terminus of VGLUT1 by yeast two-hybrid screening and found endophilin A1. As expected for the interacting molecule to VGLUT1, endophilin A1 was typically localized to VGLUT1-positive synaptic terminals in cultured hippocampal neurons. We are currently investigating physiological significance underlying their direct interaction and co-localization. The aim of this study is to investigate the molecular basis for lactate utilization. Hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from a voltage-clamped pyramidal neuron. In lactate solution, amplitude of EPSCs was decreased in 60∼90 min, followed by spontaneously recovered after120 min, while EPSC in glucose medium remained unchanged. Application of APV+Ni in lactate medium, spontaneous recovery was not observed. In neuron cultures, incorporation of 14C-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase C. In glial cell cultures, incorpotation of lactate was initially maintained. Increased expression of monocarboxylate transporter (MCT) 2 was demonstrated in the lactate medium. Results suggested that increased MCT2 expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner.
Yumei Wu 1 , Kazuhito Tomizawa 1 , Shuang Liang 2 , Iori Ohmori 1 , Teiichi Nishiki 1 , Kohji Takei 2 , Hideki Matsui 1 1 Dept. of Physiol., Okayama Univ., Okayama, Japan; 2 Dept. of Neurosci., Okayama Univ., Okayama, Japan Synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (Amph) I and dynamin I. Here, we show a novel type of regulation of vesicle endocytosis by proteolysis. In mouse hippocampal slices, Amph I was found to be cleaved by a Ca 2+ -activated protease, calpain during prolonged depolarization or stimulus trains. The calpain-cleaved N-terminal Amph I fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in COS-7 cells, indicating that the calpain cleavage of Amph I inhibits endocytosis. Amph I in hippocampus was also cleaved by calpain in vivo after kainate seizure. Although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, ALLM during the first seizure. Thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. Synaptic vesicles (SVs) are effectively recycled by endocytosis for continuous synaptic transmission. Previously, we have suggested that a high level of synaptic transmission is maintained by recycling of SVs through two types of endocytosis operating coordinately (27th this meeting). In the present study, we labeled endocytosed SVs at nerve terminals of Drosophila with fluorescence dyes, FM1-43 and FM4-64, and also measured quantatively exocytosis and endocytosis of SVs, using these dyes. EGFP-labeled Cacophony Ca2+ channels and anti HRP stained the active zone and non-active zone at synapse, respectively. Imaging analysis revealed that two distinct types of endocytosis of SVs occurred at the active zone and the non-active zone of motor nerve terminals. We have previously shown that baclofen, a GABA B receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. Syntaxin 1A is also known to modulate exocytosis. To characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin 1A were investigated by patch-clamp recording and counting the number of release sites. Transfected neurons showed higher frequency of miniature EPSCs and stronger inhibition by baclofen than controls, but no change in number of sites. Increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. These results suggest that GABA B receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin 1A in mouse hippocampal neurons. We have previously shown a transient localization of tubulin (TUB) during synaptic vesicle (SV) cycling in Drosophila nerve terminals. The TUB localization is detected during SV recycling, while microtuble (MT)-loop is observed throughout SV cycle. In this study, we characterized the two distinct TUB localizations and showed their relation with SV pool formation. Axonal MTs and MT-loops abounded in acetylated (Acetyl) TUB. The transient localization was either polymerized or depolymerized, and organized by non-Acetyl TUB. Taxol decreased the non-Acetyl TUB localization but not MT-loops, and inhibited exo/endo cycling pool (ECP) formation. In boutons containing MT-loops, ECP formation was also inhibited. Acetyl MT-loops tend to be stable whereas presynaptic non-Acetyl TUBs are either free dimers or dynamic MTs. These results suggest that presynaptic dynamic TUB, especially non-Acetyl TUB, controls ECP formation. Presynaptic TUB dynamics may regulate functional presynaptic plasticity by controlling SV pools.
Research funds: Grant-in-Aid for JSPS Fellows The mechanism by which pregnenolone sulfate (PREGS) enhances synaptic transmission was studied at the rat calyx of Held. PREGS increased the amplitude of evoked EPSCs, without affecting that of spontaneous miniature EPSCs, indicating that the site of its action is presynaptic. PREGS facilitated presynaptic voltage-gated Ca 2+ channel (VGCC) currents via accelerating their activation kinetics, but had no effect on K + currents, resting conductance, or action potential waveforms. In simultaneous pre-and postsynaptic recordings PREGS did not change the relationship between presynaptic Ca 2+ influx and EPSCs, suggesting that exocytotic machinery downstream of Ca 2+ influx was not involved in the PREGS effect. Neither BAPTA nor GTP␥S loaded into presynaptic terminals blocked the effect of PREGS. We conclude that PREGS enhances transmitter release via facilitating VGCCs by a novel mechanism, which is independent of intracellular Ca 2+ or G-proteins.
PS1A-B017 Spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus Endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic CB1 cannabinoid receptor, and induce suppression of transmitter release. To understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (DAGL␣), in the mouse brain. In the cerebellum, DAGL␣ was predominantly expressed in somatodendritic membrane of Purkinje cells, and highly concentrated at the base of spine neck. However, DAGL␣ was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGL␣ was selective to spines, but widely distributed within spines. These results indicate that DAGL␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. Synprint site of voltage-gated Ca 2+ channels interacts with synaptotagmin. However, its physiological role is not entirely clear.
Here we report that AP-2 subunit can directly bind with synprint site. This interaction was Ca 2+ -dependent, being weaker at concentrations higher than 200 nM. In contrast, the interaction of synaptotagmin with synprint was optimal at 100 M Ca 2+ , being weaker at lower or higher concentrations. The binding domain of synprint for AP-2 and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. To assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of Held. This peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. We conclude that Ca2+ channel synprint site makes Ca 2+ -dependent interactions with AP-2 and synaptotagmin thereby contributing to vesicular endocytosis.
PS1A-B019 acl-4, an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in C. elegans Naoko Hara, Takao Inoue, Yasukazu Takanezawa, Hiroyuki Arai Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
It is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. However, the physiological significance and the molecular mechanism of the remodeling are largely unknown. To address these questions, we focused on evolutionarily conserved acyltransferase like genes in C.elegans acl-1˜14, and generated their deletion mutants. The mutants of acl-4 gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. However, the mutants exhibited severe movement abnormalities in fat-3 mutant background in which long chain polyunsaturated fatty acids are depleted. Pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. These abnormalities were rescued by neuron specific acl-4 expression, suggesting that certain phospholipid species produced by acl-4 are involved in maintaining normal synaptic transmission and motility of C.elegans.
Daisaku Yokomaku 1 , Hussam Jourdi 1 , Akiyoshi Kakita 2 , Tadasato Nagano 1 , Hitoshi Takahashi 3 , Nobuyuki Takei 1 , Hiroyuki Nawa 1 1 Dept. Mol. Neurobiol., Brain Res. Inst., Niigata Univ., Japan; 2 Brain Resource Center, Brain Res. Inst., Niigata Univ., Japan; 3 Dept. Pathology, Brain Res. Inst., Niigata Univ., Japan
Scaffolding proteins containing PDZ domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. Little is known, however, about what regulates the expression of the PDZ proteins and how the levels of these proteins influence synaptic development. Here, we show that ligands for epidermal growth factor (EGF) receptors (ErbB1) decrease a particular set of PDZ proteins and negatively influence synaptic formation or maturation. In neocortical cultures, EGF decreased the expression of GRIP1 and SAP97. Moreover, EGF treatment resulted in a decrease in the frequency of pan-PDZimmunoreactive aggregates on dendritic processes. These findings revealed a novel negative effects of ErbB1 receptor ligands that attenuates the expression of the PDZ proteins and inhibits postsynaptic maturation in developing neocortex.
Takatoshi Iijima 1 , Eriko Miura 2 , Keiko Matsuda 1 , Tetsuro Kondo 1,3 , Masahiko Watanabe 2 , Michisuke Yuzaki 1 1 Dept. Physiol., Sch. Med., Keio Univ., Tokyo, Japan; 2 Dept. Anatomy, Hokkaido Univ., Sch. Med., Sapporo, Japan; 3 Mol. Neurophysiol., AIST, Tsukuba, Japan
Cbln1 is a member of the C1q and Tumor Necrosis Factor families predominantly produced in cerebellar granule cells. Recently, we have shown that Cbln1 is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of Purkinje cells.
Although other members of the Cbln family, Cbln2-4, are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. Here, we show that each Cbln member is expressed in various regions of developing and mature brains. All Cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. Like Cbln1, Cbln2 and Cbln4 were secreted as glycoproteins, whereas Cbln3 was retained in the endoplasmic reticulum. These results suggest that each Cbln member is potentially involved in synapse development and plasticity in various brain regions. S-SCAM is a synaptic membrane-associated protein with PDZ domains, a guanylate kinase domain and WW domains. It interacts with various synaptic components including NMDA receptor subunits, PSD-95 and neuroligin. As we previously reported, S-SCAM is recruited to excitatory synapses by -catenin. S-SCAM forms a ternary complex with neuroligin and PSD-95. More importantly, S-SCAM is involved in synaptic accumulation of neuroligin and subsequently affects the localization of PSD-95 at excitatory synapses. In the course of these studies, we observed signals detected by anti-S-SCAM antibody at inhibitory synapses. We have here examined whether S-SCAM is indeed localized at inhibitory synapses in hippocampal neurons. We have raised questions which molecules S-SCAM interacts with at inhibitory synapses and which role S-SCAM plays in the assembly of inhibitory synapses.
Eriko Fujita, Yuko Tanabe, Takashi Momoi Division of Differentiation and Development, Department of Inherited Metabolic Disorder, National Institute of Neuroscience, NCNP, Oawahigashi, Tokyo, Japan IGSF4/RA175 (RA175), which is a member of immunoglobulin superfamily having PDZ binding domain at C-terminals, has Ca2+independent homophilic trans-cell adhesion activity. RA175 participates in synaptic junction and epithelial junctions in various tissues including testis. Homozygous null (Ra175-/-) male is infertile and shows the defective elongating spermatids and fails to mature further. RA175 interacted with PAR-3 being involved in the polarity of epithelial cells via PDZ binding domain at C-terminals. PAR-3 was colocalized in the cell adherent region of P19 embryonal teratocarcinoma cells during RA-induced differentiation into epithelial-like cells and mainly localized in the spermatid of Ra175+/+ testis, whereas it was undetectable in the spermatid of the Ra175−/− testis. RA175 and JAM-C were localized around the head portion of spermatid and RA175 deficiency provided the abnormal polarization of the JAM-C, which is necessary for the differentiation of round to elongated spermatid. JAM-C inhibited the interaction between RA175 and PAR-3.
Research funds: 12324122
Izumi Kawabata, Shigeo Okabe Department of Cell Biology, Tokyo Medical and Dental University, Tokyo, Japan Coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. However, there have been few analyses on postsynaptic molecular assembly in interneurons during development. To address this question, we examined dynamic properties of PSD-95 clusters in cultured hippocampal interneurons. Higher density of dendritic PSD-95 clusters was observed in interneurons at 11 DIV. At 18 DIV, this difference was less prominent, mainly due to >3-fold increase of PSDs in excitatory neurons. PSD-95-GFP imaging revealed lower rate of cluster appearance/disappearance in interneurons at 11 DIV. The higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. Photobleaching of PSD-95-GFP revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. Possible involvement of other postsynaptic molecules in interneuron PSD dynamics is now being investigated.
PS1A-C025 Two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures
Hideko Nishida, Shigeo Okabe Department of Cell Biology, Tokyo Medical and Dental University, Tokyo, Japan
Several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. However, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. Here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, Cre-dependent expression of GFP with electroporation of rhodamine-dextran. Two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in 3-7DIV slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. Dendritic protrusions with astroglial contacts also showed higher tendency to form spines. Furthermore, expression of mutant Rac1 in astroglial cells induced significantly longer, non-spiny protrusions than control. These findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions.
Taiko Imura, Fusao Kato Lab. Neurophysiol., Jikei Univ. Sch. Med., Tokyo, Japan
Application of P2X receptor agonists to the neurons in the nucleus of the solitary tract (NTS) results in glutamate release facilitation (Kato & Shigetomi, 2001; Shigetomi & Kato, 2003) . Recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as ATP. This study was performed to determine whether such astrocyte-neuron interaction takes place in the NTS. First, we analyzed the spatial localization of these cells by immunohistochemistry. A large number of GFAP-positive cells with processes in the close apposition to the NeuN-positive neurons were found. Second, we analyzed the effect on synaptic activity of localized application of ATP using laser-based photolysis of caged ATP in brainstem slices. Uncaging of ATP at neuronal dendrites (2 s, 4-micrometer diameter) resulted in an immediate rise in mEPSC frequency, in a manner sensitive to P2X receptor antagonists. These results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals.
Research funds: KAKENHI (17300123) PS1A-C027 Ealy synapsin I accumulation in a granule cell axon at the filopodial attachment site of developing rodent Purkinje cell dendrites in vitro Isao Nagata, Junko Kimura-Kuroda Department of Brain Structure, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan
Synapse formation between the parallel fibers (PF) and dendrites of Purkinje cells (PC) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. However, the precise spatio-temporal PF-PC interaction has not been elucidated. We have found that growth of PC dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of 2D-and 3D-cerebellar cultures. Here, we investigated the expression of a synaptic vesicle marker, synapsin I, in granule cell axons by multiple immunofluorescence labelings in these cultures. Synapsin I was first expressed at the filopodial attachment site of a PC dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. Thus, the filopodial attachment of the juvenile PC dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles.
PS1A-C028 Integrative spike dynamics of rat CA1 neurons: an in situ multineuronal imaging study Takuya Sasaki, Rie Kimura, Norio Matsuki, Yuji Ikegaya Department of Pharmacology, University of Tokyo, Tokyo, Japan
The brain operates through a coordinated interplay of numerous neurons. Our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. In hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the Schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the CA1 stratum pyramidale. Single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. The network activity varied from trial to trial, even though given constant stimuli. This variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. Our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple CA1 circuit, neurons could collectively be engaged in complex information processing. It is assumed based on previous in vitro experiments by other researchers that mGluR6 connects with syntenin at the dendrites and mGluR7 with PICK1 at the axon terminal in ON cone bipolar cells. To prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. Next, we examined which scaffold protein would connect with mGluR7 that was known to be ectopicly expressed in the dendrites of mGluR6-deficient ON cone bipolar cells. We observed no PICK1 but only syntenin at the mGluR6-deficient dendrites, and also the syntenin immunopositivity was co-localized with mGluR7 immunopositivity. These findings suggest that mGluR7 connects with syntenin in place of mGluR6 that was knockout from the ON cone bipolar dendrites.
Noriko TRPV family is identified as thermosensitive, Ca 2+ -permeable channels. TRPV1, expressed in sensory neurons, is activated by noxious heat above 42 • C, whereas TRPV 4, expressed in keratinocytes, is sensitive to moderate temperatures (>34 • C). Here we examined the role of TRPV1 and 4 in regulation of body temperature (BT) by using infrared laser as a heat stimulus. In wild type mouse, though the laser irradiation which caused the increase in skin temperature up to 55 • C did not induce the change in BT, desensitization of TRPV1 with capsaicin resulted in the increase in BT. On the other hand, in TRPV4-knockout mouse, moderate thermal radiation (>43 • C) caused the increase in the BT. The processing of noxious and moderate thermal radiation stimuli may depend on the TRPV1 and 4 respectively.
Research funds: KAKENHI (17657052) PS1A-C032 Generation and biochemical analysis of a GluR␣2 knockout mouse Hirotsugu Azechi 1 , Manabu Abe 1 , Rie Natsume 1,2 , Kenji Sakimura 1,2 1 Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan; 2 SORST/JST, Saitama, Japan GluR␣2(GluR2) is a key subunit of AMPA receptors, since it is a critical determinant of their calcium permeability. To clarify the molecular function of GluR␣2, we generated a conditional GluR␣2 knockout mouse using the Cre/loxP recombination system. We first established a "floxed" mutant line GRa2f using C57BL/6(B6) ES cell line RENKA. The homozygous floxed mutants showed no significant abnormalities, thus our GRa2f was used as a target of GluR␣2 line. By crossing GRa2f and TLCN-Cre that expressed Cre in germ line cells, GluR␣2 null KO mice were produced, but most of them died within 3 days after birth. To overcome the lethality, the GluR␣2 mutation was transferred onto B6/129 or B6/CD-1 genetic background. Subcellular fractionation and quantitative immunoblot showed changes in the amount of AMPA receptor subunits. These results indicated a significant role of GluR␣2 in the distribution of functional AMPA receptors in vivo.
PS1A-C033 GISP: A novel brain specific protein that binds to the GABA B1 subunit and promotes its surface expression Here we report the identification and characterisation of a novel brain specific 130 kDa protein, GABA B R Interacting Scaffolding Protein (GISP), that interacts directly with the GABA B 1 subunit via a coiledcoil domain. GISP coimmunoprecipitates with GABA B 1 and GABA B 2 from rat brain. In cultured hippocampal neurons GISP displays a punctate dendritic distribution and colocalises with GABA B receptors. When co-expressed with GABA B Rs GISP increases the amount of GABA B 1 protein and also promotes GABA B 1 surface expression in the heterologous cells. Furthermore, GISP increases surface expression of GABA B 1/GABA B 2 complexes. These results suggest that GISP is involved in the forward trafficking and stabilisation of GABA B Rs. Thus GISP is an novel GABA B 1-binding protein potentially involved in the cell surface and/or synaptic targeting of the GABA B Rs. Three distinct isoforms of vesicular glutamate transporters (VGLUT1-3) have been cloned and shown to exhibit differential distribution patterns in the brain. Recent work shows the presence of VGLUTs in synaptic-like microvesicles (SLMVs) of endocrine cells. Mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous SLMVs which likely accumulate glutamate to inhibit melatonin synthesis. VGLUT1 and VGLUT2 seem to participate in this glutamate accumulation. In the present study, we found that VGLUT3 mRNA is also expressed in the adult rat pinealocytes. VGLUT3 immunoreactivity (IR) was distributed throughout the pineal gland, and was co-localized with VGLUT1-IR or VGLUT2-IR in many, but not all, processes of pinealocyte. These data indicate that there are some subpopulations of SLMVs which differ in the kind of VGLUT isoforms contained and/or in their combinations, suggesting VGLUT isoform-dependent sorting of SLMVs to pinealocyte processes.
Kenzi Saito 1,2,3 , Kenji Nakamura 4 , Toshikazu Kakizaki 1,3 , Satoe Ebihara 5 , Masakazu Uematsu 6 , Shigeo Takamori 7 , Minesuke Yokoyama 4 , Shiro Konishi 4 , Masayoshi Mishina 3,8 , Jun-ichi Miyazaki 9 , Kunihiko Obata 10 , Yuchio Yanagawa 1,3 1 Gunma Univ., Maebashi, Japan; 2 SOKENDAI, Hayama, Japan; 3 SORST, Kawaguchi, Japan; 4 Mitsubishi Kagaku INST. Life Sci., Machida, Japan; 5 Kumamoto Univ., Kumamoto, Japan; 6 Toyohashi Univ. Tech., Toyohashi, Japan; 7 Tokyo Med. Den. Univ., Tokyo, Japan; 8 Univ. Tokyo, Tokyo, Japan; 9 Osaka Univ., Suita, Japan; 10 RIKEN, Wako, Japan
The vesicular GABA transporter (VGAT) loads GABA from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing GABA and/or glycine. To assess the functional role of VGAT in development, we have disrupted the gene encoding VGAT using Cre-loxP system. Western-blot analysis showed that VGAT protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. VGAT knockout mice died around birth. All VGAT knockout mice displayed cleft palate and omphalocele. Our results suggest that VGAT plays essential roles in both palate formation and ventral body wall development.
Research funds: KAKENHI (17052002) PS1A-C036 Postnatal changes in the colocalization of VGluT1 and VGluT2 immunoreactivities at single axon terminals of the mouse neocortex Kouichi Nakamura 1,2 , Akiya Watakabe 3 , Hiroyuki Hioki 1 , Fumino Fujiyama 1 , Yasuyo Tanaka 1 , Tetsuo Yamamori 3 , Takeshi Kaneko 1,2 1 Dept. Morphol Brain Sci., Grad. Sch. Med., Kyoto Univ., Japan; 2 CREST, JST, Japan; 3 Div. Brain Biol., Nat. Inst. Basic Biol., Okazaki, Japan Vesicular glutamate transporter (VGluT)1 and VGluT2 accumulate transmitter glutamate into synaptic vesicles. The VGluTs show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. Here we quantitatively evaluated postnatal changes in the colocalization of VGluTs at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (CC) as an index of the colocalization. The CC was calculated from pixel values for VGluT1 and VGluT2 in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. In the barrels, the CC showed a prominent increase transiently around P7. The CC was higher in area S1 than areas M1 and area V1 throughout postnatal development. Our results indicate that the colocalization of VGluTs in the neocortex is regulated in an age-, area-and layer-specific manner. GABA B receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the G protein-coupled receptors.
Here we have investigated the role of 5 AMP-activated protein kinase (AMPK), as an endogenous regulator of GABA B receptor function. Site-specific mutagenesis identified multiple phosphorylation sites for AMPK within the cytoplasmic tails of both GABA B R1 and R2. The activation of AMPK regulated stability of GABA B receptors coupling with K + channels. Together highlights a novel role for AMPK in regulating the functional properties of GABA B receptors, by direct phosphorylation. Given the role of AMPK as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity.
Takao Hirai, Hiroaki Nishio Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Hiroshima, Japan Serotonin (5-hydroxytryptamine, 5-HT) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. There is increasing evidence for contribution of neuronal system to regulation of bone metabolism. This study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. RT-PCR analysis revealed constitutive expression of mRNA for several 5-HT receptor subtypes, 5-HT transporter (5-HTT) and vesicular monoamine transporter 1 (VMAT1) in primary cultured mouse osteoblasts and MC3T3-E1 osteoblastic cells. Sustained exposure to fluoxetine, a selective 5-HT reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in MC3T3-E1. These results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts.
Junko Motohashi Department of Physiology, Keio University School of Medicine, Tokyo, Japan
Hotfoot mice are spontaneous mutants with ataxic phenotype. Most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the N-terminal domain of the ␦2 glutamate receptor (GluR␦2). However, because only genomic DNA was available for most hotfoot mutants, it was unclear whether truncated forms of GluR␦2 were actually translated and involved in the ataxic phenotype. Here, we report that a newly identified hotfoot mutant, ho15J, was caused by a new type of intragenic deletion of the Grid2 gene, which was indeed translated as GluR␦2 lacking 52-amino acids in the N-terminus. Mutant GluR␦2 proteins were retained in the soma of Purkinje cells and degraded. As a result, ho15J mice exhibited a severe motor discoordination on rotarod tests. Furthermore, these mice exhibited sustained innervation of Purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. These results indicate the importance of the N-terminal domain in GluR␦2 signaling and cerebellar functions.
Research funds: KAKENHI (16200024) PS1A-D040 Role of the DRY motif in melanin-concentrating hormone receptor 1 in signaling Yumiko Saito 1 , Yoshimi Aizaki 1 , Mituse Nakano 2 , Kei Maruyama 1 1 Dept. Pharamacol., Saitama Med. Sch., Saitama, Japan; 2 International University of Health and Welfare, Tochigi, Japan
Considerable attention has been focused on the functional importance of the highly conserved DRY triplet in class A G protein-coupled receptors (GPCR). Here we investigated the role of Asp140, Arg141 and Tyr142 in the DRY of rat melanin-concentrating hormone receptor 1 (MCH1R). In transfected cells, mutation of Asp (D/A) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to MCH. D/A mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. Y/A mutation also yielded a loss of function phenotype that is similar to D/A mutation. Mutation of the Arg (R/A) showed higher EC50 value in signaling with a decrease in MCH binding, while the level of cell surface expression exhibited only moderate decrease. These data suggest that a function for DRY motif different from that widely accepted for class A GPCRs in regulating MCH1R-mediated signal pathway. In this study we confirmed functional heteromultimerization between A 1 R and P2Y 1 R electrophysiologically using Xenopus oocyte expression system. When A 1 R and P2Y 1 R were coexpressed, application of non-hydrolyzable ATP analogue induced G i/o response, showing formation of functional heteromultimers with a unique phenotype. It was also observed that the heteromultimers can activate G q/11 pathway by ATP analogue and also G i/o pathway by adenosine analogue, maintaining the features of the original subunits.
PS1A-D043 Dual signaling via metabotropic glutamate receptor1␣ is regulated by a cytoskeletal protein 4.1G
Michihiro Tateyama 1,2 , Yoshihiro Kubo 1,2 1 Department of Biophysics and Neurology, NIPS, Aichi, Japan; 2 SORST, JST, Saitama, Japan
The G protein-coupled metabotropic glutamate receptor 1␣ (mGluR1) is known to functionally couple to different types of G proteins. Recently we have reported that the signaling pathways through mGluR1 are differentially regulated by different types of ligands, glutamate and Gd 3+ . On the other hand, several cytoskeletal proteins have been reported to interact with the C-terminal cytoplasmic tail of mGluR1. These proteins, such as homer and 4.1G, are also known to change the membrane expression of and modulate the function of mGluR1. Here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mGluR1. Interestingly, the functional couplings of mGluR1 to Gq and Gs pathways were altered by co-expression of 4.1G, but not by homer. Deletion of the C-terminal tail abolished the effect of 4.1G, indicating that the interaction of 4.1G with the C-terminal tail of mGluR1 regulates the multi path signaling.
PS1A-D044 Modulation of the EAAC1-mediated glutamate uptake by the addicsin mutant Mitsushi J. Ikemoto 1,2 , Saori Akiduki 1,2 1 Age Dimension Research Center, AIST, Ibaraki, Japan; 2 Graduate School of Science, Toho University, Chiba, Japan
Addicsin is a murine homologue of rat glutamate-transporterassociated protein 3-18 (GTRAP3-18), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier 1 (EAAC1). It contains two potential PKC phosphorylation motifs at positions 18-20 and 138-140. However, its physiological function remains almost unknown.
To clarify a significance of these PKC phosphorylation motifs, we investigated EAAC1-mediated glutamate transport activity in C6Bu-1 cells provided with a mifepristone-inducible expression of addicsin (WT), its mutants mutated at Serine 18 into Alanine (S18A) or at Serine 138 into Alanine (S138A). As compared with WT, S18A had no inhibitory effect on glutamate transport activity under exposure to 100 nM PMA, and had increased glutamate transport activity under normal condition. By contrast, S138A had the same glutamate transport activity as that of WT. Thus, the EAAC1-mediated glutamate transport activity may be regulated by a PKC-dependent phosphorylation at Serine 18 in addicsin.
Kaori Akashi 1 , Manabu Abe 1 , Toshikazu Kakizaki 1 , Rie Natsume 1,2 , Kenji Sakimura 1,2 1 Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Japan; 2 SORST-JST, Saitama, Japan
Kainate type glutamate receptors are composed of various combinations of GluR1-3(GluR5-7) and GluR␥1-2(KA1-2) subunits. Although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. To clarify the functions and subunit dynamics of kainate receptors, we generated GluR2KO mice from C57BL/6 ES cell line. The GluR2KO mice were viable, fertile, and displayed no overt phenotype. On the other hand, the amounts of GluR␥1 and GluR␥2 proteins were significantly decreased in the crude fraction of CA3 region of GluR2KO. Furthermore, subcellular localizations of both subunits were also changed in GluR2KO. These results suggested that native kainate receptors might function as heteromeric channels (GluR/␥) and the GluR2 subunit might determine subcellular localization of the GluR␥ subunits, similar to the roles of NMDA receptor GluR subunits determining stability and distribution of the GluR subunits.
PS1A-D046 Sema4D/Plexin-B1 activates GSK-3 via R-Ras GAP activity, inducing growth cone collapse Yuri Ito, Izumi Oinuma, Hironori Katoh, Manabu Negishi Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
Plexins are receptors for repulsive axonal guidance molecules semaphorins. We have recently reported that semaphorin 4D (Sema4D) receptor Plexin-B1 induces growth cone collapse by functioning as an R-Ras GAP. Here we characterized the downstream signaling of Plexin-B1-mediated R-Ras GAP activity, leading to growth cone collapse. Sema4D suppressed the endogenous R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of GSK-3. Ectopic expression of the constitutively active mutant of Akt, myr-Akt, or treatment with GSK-3 antagonist suppressed the Sema4D-induced growth cone collapse. The R-Ras GAP activity was necessary for Plexin-B1-induced dephosphorylation of Akt and GSK-3. Plexin-A1 also induced dephosphorylation of Akt and GSK-3 through its R-Ras GAP activity. Thus, we conclude that Plexin-B1 dephosphorylates Akt and GSK-3 through R-RasGAP activity, inducing growth cone collapse. To find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (HOPS) as a candidate gene. HOPS had three transmembrane domains, and expressed abundantly on a brain tissue. HOPS was detected in membranous regions from subcellular fractionation and immunohistochemistry. HOPS was recruited to membranous structures when overexpressed in COS7 cells. When expressed in hippocampal cultures, HOPS enhanced the amplitude of mEPSC. From antibody feeding assay, we discovered that HOPS enhanced the recycling of GluR2. HOPS was co-immunoprecipitated with GRIP1 (Glutamate receptor interacting protein 1) when they were co-transfected to HEK cells. Thus, it was suggested that HOPS had roles in synaptic transmission enhancement by stabilization of surface GluR2 via GRIP1 binding. Serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. We have recently identified a serine transporter Asc-1. A neural amino acid transporter SNAT2/ATA2 also transports serine. We investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. The Asc-1 immunoreactivity (Asc-1-ir) was detected in dendrites and somata of pyramidal neurons. The SNAT2-ir was widely detected in neurons, whose intracellular localization was similar to that of Asc-1-ir. Deferent from Asc-1-ir, SNAT2-ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. These results suggest the significant contribution of Asc-1 and SNAT2 to the neuronal uptake of L-serine. SNAT2 might also accumulate L-serine in astrocytes from the extracellular spaces including blood and CSF.
PS1A-D049 Neuronal glutamate transporter EAAT4 controls climbing fiber-mediated presynaptic inhibition of GABAergic transmission at cerebellar interneuron-Purkinje cell synapses
Shin'Ichiro Satake 1 , Si-Young Song 2 , Shiro Konishi 3 , Keiji Imoto 1 1 Natl. Inst. Physiol. Sci. (NIPS), Okazaki, Japan; 2 Mitsubishi Kagaku Inst Life Sci, Tokyo, Japan; 3 Tokushima Bunri Univ., Sanuki, Japan
through extrasynaptic diffusion and activation of presynaptic AMPA receptors in BC terminals. We here examined possible roles of glutamate transporters in this CF action. The EAAT4/GLT-1 blocker threo-3-methylglutamate, but not the GLT-1 blocker dihydrokainate, augmented the CF-induced inhibition. CF stimulation obviously inhibited GABAergic transmission onto PCs in the lobule III, where EAAT4 expression was low, whereas the CF-induced inhibition was minimal in the lobule X, where EAAT4 was abundant. The results suggest that EAAT4 plays a major role in regulating the concentration of CF transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of CF-induced inhibition of GABA release from BCs depending on the regional difference of EAAT4 expression in postsynaptic PCs.
Chitoshi Takayama 1 , Yoshiro Inoue 1 1 Department of Molecular Neuroanatomy, Hokkaido University School of Medicine, Sapporo, Japan GABA mediates inhibitory transmission in the adult central nervous system (CNS). In contrast, GABA induces depolarization in the immature CNS. This developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, Na-K-2Cl co-transporter 1 (NKCC1) and K-Cl co-transporter 2 (KCC2). In this study, we focused on KCC2, which lowers the intracellular chloride ion concentration, and examined the developmental localization of the KCC2 with special reference to the neuronal development in the cerebellum. KCC2 was negative in the proliferating and migrating neurons. Post-migratory neurons, which formed synapses, expressed the KCC2. The KCC2-protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. These results suggested that formation of synapses might induce KCC2-expression and localization, and GABAergic transmission might shift from excitation to inhibition after synapse formation.
Akinori Nakajima 1 , Hisashi Mori 1 1 Molecular Neuroscience, University of Toyama, Toyama, Japan
The actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (G-proteins). The dopamine receptors are classified into two categories, D1-like and D2-like according to their pharmacological properties. The D1-like receptors consist of D1 and D5 receptor, and are coupled to the adenylyl cyclase activating G proteins (Gs). In the present study, we have generated a series of D1 receptor mutants and examined the effect on the Gs coupled receptor signaling. We found that the expression of the third intracellular loop (3i loop) domain of D1R fused with EGFP effectively reduce cAMP production mediated by D1 and D5 receptors. Interestingly, we also identified that the 3i loop domain of D1R interfere with Gs coupled beta2 adrenergic receptor signaling. These results suggest that the third intracellular loop of the D1 receptor is a primary determinant in its coupling to Gs signaling.
The activation of phosphatidylinositol-linked D1-like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus Yoshinobu Noriyama 1 , Yoichi Ogawa 2 , Hiroki Yoshino 1 , Masayuki Yamashita 2 , Toshifumi Kishimto 1 1 Dept. Psychiatr.; 2 Dept. Physiol I, Nara Med. Univ., Kashihara, Japan
We studied the effect of dopamine (DA) on GABAergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from CA1 pyramidal cells. DA (100 M) profoundly decreased GABA A receptor-mediated postsynaptic currents to 32% in the first postnatal week, when GABA provides excitatory drive. DA also decreased AMPA receptor-mediated excitatory post synaptic currents to 29% in the second postnatal week, when glutamate responses first appear. The DA-induced inhibition declined after these periods. The receptor subtype involved in the DA-induced inhibition was phosphatidylinositol (PI)-linked D1-like receptor, since SKF 83959, a selective agonist for PI-linked D1-like receptor, clearly mimicked the action of DA. These results suggest that the activation of PI-linked D1-like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus.
Ayuka Ina 1 , Jinko Konno 1 , Sachine Yoshida 1 , Hideki Ohmomo 1 , Hitoshi Kawano 2 , Fumihiro Shutoh 1 , Haruo Nogami 1 , Setsuji Hisano 1 1 Graduate Sch., Comprehensive Human Sci, Univ. Tsukuba, Tsukuba, Japan; 2 Tokyo Metro Inst Neurosci, Tokyo, Japan
Supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express VGLUT1 or -2 mRNA at early fetal ages. To know roles of fetal VGLUT in cortical development, we studied expressions of VGLUT proteins in mouse fetuses by immunohistochemistry. On embryonic day 13 (E13), VGLUT1 immunoreactivity (ir) was first detected in the marginal zone (MZ), subplate (SP) and intermediate zone (IMZ). On E15, VGLUT1-ir was seen as puncta close to L1-ir thalamocortical fiber tracts in the SP and also localized to fiber tracts expressing L1or TAG1-ir in the IMZ, whereas VGLUT2-ir was first observed in the SP and upper IMZ where L1-ir existed. These results show that VGLUT1ir corticofugal fibers appose to elongating VGLUT2-ir thalamocortical fibers, suggesting that VGLUT may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex.
PS1A-D054 Developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal CA1 regions
Michie Kon 1 , Yoichi Avil 2 , Hiroshi Tsubokawa 1,2 1 Dept. of Information Engineering, Tohoku Univ., Sendai, Japan; 2 Grad. Schl. of Information Sciences, Tohoku Univ.
To investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. Swelling of several areas within the CA1 region were detected as increases in transmittance of near infrared light (IRT). Field EPSPs (fEPs) were recorded simultaneously from the stratum radiatum of CA1 region. In adult mice, repetitive stimulation of afferent fibers induced transient increases in IRT at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with fEPs. Application of the Bicuculline, a GABA-A receptor antagonist, reduced these optical signals. However, in mice under 15 days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. These results suggested that GABA-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal CA1 region. In the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal CA1 region on morphine-induced conditioned place preference (CPP) were investigated in rats. Subcutaneous administration of different doses of morphine (2.5-10 mg/kg) produced a dose-dependent CPP. Using a 3-day schedule of conditioning, it was found that intra-CA1 administration of NMDA receptor antagonist, MK-801 (2 and 4 g/rat) significantly attenuated the morphine (7.5 mg/kg)-induced CPP. Moreover, NMDA receptor agonist, NMDA (0.1, 0.2 and 1 g/rat) significantly potentiated the morphine (2.5 mg/kg)-induced CPP. These results suggest that the development of morphine-induced CPP may be related to NMDA and MK-801 receptors in that the glutamatergic system can modulate opiate reward.
Takahiro Sonomura 1 , Kouichi Nakamura 2,3 , Hiroyuki Hioki 2 , Masanori Uemura 1 , Takeshi Kaneko 2,3 1 Dept. Anatomy for Oral Sciences, Grad. Sch. Med. and Dent., Kagoshima Univ., Kagoshima, Japan; 2 Dept. Morphological Brain Sciense, Grad. Sch. Med., Kyoto Univ., Kyoto, Japan; 3 CREST, JST
The majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing PPD, and striatopallidal neurons producing PPE, and striatoinnominatal neurons producing PPTB. These projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. It has been assumed that D1 receptor are expressed in striatonigral neurons and D2 receptor are expressed in striatopallidal neurons. In recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (D1, D2, D3, D4, D5). In this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. The cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. In the present study, we examined the localization of chondroitn sulfate proteoglycans (CSPGS) by CSPG-specific antibodies and lectins. CSPGs are mainly observed at molecular layer in developing cerebellum (P10-15) but they scarcely seen at external granular layer. Electron microscopic observation demonstrated that phosphacan, one of CSPGs, is localized at axonal membrane of parallel fibers. Moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. Thus, CSPGs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. The vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading.
In the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (CSPGs) in vasopressin neurons. This downregulation is possibly due to proteolysis by tPA, since (1) tPA immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, (2) Salt loading increased protein and mRNA levels of tPA in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, (3) Depolarizing agent released tPA from isolated neurosecretosomes, (4) tPA knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. Thus, it is probable that tPA is participated in regulating structural plasticity of vasopressin neurons by degrading CSPGs. Chondroitin sulfate (CS) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. CS chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. In this study, we analyzed the expression patterns of several CS sulfotransferases in the developing mouse cerebrum. Using in situ hybridization analysis, it was revealed that CS sulfotransferase mRNAs (U2ST, GalNAc4-6ST, D4ST) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. Immunohistochemical analysis with anti-CS antibodies revealed that CS were highly expressed in the ventricular zone and the marginal zone. These results suggest that the CS structural domains generated by these CS sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration.
Nobuaki Maeda 1 , Maki Ishii 1 , Isao Nagata 2 , Yumiko Shimazaki 1 1 Dept. of Dev. Neurosci., Tokyo Metro. Inst. for Neurosci., Tokyo, Japan; 2 Dept. of Brain Structure, Tokyo Metro. Inst. for Neurosci.
Chondroitin sulfate (CS) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. Previously, we revealed that CS is involved in the morphogenesis of the Purkinje cell dendrites. In this study, we analyzed the expression of CS in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in CS. Among the epitopes recognized by these antibodies, the expression of MO-225 epitopes, GlcA(2S)1-3GalNAc(6S) (D unit)containing structures, remarkably increased during development. Detailed immunohistochemical analysis indicated that D unit-rich CS was deposited between Purkinje cell surface and the processes of Bergmann glia. Furthermore, it was found that pleiotrophin bound to D unit-rich CS on phosphacan distributed around Purkinje cells. These observations suggest that D-type structure in CS is important for the signaling of pleiotrophin, which play roles in Purkinje cell-Bergmann glia interaction.
Nobuna Fukazawa 1 , Mineko Kengaku 2 , Nobuaki Maeda 1 1 Dept. of Dev. Neurosci., Tokyo Metro. Inst. for Neurosci., Tokyo, Japan; 2 Lab. for Neuronal Cell Polarity, Riken BSI, Wako, Japan PTP is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-PTP signaling regulates the morphogenesis of Purkinje cell (PC) dendrites. We previously revealed that PTP associated with Delta/Notchlike EGF-related receptor (DNER), which mediates the PC-Bergmann glia (BG) interaction and regulates morphological differentiation of these cells. Here, we found that PTP was expressed by both PCs and BGs and the expression by PC occurred at relatively late developmental stage. PTP showed patchy distribution in the dendritic shafts of PCs, which partially overlapped with the localization of DNER. Furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of DNER were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the PTP catalytic domain. These results suggested that PTP participate in the PC-BG interaction by regulating tyrosine phosphorylation level of DNER.
Masahiko Tanaka 1 , Tohru Marunouchi 1 1 Division of Cell Biology, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan Cerebellar Purkinje cells have the most elaborate dendritic trees among the neurons in the CNS. To investigate the cellular and molecular mechanisms of dendrite development of Purkinje cells, we cocultured Purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of 3 m pore size. When Purkinje cells were co-cultured with granule cells, dendrite development of Purkinje cells was promoted in comparison with that in control conditions. This co-culture effect was abolished by addition of a glutamate antagonist in the cultures. In contrast, dendrite development of Purkinje cells was inhibited when Purkinje cells were co-cultured with astrocytes. We propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of Purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. Heparan sulfate (HS) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. An extracellular endosulfatase, SulfFP1, has been implicated in the regulation of growth factor/morphogen signaling through HS remodeling in vitro, but its physiological roles remain unknown. Here we generated knockout mice lacking the SulfFP1 gene, and examined the motor control. Homozygotes appeared to be normal, showing no sign of ataxia. Performances of the rotarod and beam-walking tests were normal compared with the control mice. Both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. Heparan sulfate (HS) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. These interactions are mediated by the specific sulfation patterns in HS, but the mechanism generating such modifications has not been fully elucidated. Here we show that a new class of HS endosulfatases plays an important role in brain development. The mice deficient in either SulfFP1 or SulfFP2 appeared to be normal, while most of the double knockout mice died soon after birth. Mutant brains had higher content of 6-O-sulfated disaccharide units in HS, suggesting a role of SulfFPs in heparan sulfate remodeling in vivo. The double mutant brains were smaller than the controls and showed some axon guidance defects. These data demonstrate that specific HS modification generated by SulfFPs is important for normal brain development. Recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. Here, we examined roles of 5-hydroxytryptoamine (5-HT), noradrenaline (NA) and dopamine (DA) in the formation of dendrites and synapses by dissociation culture. Embryonic day 16 or 18 rat cerebral cortex was cultured in the presence of 5-HT, NA or DA. After 4 days, we analyzed dendrite formation using anti-MAP2 antibody. After 14-21 days, we analyzed synaptogenesis with anti-PSD-95, anti-synaptophysin, and anti-MAP2 antibodies. The addition of 5-HT (100-10000 nM), NA (100-1000 nM) or DA (10-1000 nM) increased dendritic length of pyramidal neurons. 5-HT (100-1000 nM) also increased the synaptic density. By using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by 5-HT 1A receptor, ␣ 2A receptor and D 2 receptor, while inhibited by 5-HT 2A and receptors. In addition, synaptogenesis was promoted by 5-HT 2A and 5-HT 2C receptors, whereas inhibited by 5-HT 1A receptor.
Tatsuya Mori, Tomoe Wada, Takahiro Suzuki, Naoyuki Inagaki Department of cell biology, Nara institute of science and technology, Nara, Japan
Most neurons have polarized shape consisting of a single long axon and multiple dendrites. Several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. In this study, with proteomic approach, we identified a novel protein, Singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. Singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. In 293T cell, Singar associated with p85 and p110, the subunits of PI3Kinase which is considered as one of the key molecules in neuronal polarization. Moreover, inhibition of Singar by RNA interference induced the formation of multiple axon-like neurites. These data suggest that Singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons.
PS1A-E068 Lrfn2, a neuronal leucine-rich repeatcontaining transmembrane protain can interact with PSD-95
Naoko Morimura, Takashi Inoue, Kei-ichi Katayama, Jun Aruga Laboratory for Comparative Neurogenesis, RIKEN BSI, Saitama, Japan
In a variety of organisms, proteins with leucine-rich repeat domain (LRR) function significantly in neural development. Lrfn, a neuronal LRR transmembrane family, was expressed in the brain specifically. Expression of Lrfn2 was low in embryonic brain, and increased dramatically after birth. In the rat dissociated hippocampal neurons, Lrfn2 protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with PSD-95, a postsynaptic scaffold protein. We examined the physical interaction between Lrfn2 and PSD-95 by immunocrecipitation and pull-down assay, since Lrfn2 contains Class I PDZ domain-binding motif at its C-terminal tail. We revealed that Lrfn2 associated with PSD-95/NMDA receptor complex in the brain extracts and Lrfn2 directly bound to PSD-95 via its PDZ domain-binding motif. In this study, we suggest that Lrfn2 may play an important role in the regulation of synaptic functions.
Tsuya Taneda, Shingo Miyata, Hiroaki Okuda, Masaya Tohyama Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan
Protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine N-methyltransferases (PRMT1-8). Among the PRMT proteins, the PRMT3 has some characteristic motifs in the N-terminal tract which follows its active methyltransferase site. Although little attention has been paid to protein methylation in the nervous system. First of all, we have examined the distribution of the PRMT3 in the rat brain. The PRMT3 was expressed in the cell bodies and dendrites in the hippocampal neurons. Further, the ontogenetic analysis revealed the PRMT3 expression increased from the perinatal stages to the adulthood. These findings suggest that the PRMT3 relates to the neural function in the young and adult brain. Furthermore in order to study the role of the PRMT3 in the brain, we tried to identify novel interacting proteins with the PRMT3 in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry.
PS1A-E070 Proteomics of the growth cone: II. The systematic immunostaining analysis of the growth cone proteins identified by the proteomic research
Motohiro Nozumi 1,2 , Michihiro Igarashi 1,2 1 Div Mol Cell Biol, Grad. Sch. Med Dent Sci; 2 Trans-diciplinary Res Program, Niigata Univ., Niigata, Japan
Proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. In the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. Although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. Currently, we have already performed the immunocytochemistry concerning more than 150 identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. By quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. We have found more than twenty proteins specifically localized in the growth cone by this analysis.
Research funds: KAKENHI 17023019; Project-promoting grant from Niigata Univ PS1A-E071 Netrin-1 is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue
Tomoyuki Masuda 1 , Keisuke Watanabe 2 , Kazuhiro Ikenaka 2 , Katsuhiko Ono 2 , Hiroyuki Yaginuma 1 1 Dept. Anat., Fukushima Med Univ. Sch. of Med., Fukushima, Japan; 2 Div Neurobiol Bioinfo, Nat Inst Physiol Sci, Aichi, Japan
In higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (DRG) axons to orient them toward their targets. Netrin-1 is known to be a chemorepellent for a subset of axons, the role of netrin-1 for ventral spinal cord-derived repulsion is, however, unknown. By employing culture assays, we report here the involvement of netrin-1 in this repulsion. In the mouse embryo at E11, netrin-1 is expressed in the floor plate and the dermamyotome, and the netrin-1 receptor unc5c is expressed in DRG neurons. We show that HEK-cell aggregates secreting netrin-1 repelled chick E5 DRG axons. Moreover, using function-blocking antibody against netrin-1, we revealed the fact that netrin-1 plays an important role in ventral spinal cord-derived repulsion. Together, these findings suggest that the ventral spinal cord repels DRG axons by secreting netrin-1 to shape the initial trajectories of DRG axons.
Research funds: Grants-in-Aid on Priority Area (C) (MECST 17590166 to T.M.)
Hitoshi Maeda, Masaki Sakurai Department of physiology, Teikyo University school of medicine, Tokyo, Japan
In the previous reports, we showed that in the early development, corticospinal synapses (CS) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. However, the property of postsynaptic cells to CS input is poorly understood. In the present study, we investigated the electrophysiological and morphological properties of the neurons that receive CS synapses in the acute spinal cord slices of neonatal rat. The postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the CS tract is located in rodents, were whole cell patch clamped and labeled by Neurobiotin TM . Responsive neurons are widely distributed in the P6 neonates, but the ventral neurons became unresponsive after P9. The majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns.
Keisuke Watanabe 1 , Hirohide Takebayashi 1,2 , Kazuhiro Ikenaka 1 , Katsuhiko Ono 1 1 Div. Neurobiol. Bioinfo., Natl. Inst. Physiol. Sci., Okazaki, Japan; 2 Dev Stem Cell Biol. Program, UCSF, USA Netrin-1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. However, it is not known whether netrin-1 also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. To test this hypothesis, we examined dorsal interneuron migration in netrin-1 −/− background, using Olig3-lacZ knockin allele, which marks most of ventral-ward migrating dorsal interneurons. In the embryonic spinal cord of Olig3 +/lacZ ;netrin-1 −/− mice, ventral migration of Olig3 cells was significantly impaired. Furthermore, a netrin receptor, Dcc was expressed in Olig3-positive cells. These results suggest that netrin-1 exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. Netrin-G1 and netrin-G2 are vertebrate-specific membrane-anchored members of the UNC-6/netrin family that have no affinity to classic netrin receptors and their function is unknown. Here we show that netrin-G1 and netrin-G2 proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. Netrin-G1 and netrin-G2 differentially bind to LRRcontaining proteins, NGL-1 and a related molecule NAG14, in vitro. NGL-1 and NAG14 in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-G1 and netrin-G2, respectively. Furthermore, in netrin-G1 and netrin-G2 deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. Together, these results suggest that axonal netrin-G proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments.
Jinhong Huang 1 , Ryuichi Sakai 2 , Teiichi Furuichi 1 1 Lab. Molecular Neurogenesis, Brain Science Institute of RIKEN, Saitama, Japan; 2 Division of Cell Growth Factor, National Cancer Center of Japan, Tokyo, Japan
Cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. The neuronal function of Cas, however, is poorly understood. Here we report that Cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. During cerebellar development, Cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. Cas coimmunoprecipitates with Src family protein tyrosine kinases, Crk, and cell adhesion molecules. The axon extension of granule cells is inhibited by either RNA interference knockdown of Cas or overexpression of the Cas mutant lacking the Crk binding motifs. These results demonstrate that Cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation.
Research funds: Huang was a postdoctoral fellowship recipient of JSPS In in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on P4-P8, stimulation of parallel fibers produces excitation of Purkinje cells lasting for 600-800 ms. This unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where Purkinje cells develop primary dendrites on P5-P7. Since -Agatoxin IVA and -conotoxin MVIIC abolished the prolonged response, we suggest the involvement of P/Q type Ca channels. Immunohistochemical labeling revealed that P/Q type Ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on P4 and that they then locate in Purkinje cells, in cell body on P5 and in primary dendrites on P6-P7. The parallel development of P/Q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-Purkinje cell transmission suggests their causal relationships.
Naoya Ichikawa, Yasuo Kitagawa, Tatsuhiko Kadowaki Graduate School of Bioagricultural Sciences, Nagoya University, Aichi, Japan
We have recently identified a novel gene, Mahya, which is specifically conserved between Hymenoptera and Deuterostome. Mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a C-terminal novel domain. Mouse Mahya genes (mMahya-1 and mMahya-2) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. We have found that mMahya-1 protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. These results suggest that mMahya-1 is involved in either the migration of granule cells or the dendritic maturation of Purkinje cells. We will further report the analysis of the functions of mMahya-1 for the early cerebellum development.
Toshitaka Morishima 1 , Erina Fukushi 1 , Kazuto Kobayashi 2 , Naohiro Hozumi 1 , Sachiko Yoshida 1 1 Toyohashi University of Technology, Toyohashi, Japan; 2 Honda Electronics Co. Ltd., Toyohashi, Japan
In cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. Although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. We have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. An acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. The developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature EGL from neonatal rat. The reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. This imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required.
Tatsuro Yamamoto 1 , Hideyuki Dekimoto 1 , Tomiyoshi Setsu 1 , Masahiko Watanabe 2 , Mikio Hoshino 3 , Yo-Ichi Nabeshima 3 , Toshio Terashima 1 1 Dept. of Anat., Kobe Univ. Grad. Sch. of Med., Kobe, Japan; 2 Dept. of Anat., Hokkaido Univ. Grad. Sch. of Med., Sapporo, Japan; 3 Dept. of Pathol and Tumor Biol, Grad. Sch. of Med., Kyoto Univ., Kyoto, Japan A mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. The responsible gene for this mutation is Ptf1a, whose expression is lost in this mutant. In the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. The injection of Fluoro-Gold (FG) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. These FG-labeled neurons were glutaminase-positive. After the injection of BDA into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. These findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex.
Kumiko Ishida 1 , Tomoko Nishiyama 1 , Hitoshi Tatsumi 1 , Masahiro Sokabe 1,2 1 Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2 ICORP, Cell Mechanosensing Project, Japan Science and Technology Corporation
Sprouting and synaptic reorganization of the mossy fiber (MF) are commonly found in the hippocampus of temporal lobe epilepsy patients. As the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. We found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately 12 h and the elongation started at approximately 24 h after the drug application. Tetrodotoxin strongly inhibited both the retraction and elongation, while the BDNF sequestering protein, TrkB/FC, retarded only the elongation. This result suggests that Na + channel dependent neuronal excitation and following activitydependent BDNF releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices.
Rieko Muramatsu, Yuji Ikegaya, Maki K. Yamada, Norio Matsuki, Ryuta Koyama Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo
Hippocampal granule cells extend their axons, i.e. the mossy fibers (MFs), from the dentate gyrus (DG) to the area CA3. Once this oneway projection is disrupted, the MFs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the DG. To clarify the mechanism that regulates normal, anterograde MF projections, we used a co-culture system of hippocampal slices. When a DG slice from a GFP(+) rat was juxtaposed to the CA3 region of a host hippocampal slice from a wild type rat, the GFP(+) MFs ran through the host CA3 toward the host DG but failed to invade it even after ten days in vitro. Thus the DG seemed to serve as a barrier that blocks retrograde projections of MFs. However, the MFs extended into the DG when forskolin, an activator of adenylate cyclase, was chronically applied. These results suggest that the DG has a mechanism supporting anterograde MF projections to CA3, which is regulated by the levels of adenylate cyclase activation. Calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. CGRP is expressed from early developmental stage in rat brain, suggesting that CGRP may be involved in not only neurotransmission but also neural development. But roles of CGRP in neuronal development of cerebral cortex and hippocampus remain unclear.
In the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (E) 16 or E18 rat. Dendritic outgrowth of pyramidal neurons was analyzed after 4 days using anti-MAP2 antibody. Synapse formation was analyzed after 2-3 weeks, using anti-PSD-95 and anti-synaptophysin antibodies. In the presence of CGRP (10-1000 nM), both dendritic length and synaptic density were increased. However, the number of dendritic branching was not affected. These results suggest that CGRP promotes dendritic outgrowth and synapse formation.
Chisako Kanamaru, Kazunori Suda, Kouji Senzaki, Takashi Shiga University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Japan
Recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. In this study, we examined roles of 5hydroxytryptoamine (5-HT) and noradrenaline (NA) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. Embryonic day 18 rat hippocampus was cultured in the presence of 5-HT or NA. After 5 days, we analyzed formation of dendrites using anti-MAP2 antibody. After 21 days, we analyzed formation of synapses using anti-PSD-95, anti-synaptophysin, and anti-MAP2 antibodies. The addition of 5-HT (500 nM) or NA (500 nM) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites 5-HT (100-1000 nM) and NA (10-1000 nM) also increased the synaptic density. By using receptor agonists and antagonists, it was suggested that ␣ 2A receptor promotes dendritic outgrowth, while  receptor suppress dendritic outgrowth and branching. In addition, 5-HT 2A receptor and ␣ 2A receptor promote synapse formation.
Kenji Amano Down syndrome cell adhesion molecule (Dscam) knock-out (KO) mouse died within 24 h after the birth. To investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the C4 inspiratory activity using brainstem-spinal cord preparation. The respiratory activity of Dscam-KO mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. Furthermore, C4 inspiratory activity also showed irregular frequency and narrow duration of the bursting. We then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (Di-2 ANEPEQ) and the imaging system (MiCAM02). In Dscam-KO mice, the optical signal which precedes C4 inspiratory activity was depressed. These results suggest that Pre-Inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in Dscam-KO mice and causes lethal respiratory dysfunction.
PS1A-F088 Hippocampal cells cultured on 3D collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth Shantanu Sur, Thomas Launey, Masao Ito Brain Sc. Inst., RIKEN, Japan
The brain extracellular matrix (ECM) influences neuronal migration and morphogenesis. We explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ECM. After 2 weeks in vitro, neurons formed a dense layer, >0.1 mm below the gel surface, with neurite outgrowth toward the surface, within the Top Gel Layer (TGL). Initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. However, (1) collagenasespecific inhibitor did not affect cell depth, (2) limiting gliosis by antimitotics reduced the thickness of the TGL by 40%, (3), neither glial nor neuronal cell body were found in the TGL by GFAP/MAP2 detection, (4) neurite outgrowth was observed only within this TGL, but not toward the bottom of the gel. To see whether the TGL is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. The TGL was completely devoid of beads after 2 weeks, suggesting that the TGL is newly formed by ECM material, largely secreted by glial cells.
Emi Kumamaru, Tadahiro Numakawa, Yuki Yagasaki, Hiroshi Kunugi Disorder Research, National Institute of Neuroscience, NCNP, Tokyo, Japan
The level of glucocorticoid is regulated through HPA axis, and glucocorticoid itself has a negative feedback effect on HPA axis. However, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. On the other hand, it is possible that the reduction of neuronal function mediated by BDNF is partly related to the cause of the disorder. Therefore, in the present study, we investigated the effect of glucocorticoid (Dexamethasone, DEX) on synaptic maturation and function enhanced by BDNF in early developing hippocampal neurons. We found that BDNF increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with DEX significantly inhibited the up-regulation of these proteins by BDNF. Further, increase in release of glutamate and in intracellular Ca2+ by BDNF was suppressed after DEX pretreatment, suggesting that DEX inhibits the maturation of synaptic function mediated by BDNF.
Takashi Ueyama 1 , Kazuto Kujira 1 , Tetsuya Kawabe 2 , Takao Ito 1 , Yoshihiro Tsuruo 1 1 Department of Cell Biology and Anatomy, Wakayama Medical University, Wakayama, Japan; 2 Department of Cardiovascular Medicine, Wakayama Medical University, Wakayama, Japan
In this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-Fos expression in response to immobilization stress (IMO) between castrated rats (Cast) and sham-operated rats (Sham). Increased c-Fos immunoreactive cells in response to IMO were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. In Cast compared with Sham, the numbers of c-Fos-IR cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. These data suggest that neuronal activity in these areas is influenced by systemic androgen level. This may underlie the pathophysiology of partial androgen deficiency in aged men (PADAM).
Research funds: Grant-in-Aid for Scientific Research (C) (17590600) PS1A-F091 Metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold Fujiya Furuyama 1 , Hitoo Nishino 1 , Takehiro Yahata 2 1 Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 2 Nayoro City College, Nayoro, Japan
The inbred FOK rat was developed by us using heat selection and inbreeding for generations. FOK rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. Saliva spreads widely over the whole ventral body surface in FOK rats. However, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. On the other hand, body temperature of the FOK rats did not decreased in a extream cold environment as those in control rat strain. Thermogenesis induced by cold in FOK rats was larger than those in control rat strains. The larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. Blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in FOK rats than those in control rat strains. These changes can be considered to be results of genetically acquired heat-tolerance. Oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in Parkinson s disease (PD). Vitamin E is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (TTP) in brain. Dysfunction of TTP has been shown to result in systemic deficiency of Vitamin E in human and mice. In the present study, we using the TTP knockout mice, investigated the effect of Vitamin E deficiency in PD development by generating MPTP mouse model of PD. We confirmed that Vitamin E depleted in the brain of TTP knockout mice completely. While the MPTP treatment decreased striatal dopamine in the all three TTP genotypic groups, there were no significant differences among them. Our results suggest that Vitamin E does not play a major protective role in MPTP-induced nigrostriatal dopaminergic neurodegeneration in the brain.
Priyanka Dikshit 1 , Anand Goswami 1 , Nobuyuki Nukina 2 , Nihar Ranjan Jana 1 1 National Brain Research Centre, India; 2 Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, 2-1 Hirosawa, Wakoshi, Saitama 351-0198, Japan
A major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (NIIs) of the disease proteins, often associated with various chaperones and proteasome components. But, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. Here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. Secondly, we identified CHIP ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin-3 for the misfolding-dependent ubiquitination and degradation by the proteasome. The over expression of CHIP reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when CHIP is over expressed along with Hsc70. Finally, we show that the expression of CHIP is increased in the expanded polyglutamine protein expressing cells. Hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. For the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary DNA sequences (expressed sequence tags (ESTs)) from rat hypothalamus.
We have identified the total of 11,092 ESTs (5,442 non-redundant sequences). 2858 of them matched known genes of rodents in the Genbank databases, but 2584 remained unknown. Now we classified a full set of hypothalamic ESTs on the basis of their functional domains. Complete profile of them will be presented in the meeting.
These ESTs will also be applied to a cDNA microarray for stress experiments. The present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder.
Research funds: Grants-in-Aid from the Ministry of Health, Labor and Welfare
Shinya Yanagita, Seiichiro Amemiya, Satoko Suzuki, Ichiro Kita Graduate School of Science, Tokyo Metropolitan University, Japan
Our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN). Many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. It is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. In this study, we examine the effects of 0, 2, or 4 weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as PVN, central nucleus of amygdala (CeA), locus coeruleus, dorsal raphe, ventral tegmental area (VTA), and prefrontal cortex during acute running using immunohistological methods in rats. Prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of CeA, and increased that of VTA. These results suggest that prior wheel running influences stress-related neuronal activity during acute running.
PS1A-F096 Transforming growth factor- in the brain regulates fat metabolism during exercise Kazuo Inoue, Toma Ishikawa, Wataru Mizunoya, Tetsuro Shibakusa, Tohru Fushiki Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
We have previously reported that the concentration of transforming growth factor- (TGF-) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of TGF-. These results led us to postulate that TGF- in the brain regulates the enhancement of fatty acid oxidation during exercise. To test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of TGF- in the brain using intracisternal administration of anti-TGF- antibody or SB-431542, an inhibitor of the type 1 TGF- receptor (TR1). We found that each reagent blocked the increase in fatty acid oxidation. These results suggest that brain TGF- has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the TR1 signal transduction system.
Yoshii Takanobu It has been demonstrated that vasopressin (AVP) might play a role in anxiety-related behavior. We hypothesized that traumatic stress changes AVP activity and AVP contribute to the symptom of PTSD. We carried out in situ hybridization (ISH) for AVP mRNA expression and AVP immunohistochemistry (IHC) with an experimental paradigm of single prolonged stress (SPS) as PTSD model. SD male rats were exposed to SPS (2 h restraint; 20 min forced-swimming; ether anesthesia) then they were put in untouchable situation for 7 days. AVP mRNA expression significantly decreased in the SON. IHC showed no significant change in AVP-ir, but after additive stress (forced swimming 15 min), AVP-ir in the SON was significantly diminished. We considered that the stress decrease AVP synthesis, but has little effect to the storage of AVP.
Mumeko Tsuda, Takaaki Ozawa, Aosa Fukushi, Sonoko Ogawa Kansei, Behavioral and Brain Sciences, University of Tsukuba, Tsukuba, Japan
Neonatal maternal separation (MS) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. In the present study, we examined the effect of MS on an array of emotional and socio-sexual behaviors in both sexes of C57BL/6J mice. Pups were separated from mothers daily (3 h) on postnatal days 1 through 14. Starting at 13 weeks of age they were tested for (1) emotionality and anxiety levels in open field (OFT), light-dark transition (LDT), and elevated plus maze tests; (2) responses to social stimuli in social investigation (SIT) and social preference tests; and (3) socio-sexual behaviors in aggressive and sexual behavior tests. Overall, there was no apparent effect of MS on behaviors measured in the OFT and LDT except for higher levels of exploration in the MS group compared to the non-stressed (NS) group in both sexes. During the SIT, social investigation time and general activity in MS females were much lower than those in NS females suggesting MS females may be more fearful to social stimuli. In the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by Morris water maze test. Mice were exposed to the noise of 90 dB (SO), or were forced to swim (SW). These manipulations were performed for 15 min once a day at 2 weeks after birth (from postnatal days 14 to 18) or 3 weeks after birth (from postnatal day 21 to 25). Normal mice were left undisturbed (NO). The spatial learning activity was tested at the age of 6 weeks. It was found that the spatial learning activity of both SO and SW mice manipulated 2 weeks after birth was impaired as compared to NO mice. SO mice manipulated 3 weeks after birth exhibited the same learning behavior as NO mice, while that of SW mice manipulated 3 weeks after birth was impaired. Present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation.
Kin-ya Kubo 1 , Yukiko Yamada 2 , Mitsuo Iinuma 2 , Yasuo Tamura 2 , Fumihiko Iwaku 1 , Kazuko Watanabe 3 , Minoru Onozuka 4 1 Dept. Oral Anat., Asahi Univ. Sch. Dent., Japan; 2 Dept. Ped. Dent., Asahi Univ. Sch. Dent.; 3 Dept. Physiol., Gifu Univ. Sch. Med., Japan; 4 Dept. Physiol. and Neurosci., Kanagawa Dent. Coll., Japan
Recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (HPA) axis. In addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. In this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in SAMP8 mice. This treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal CA3 region. The results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory.
Yurie Nakamoto, Go Mugishima, Mitsuko Sato, Masako Miwa, Mitsunobu Yoshii Division of Psychobiology, Tokyo Institute of Psychiatry, Tokyo, Japan
It has been shown that PBR are increased after acute stress and decreased under chronic stressful conditions. In our previous studies, expression of PBR was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the PBR gene. In addition, males appeared to have higher PBR densities than females in their prime lives. The present study was designed to analyze these sexual differences in rats. Blood samples were obtained from adult male and female Slc Wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for 12 weeks after weaning). In naïve, male rats expressed higher densities of platelet PBR than females. Chronic social isolation caused a marked increase in platelet PBR in male rats compared to female. The results indicate that PBR responses to environmentally induced stress are much less in female, probably under the influence of estrogen.
Kanako Tambara 1 , Yayoi Kitamura 1 , Junichi Tanaka 2 , Yukio Hattori 1 , Yasushi Hayashi 1 1 Department of Human Nutrition, Notre Dame Seishin University, Okayama, Japan; 2 Department of Curriculum, Teaching and Memory, Naruto University of Education, Tokushima, Japan
We investigated the effects of exogenous putrescine on stressinduced hyperthermia (SIH) in male C57BL/6J mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. In addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on SIH. The rectal temperature of singly housed mice was measured twice at a 10-min interval, to measure the basal temperature (T 1 ) and stress-enhanced temperature (T 2 ), respectively. The difference ( T = T 2 − T 1 ) gives the SIH. In control mice, T was approximately 1 • C. Pretreatment with diazepam caused dose-dependent inhibition of the SIH. Similarly, putrescine reduced T, although it caused a dose-dependent decrease in T 1 . Furthermore, spermidine and spermine also lowered T and T 1 at doses lower than that of putrescine. These results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions.
Eriko Iguchi, Yasuhiro Tanaka, Toshiyuki Matsuoka, Shuh Narumiya Department of Pharmacology, Kyoto University, Kyoto, Japan
Prostaglandins (PGs) are synthesized in many organs including the brain. Of their synthesis, the rate limiting step depends on cyclooxygenase (COX), which has two subtypes, COX-1 and COX-2. It has been known that, under some stressful conditions, COX-2 is induced in some neurons and increases PGs production. But the roles of the increased PGs under stress are not fully elucidated. In this study, we restrained mice in small tubes individually for 6 h and subjected them to the elevated plus maze task 24 h later. These mice showed more anxiety. Immunohistochemistry showed significant induction of COX-2 by restraint in some parts of the brain, such as cerebral cortices and amygdala. Next, we examined the effect of indomethacin on this stress-induced anxiety. Indomethacin is expected to reduce PGs production. Mice treated with indomethacin stayed on open arms longer than control mice. These data suggest that PGs synthesized during stress may have anxiety-increasing effect.
PS1A-G104 Imaging brain and immune association accompanying cognitive appraisal of acute stressor To investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded 15O-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when 11 male subjects conducted a mental arithmetic task in a high controllability (HC) condition and a low controllability (LC) condition. Activation in the orbitofrontal (OFC) and medial prefrontal (MPFC) cortices was observed in the LC compared to the HC. Furthermore, significant correlations between brain activation and HR, HRV, BP, and NK cells were found commonly in the OFC in the LC, but not in the HC. Thus, the OFC is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor.
Wei Zhang 1 , Takesi Sakurai 2 , Yasuitirou Fukuda 3 , Tomoyuki Kuwaki 1,3 1 Dept. Molec. Integ. Physiol., Chiba Univ., Japan; 2 Dept. Pharmacol., Univ. Tsukuba, Japan; 3 Dept. Autonom. Physiol., Chiba Univ., Japan
We have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. It is still open question, however, how information of stressor activates the orexinergic neurons. In this study, we examined possible afferent nuclei to activate orexinergic neurons. In urethane-anesthetized mice, a GABA-A receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (BNST), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. Bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. However, dose-response curve was rightward shifted in the former. We conclude that the amygdala and BNST constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. In this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male Balb/C mice. The early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of 3-8 weeks, indicating persistent higher anxiety. High performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. The early-weaned mice had higher levels of galactosyl ceramide at the age of 5 weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. These results suggest that the early weaning induces precocious myelin formation in the amygdale between 3 and 5 weeks of age, which would be related to higher anxiety state in the early-weaned mice.
Research funds: SASAKAWA SCI. RES. GRANT
Takefumi Kikusui, Yuji Mori Veterinary Ethology, University of Tokyo, Tokyo, Japan
We previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. In this study, developmental changes of brain derived neurotrophic factor (BDNF) protein levels were investigated in early-weaned ICR mice. The early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of 3 weeks, and this change was persistently observed in males. Concurrently, the early-weaned males showed decrease of BDNF in the prefrontal cortex between 3 and 8 weeks of age, and in the hippocampus at the age of 3 weeks. However, there was no difference of BDNF expression in females. In addition, the early-weaned males, but not females, showed reduced BrdU immunoreactivity in the dentate gyrus. These results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of BDNF in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress.
Research funds: KAKENHI #14760187
PS1A-G108 A systematic analysis of genetic factors associated with behavioral diversity between MSM and C57BL/6
Koide Tsuyoshi 1,2 , Aki Takahashi 1,2 , Toshihiko Shiroishi 2,3 , Akinori Nishi 1 1 MGRL, National Institute of Genetics, Mishima, Japan; 2 SOKENDAI, Hayama, Japan; 3 Mammalian Genetics Lab, NIG, Mishima, Japan
In the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, MSM and C57BL/6. In order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of MSM strain. The behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. One of the consomic strains, B6-6CMSM, which carries chromosome 6 of MSM, showed extreme behavioral differences from C57BL/6. The strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. By conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype.
Tomoko Soga 1 , Yu Kajiyama 2 , Shigenobu Shibata 2 , Hiroshi Kunugi 1 1 Department of mental disorder research, National Institute of Neuroscience, Center of Neurology and psychiatry, Tokyo, Japan; 2 Department of Electrical Engineering and Bioscience, Waseda University
The hypothalamus-pituitary-adrenal (HPA) axis plays an important role in the pathophysiology of depression. Alterations of brain derived neuronal factors (BDNF) have been implicated in depression. We examined the effects of synthetic glucocorticoid (dexamethasone; DEX) on emotional behavior and gene expression of HPA-related molecules and BDNF in mice. DEX treatment for 4 days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. DEX treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. There was no change in gene expression of HPA-related molecules in DEX-treated group. BDNF gene expression decreased significantly in DEX-treated group, which showed behavioral abnormalities. Our results lend further support for the involvement of glucocorticoid and BDNF in depression-related behavior.
Sachiko Chikahisa, Hiroyoshi Sei, Atsuko Sano, Kazuyoshi Kitaoka, Yusuke Morita Department of Integrative Physiology, The University of Tokushima Graduate School, Tokushima, Japan Music is known to be able to elicit emotional changes including anxiolytic effect. The gonadal steroid hormone estrogen (E2) has been associated with anxiety levels. In this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. Behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (OVX) female mice treated with placebo (OVX + placebo) or chronic estradiol (OVX + E2) replacement. In three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while OVX + placebo mice did not show these effects at all. OVX + E2 mice showed the anxiolytic effect of music only in the marble burying test. These results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice.
Tatsuhiro Yasuda Free, Tokyo, Japan
Strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. The air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. Simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. The stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. Continuation of this may induce hyper ventilation or tension. If the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. Lasting this situation may fall into depression. Suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. Body system is suggested to own inherent observer that detects alerting or safe state so called homunculus.
Kenichi Sasaguri 1 , Takero In general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. We determined whether the malocclusion model influences brain activities by using fMRI study. The results indicated that in some of volunteers, significantly BOLD signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. It is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains.
PS1A-G113 Synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat Toshiko Tsumori, Yi Qin, Shigefumi Yokota, Tatsuro Oka, Yukihiko Yasui Dept. Anat. & Morphol. Neurosci., Shimane Univ. Sch. Med., Izumo, Japan
The retrorubral field (RRF) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. In the present study, we showed that the axon terminals from the central amygdaloid nucleus (ACe) made synaptic contacts with non-dopaminergic RRF neurons sending their axons to the parvicellular reticular formation (RFp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. The ACe axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the RFp-projecting RRF neurons. Moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. The present study suggests that the ACe exerts inhibitory influences upon the non-dopaminergic RFp-projecting RRF neurons to control orofacial movements closely related to emotional behavior.
Research funds: KAKENHI (15500238) PS1A-G114 Involvement of NR2B tyrosine-phosphorylation in emotional responses mediated at the amygdala Mina Delawary 1 , Takanobu Nakazawa 1 , Yuji Kiyama 2 , Toshiya Manabe 2 , Tadashi Yamamoto 1 1 Div. of Oncology, IMS, Univ. of Tokyo, Tokyo, Japan; 2 Div. of Neuronal Network, IMS, Univ. of Tokyo, Tokyo, Japan NR2B is tyrosine-phosphorylated, with Tyr-1472 being its major phosphorylation site. To investigate the role of Tyr-1472 phosphorylation, we generated mice with a Tyr1472Phe knock-in mutation (YF/YF mice). In the elevated plus-maze test, time spent in open arm was reduced in YF/YF mice as compared to that in wild-type mice. Similar phenotype was seen in the corticotropin-releasing factor (CRF) overexpressing mice. This phenotype of YF/YF mice was canceled by the administration of CRF receptor antagonist. As expected, in YF/YF mice, the expression level of CRF in the amygdala was increased compared with that in wild-type mice. In the slice of amygdala from wild-type mice, NMDA application induced de-phosphorylation of Tyr-1472 and up-regulation of CRF mRNA level. Given that CRF is important in emotional responses, these data strongly argue that phosphorylation of NR2B is involved in the control of emotional responses by regulating CRF content.
PS1A-G115 Increase in anxiety in transgenic mice overexpressing CaMKII in forebrain Previous studies have shown that ␣Calcium/Calmodulin dependent protein kinase II (␣CaMKII) plays important roles in aggressive and fear response in mice. To understand roles of alpha CaMKII in emotional behaviors, we have generated transgenic mice overexpressing ␣CaMKII in forebrain. Because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (SSRI) on anxiety-related behavior of these mutant mice. Treatment with SSRI suppressed anxiety-related behavior of CaMKII mutant mice, suggesting that CaMKII mutant mouse is a mouse model of anxiety disorder. To investigate the mechanisms for increase in anxiety led by overexpression of CaMKII, we next compared the expression profiles between wild and mutant mice using DNA micro array. These mutant mice showed abnormal changes in expression levels of genes related to Ca 2+ signal transduction in hippocampus.
Yumiko Ikeda, Katsunori Kobayashi, Hidenori Suzuki Department of Pharmacology, Nippon Medical School, Tokyo, Japan
Environment is known to influence behavior of animals. However, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. We examined effects of changes in environment on locomotor activity and mossy fiber (MF) synaptic transmission in hippocampal slices. In mice housed in enriched condition for 5 weeks, locomotor activity and longinterval (200, 1000 and 5000 ms) paired-pulse facilitation (PPF) at MF synapses were reduced. In contrast, in mice housed in isolated condition for 5 weeks, there was no detectable change in either the total ambulation distance or the magnitude of PPF. We compared properties of the MF synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. Our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity.
Yilong Cui 1,3 , Yosky Kataoka 1,2 , Yasuhisa Tamura 3 , Yasuyoshi Watanabe 2,3 , Hisao Yamada 1 1 Department of Anatomy and Cell Science, Kansai Medical University, Osaka, Japan; 2 Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan; 3 Molecular Imaging Research Program, RIKEN Frontier Research System, Saitama, Japan During long-term intracranial self-stimulation (ICSS; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at 50-60 times/min), inhibition periods (less than 10 times/min) were often observed 2 h after start of ICSS. We have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. Furthermore, the inhibition period was significantly decreased by pre-treatment with NS-398, a selective COX-2 inhibitor. These observations indicate that the arachidonic acid cascade is involved in inhibition of long-term ICSS and would be in weariness or fatigue sensation. Male bluegill, Lepomis macrochirus, is known to display alternative reproductive tactics. "Parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. In teleost and other non-mammals, arginine vasotocin (AVT), the homologue of mammalian AVP, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. Behavioral evaluation and immunohistochemical study in preoptic area (POA) were conducted in parental and satellite bluegills to clear the role of AVT in teleost reproductive tactics. Parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of AVT cells showed no difference between the male morphs. These results suggested that hypothalamic AVT might play some part in the central control of reproductive behavior in teleost.
PS1A-G119 Impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost Toshiya Matsushima 1 , Naoya Aoki 2 , Andras Csillag 3 1 Biology, Hokkaido Univ., Sapporo, Japan; 2 Agriculture, Nagoya Univ., Nagoya, Japan; 3 Anatomy, Semmelweis Univ., Budapest, Hungary Choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. To study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. In task 1, chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to 3 s. In task 2, chicks chose between small reward located at 20 cm and large reward at −140 cm, where cues signaled the distance of invisible food. Task 3 was similar to the task 2, except that cues signaled the food quantity. In task 4, total handling time differed due to lowered food accessibility, while the delay was kept identical. Lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost.
Research funds: KAKENHI (15370033, 17021018)
PS1A-G120 Analysis of the brain regions associated with the dance language of the honeybees Taketoshi Kiya, Takekazu Kunieda, Takeo Kubo Dep. Biol. Sci., Univ. Tokyo, Tokyo, Japan Social animals have highly developed communicative abilities. The worker honeybees (Apis mellifera L.) can transmit location of food sources by the dance language. In spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. Previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type Kenyon cells (sKCs) of the mushroom bodies. Here, we report that kakusei was similarly expressed in the sKCs of the foraging workers (foragers), which do not always show the dance behavior. In contrast, the sKCspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. These results imply that the activities of the sKCs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. C. elegans wild type animals are usually attracted to NaCl, but show avoidance behaviors after being conditioned with NaCl and starvation (food−/NaCl+). This behavioral plasticity is not induced under the food−/NaCl− or food+/NaCl+ conditions.
We isolated learning-defective mutants including pe401, which had a missense mutation in the casy-1 gene. Several casy-1 deletion mutants also showed learning defects. CASY-1 has an extensive similarity to human Calsyntenin/Alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of CNS synapses. Alcadein forms a stable tripartite complex with APP and X11L/Mint2. However, after dissociation of X11L, Alcadein is susceptible to cleavage by protease(s). We found that CASY-1 was expressed mainly in neurons and functioned at the adult stage. We are now investigating the localization pattern of the GFP-tagged protein, and whether CASY-1 can also be proteolytically cleaved.
PS1A-G122 Insulin-like signaling is required for association between temperature and feeding state in C. elegans
Eiji Kodama 1 , Atsushi Kuhara 1 , Akiko Mohri 1,2 , Kotaro Kimura 1,3 , Masatoshi Okumura 1 , Masahiro Tomioka 4 , Yuichi Iino 4 , Ikue Mori 1,5 1 Div. of Biol. Sci., Nagoya Univ., Japan; 2 Present address: Univ. of Texas, Health Sci. Cent., USA; 3 Present address: Natl. Inst. of Genet., Japan; 4 Mol. Genet. Res. Lab., Univ. of Tokyo, Japan; 5 Inst. for Advanced Res., Nagoya Univ., Japan C. elegans can associate cultivation temperature with feeding state.
Mutations in ins-1 encoding insulin homologue caused defective associative learning, mutations in daf-2 and age-1 encoding the homologues of insulin receptor and PI 3-kinase, respectively, suppressed the defect of ins-1, and the mutation in daf-16 encoding forkhead transcriptional factor caused the learning defect. This suggests that INS-1 antagonizes DAF-2 insulin-like signaling for associative learning. Interestingly, age-1 animals associate their cultivation temperature with feeding-state quicker than wild type. This defect was rescued by expressing AGE-1 in some head interneurons. In addition, the activity of these interneurons were down-regulated by starvation through INS-1. We suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning.
PS1A-G123 Analysis of ttx-8: Novel thermotaxis gene conserved among various organisms Akiko Miyara, Akane Ohta, Yoshifumi Okochi, Masatoshi Okumura, Ikue Mori Laboratory of Molecular Neurobiology and Institute for Advanced Research, Nagoya University, Nagoya, Japan C. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. This response to temperature is called thermotaxis. Several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood.
The ttx-8(nj21) and ttx-8(nj34) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. We revealed that ttx-8 encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior.
The predicted protein structure of TTX-8 is similar to RIC-3, identified in C. elegans at first and conserved among several species (Halevi et al., 2002 (Halevi et al., , 2003 . RIC-3 is thought to be required for the maturation of acetylcoline receptor (Halevi et al., 2002) , so TTX-8 may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein.
PS1A-G124 Analysis of aho-3 mutant that cannot associate cultivation temperature with feeding state in C. elegans Nana Nishio 1 , Akiko Mohri 1,2 , Eiji Kodama 1 , Atsushi Kuhara 1 , Mizuho Koike 1 , Kotaro Kimura 1,3 , Ikue Mori 1,4 1 Div. of Biol. Sci., Nagoya Univ., Japan; 2 Present address: Univ. of Texas, Health Sci. Cent., USA; 3 Present address: Natl. Inst. of Genet., Japan; 4 Inst. for Advanced Res., Nagoya Univ., Japan
The nematode C. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. To identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. We isolated aho-3(nj15) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (Sawin et al., 2000) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho-3 gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although AHO-3 is a highly conserved protein throughout yeast to human. Currently, we are trying to dissect the molecular and cellular analysis of aho-3 gene further. We have demonstrated aversive conditioning in Lymnaea using 100 mM sucrose presentation as the appetitive stimulus (CS) and mechanical tactile stimulation to the head as the noxious stimulus (UCS). We measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. We also measured the neuronal activity of B3, located in the buccal ganglion. An associative memory, lasting 24 h, was produced with 20 pairings of CS and UCS. The learning was characterized by a shift in the response to the UCS from a whole body withdrawal response to the cessation of feeding behavior. B3 neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand CS application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. This can interpret that the conditioning decreased the excitability of B3 neuron activity thus to decrease the fictive feeding behavior.
Yasutaka Nomura 1 , Dai Hatakeyama 1 , Tetsuro Horikoshi 1 , Etsuro Ito 2 , Manabu Sakakibara 1 1 Lab. Neurobiol. Engr, Sch. High-Tech, Tokai Univ., Numazu, Japan; 2 CRIS, Hokkaido Univ., Sapporo, Japan
Calexcitin, low molecular weight GTP-binding protein is found to be phosphorylated in the visuo-vestibular conditioned Hermissenda at the type B photoreceptor. We found positively stained neurons to anti-calexcitin antibody (gift from Dr. Kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned Lymnaea with two different ways. One was the same conditioning paradigm as Hermissenda and the other was taste aversion conditioning. Both of these conditioning response is the whole-body withdrawal. No positive neuron was found in naïve animal. Neurons in CB cluster and PeA cluster showed both positivity to calexcitin and serotonin. This suggested the functional role in conditioning.
Ken Honjo, Katsuo Furukubo-Tokunaga Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan
The fruit fly Drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. We have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (MBs). We found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. By expressing shi ts1 in larval MBs, we demonstrate that disruption of neural output from MBs abolishes both appetitive and aversive memory indicating that both memories are stored before the MB output synapses. Moreover, we show that disruption of either CREB or amnesiac functions abolishes appetitive but not aversive memory. Thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in MBs.
Motomi Matsuno 1 , Minoru Saitoe 1,2 , Tim Tully 3 1 Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan; 2 Department of Biology, Tokyo Metropolitan University, Japan; 3 Cold Spring Harbor Laboratory, USA
We identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). Klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. We report here that klg is necessary for long-term memory as well as early-phase memory. We show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, CREB and the cell surface receptor Notch, both of which are well known to function in LTM formation. Transgenic expression of klg improves memory of a klg mutant. Since Klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. We have investigated the ability of context-dependent olfactory learning in the cockroach, Periplaneta Americana. We trained one group of cockroaches to associate peppermint odor (conditioned stimulus, CS, P) with sucrose solution (appetitive unconditioned stimulus, US+), and vanilla odor (CS, V) with saline solution (aversive US, US−) under illumination (L), and to associate P with US− and V with US+ in the dark (D). Another group received training with opposite stimulus setup (L: V+/P−, D: V−/P+). Before training, cockroaches preferred V over P. 1 day after training, the former group significantly preferred P over V under illumination but preferred V over P in the dark, and the latter group displayed the invert odor preference. Result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of CSs. Thus cockroaches are capable of disambiguating the meaning of CS odors according to the visual context.
Hidehiro Watanabe, Makoto Mizunami Graduate School of Life Sciences, Tohoku University, Sendai, Japan A century had passed since Pavlov reported classical conditioning of salivation in dogs. However, the cellular mechanisms underlying this conditioning remain obscure. In insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. Here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, Periplaneta americana. In insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. We found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. The level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. These results are the first to suggest the classical conditioning of salivation in non-mammalian species. These results are useful to study neural mechanisms underlying classical conditioning of salivation.
Research funds: KAKENHI 17005050
Ke Zhang 1 , Jian Z. Guo 1 , Ai K. Guo 1,2 1 Institute of Neuroscience, Chinese Academy of Sciences, China; 2 Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
The cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. Drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow.
Here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (MB), was genetically silenced by Gal4/UAS-shi ts1 system, suggesting that this behaviour is mediated by dopaminergic system acting through MB, a structure shown to be densely innervated by dopaminergic fibers. However, the dopaminergic and MB synaptic activities were required only during the early choice period (<4 min), but not for the sustenance of the chosen flight path. Thus the dopaminergic system and MB are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in Drosophila may help to understand the neural basis of higher cognitive functions.
Sae Unoki, Yukihisa Matsumoto, Makoto Mizunami Graduate School of Life Sciences, Tohoku University, Sendai, Japan
In mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. Previous studies in insects suggested that octopamine (OA) and dopamine (DA) mediate various kinds of reward and punishment signals in olfactory learning. However, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. We pharmacologically studied the roles of OA and DA in appetitive and aversive forms of visual pattern learning in crickets. Crickets injected with OA receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. The opposite influences were observed by injection of DA receptor antagonists. Our finding that OA and DA participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. This suggests conserved roles of aminergic reinforcing systems among different phyla.
Aiko Watanabe, Neal A. Hessler Laboratory for Vocal Behavior Mechanisms, RIKEN Brain Science Institute, Saitama, Japan
In adult songbirds, neural turnover occurs in HVC, a forebrain motor control nucleus. Cells labeled by bromodeoxyuridine (BrdU), a cell birth marker, appear in the ventricular zone, migrate into HVC, and some of them mature into projection neuron. To assess the role of neurogenesis in adult song plasticity, we deafened adult Bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. Deafened birds had more BrdU-labeled cells in HVC than control birds within the first month. More TUNEL-stained apoptotic cells also tended to be seen in HVC of deafened birds. However, number of the BrdU-labeled cells decreased 2 months after deafening, when the songs had stabilized. Most of the BrdU-labeled cells in HVC of deafened birds were immunoreactive for a neuron-specific marker, Hu. Additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. These results suggest that the amount of neurogenesis is related to adult song plasticity.
Yasko Tobari 1,2 , Kazuo Okanoya 1,2,3 1 Lab. for Biolinguistics, RIKEN-BSI, Wako, Japan; 2 Grad. Sch. of Sci. and Tech., Chiba University, Chiba, Japan; 3 PRESTO, JST. Kawaguchi, Japan A set of brain nuclei controls song production in songbirds. Among these nuclei, the robust nucleus of arcopallium (RA) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. The projections of RA to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (XIIts), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (Ram) were present in Bengalese finches. In this study, we have focused our attention on the descending projections of RA, with a view to the presence of contralateral projections to XIIts and Ram, using in vivo tract-tracing technique.
The results indicated that Ipsilateral and contralateral projections of RA to respiratory-vocal nuclei in the brainstem were defined in adult male Bengalese finches. Birdsong is composed of various song elements that have typical frequency modulation. Each element is aligned in own sequential rule. Especially in Bengalese finches, the sequential rule obeys finite state grammar. It has been focused what neural mechanism enables such a complex sequential rule. In order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area HVC. We collectively recorded the activities of HVC neurons driven by all possible element pair stimuli. The results show that most of neurons in HVC respond not only the sequence included in their own song but also the sequence not included. Each neuron has typical response distribution toward the whole element sequence. In addition, the distribution property is different among neurons in same individual. Taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding.
Hironobu Sakaguchi Department of Physiology and Biological Information, Dokkyo University, School of Medicine, Japan
Avian vocal learning provides a good model for human speech learning. Young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. Many behavioral studies have shown that vocal learning is affected by a song template and social factors. If a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. Here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. Consequently, isolated birds, exposed to new tutors from day 120, developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. However, they were not able to imitate the syllable phonology from new tutors. The differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax.
PS1A-H137 Facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats Fumino Okutani, Jing-Ji Zhang, Guang-Zhe Huang, Hideto Kaba Department of Integrative Physiology, Kochi Medical School, Nankoku, Japan
Oxytocin (OT) within the olfactory bulb (OB) has been reported to be important for the induction of maternal behavior and recognition of offspring. The activity of mitral cells, olfactory relay neurons in the OB is inhibited by granule cells via reciprocal dendrodendritic synapses. Electrophysiological studies have revealed that OT modulates mitral cell activity by acting on mitral and granule cells. In a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. Our studies have shown that plasticity in the OB is critical for this olfactory learning. Pups that received OT infusion into the OB in the presence of citral odor developed an aversion to the odor without shock, suggesting that OT infusion has a facilitatory effect on olfactory learning. Using OB slices, long-term potentiation (LTP) was induced in field EPSPs recorded in the granule cell layer. OT administration also facilitated LTP. These results demonstrate that OT is involved in olfactory learning in young rats.
Research funds: KAKENHI 17023034
PS1A-H138 The GABA A receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats
Tadashi Inui, Tsuyoshi Shimura, Takashi Yamamoto Div. Behav. Physiol., Dept. Behav. Sci., Grad. Sch. Human Sci., Osaka Univ., Japan
We examined the effects of microinjections of GABA A receptors antagonist bicuculline into the ventral pallidum (VP) on the retrieval of conditioned taste aversion (CTA). In Experiment 1, rats received a pairing of saccharin or quinine hydrochloride (CS) with an i.p. injection of 0.15 M lithium chloride (US). After this conditioning, vehicle or bicuculline was bilaterally infused into the VP just before the re-exposure to the CS. The microinjections of bicuculline significantly increased the intake of saccharin CS, but not quinine hydrochloride. In Experiment 2, rats were presented with saccharin as CS via an intraoral cannula. The microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. These results suggest that the GABA A receptors in the VP play an important role in the expression of ingestive and/or aversive responses to saccharin CS during the retrieval of CTA so that the microinjection of bicuculline might increase the intake of saccharin CS.
Research funds: KAKENHI (17730431) PS1A-H139 Transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning Roles of c-fos gene expression and its protein product, Fos, in conditioned taste aversion (CTA) learning were examined using the antisense oligodeoxynucleotide (ODN) method in rats and in mice carrying c-fos gene deficiency. Infusion of antisense ODN (AS-ODN) directed against c-fos mRNA into the parabrachial nucleus (PBN), but not into the amygdala or insular cortex (IC), impaired the acquisition, while infusion of randomized and inverted control ODNs had no effect. Suppression of Fos synthesis in the amygdala or IC impaired the retention. Retrieval of an acquired CTA was not impaired by AS-ODN infusion into the PBN or amygdala. In contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. The present results suggest that the Fos-mediated signals in the PBN, amygdala or, IC plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term CTA memory.
PS1A-H140 GABA receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning
Classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. The knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. We examined the role of the deep cerebellar nuclei (DCN) during delay eyeblink conditioning in C57BL/6 mice by using GABA A receptors agonist and antagonist. In the acquisition tests, in which muscimol (MSC) or picrotoxin (PTX) was injected from beginning of training, aCSF-injected control mice learned this task, but both MSC-and PTX-injected mice showed a significant impairment in acquisition of conditioned response (CR). In the retention tests, in which the drug was injected after acquisition of training, CR % in aCSF-injected mice were kept over 80%, while those in the MSCand PTX-injected mice decreased to 30%. These results revealed that GABAA receptors in the DCN play important roles in acquisition and retention of mouse eyeblink conditioning. Various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. In the cerebellum, Delphilin is expressed selectively in Purkinje cells (PCs) and localized exclusively at parallel fiber (PF) synapses, where it interacts with glutamate receptor ␦2 that is essential for long-term depression (LTD) and motor learning. Here, we showed that ablation of Delphilin proteins facilitated LTD induction at PF-PC synapses and enhanced optokinetic response adaptation without affecting histology. This finding suggests that threshold regulation of LTD at PF-PC synapses is a limiting step for motor learning efficiency.
PS1A-H143 Post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei Soichi Nagao 1,2 , Takehito Okamoto 1 , Fumihiro Shutoh 1,3 1 Lab for Motor Learning Control, RIKEN BSI, Saitama, Japan; 2 SORST, JST, Saitama, Japan; 3 Dept. Anat., Grad. Univ. Tsukuba, Ibaraki, Japan
One-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (HOKR) gains in mice. Succession of 1 h daily training for 1 week induces long-term adaptation. We recently reported that the memory trace of adaptation of HOKR is initially acquired within the cerebellar flocculus through long-term depression (LTD), and later transferred to the vestibular nuclei for consolidation. In order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. Mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei.
Research funds: KAKENHI (16500204) PS1A-H144 Modification of gene expression in the cerebellar cortical neurons related with long-term motor learning Yuji T. Katagiri 1,2 , Takehito Okamoto 1 , Shin-ichi Nishimura 1 , Fumihiro Shutoh 1,3 , Soichi Nagao 1,4 1 Lab. for Motor Learning Control, RIKEN BSI, Saitama, Japan; 2 Univ of the Air, Chiba, Japan; 3 Dept. of Anatomy, Human Comprehensive Science, Grad. Univ. Tsukba, Japan; 4 SORST, JST, Japan
We recently reported that the cerebellar LTD plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (Shutoh et al., 2006) . In order to listup the molecules involved in the motor learning, we sampled total RNA from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. We found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. We furthermore examined the gene expression of Purkinje cells by the laser micro-dissection and quantitative RT-PCR methods.
PS1A-H145 Influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice Hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. The influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. Place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. These results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning.
Son Ho 1,2 , T Kobayashi 1,2 , E Hori 1,2 , K Umeno 1,2 , T Ono 1 , H Nishijo 1,2 1 System Emotional Science, Univ. of Toyama, Toyama, Japan; 2 CREST, Tokyo, Japan
We investigated a role of the hippocampal formation (HF) in encoding of a moving object in an open field. Rats acquired ICSS rewards if they moved freely. Then, a remote-controlled car was placed inside the open field. The rats could receive ICSS if it chased and approached the car. Of a total of 133 place cells recorded, activity of 40 was significantly modulated by the car speed and/or distance between the car and rat; 21, 12 and 7 cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. Furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. In a control experiment, the same car was introduced, but the rats could receive ICSS rewards without relation to relative distance between the rat and car. So far, 16 place cells were recorded in this experiment. Of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. The results suggest that HF encodes not only spatial information of own location, but also that of other moving object in an environment.
Hisae Gemba, Kazuko Nakao, Ryuiti Matsuzaki, Yusaku Amaya Department of Physiology, Kansai Medical University, Moriguchi, Japan
Cortical field potentials were recorded by electrodes implanted on the surface and at a 2.0-3.0 mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. It was found that an s-N, d-P potential, at about 40 ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of Broca's area) was related to recognition learning (association of stimulus with movement), and that an s-N, d-P potential in the motor and somatosensory cortices, and areas 5 and 7, contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). In visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area 5 to skill learning, as previously reported. This indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. Computational studies of hippocampal function generally assume that CA1 performs a match-mismatch comparison of memory retrieval with sensory input. Here we investigated this comparator model using an ensemble recording during task behaviors in the rat. We employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. After training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. During this task, we found that some CA1 neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. These data suggest that CA1 plays an important role in the mismatch detecting and correcting process of behavior. N-Methyl-d-aspartate (NMDA) receptor has high permeability to Ca 2+ but is blocked by Mg 2+ in a voltage-dependent manner. This property is a molecular basis of NMDA receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. We have generated the genetically engineered mice in which mutated NMDA receptors defecting in Mg 2+ binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. The mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. To elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal CA1 cells, the output stage of hippocampal circuit. The link between the behavioral anomaly and the hippocampal activity is discussed.
Mikako Sakurai, Ko Zushida, Masayuki Sekiguchi, Keiji Wada Department of Neurodegenerative Diseases, National Institute of Neuroscience, NCNP, Tokyo, Japan UCH-L1 is a component of the ubiquitin system. UCH-L1 is expressed at high levels in the hippocampal neurons. However, the functional role of UCH-L1 in synaptic plasticity and behavior is not understood. We examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding Uchl1. Gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. Theta burst stimulation (TBS) of Shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (LTP) in the area CA1. In contrast, non-decremental LTP was induced in control wild-type mice. The maintenance of TBSinduced LTP in the wild-type mice was impaired by actinomycin D, an inhibitor of transcription, whereas TBS-induced LTP in gad mice was insensitive to actinomycin D. These results suggest that UCH-L1 is a molecule participating in the synaptic plasticity elicited by TBS and the memory function.
PS1A-I151 Learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers Yoshinori Izaki, Tatsuo Akema Department of Physiology, St. Marianna University School of Medicine, Japan
To investigate whether the relationship between hippocampus (Hip) and prefrontal cortex (PFC) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (CC) of these local field potential powers during operant reversal training sessions. Rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage 0). Then the rats received the reversal training. Learning stages examined were as following: the first training session (stage I), leaning stage for S+ (stage II) and for S− (stage III). Different changes of the CC in some frequency-band powers with learning stages were observed. The CC in higher gamma-band (64-120 Hz) was strong at stage 0 and changed with leaning stages. Particularly, the CC decreased to almost zero at stage II. These results suggest that functional connection between Hip and PFC is reflected in this frequency-band and changes with learning stages.
PS1A-I152 Longitudinal fiber systems in the dentate gyrus of the rat Norio Ishizuka, Yoshitomo Umitsu Department of Brain Structure, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan
Longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of PHA-L and retrograde labeling method with fluorescent dyes. The flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. Injection of PHA-L into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. The first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about 1.5 mm from the injection level. In the second fiber system, axonal terminations began to appear at the level of 1 mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. The axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. In the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled.
Ryoichi Moki, Ryang Kim, Hisahiro Umeeda, Akinobu Suzuki, Satoshi Kida Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan
Recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. In addition, previous our study using conditional CREB mutant mice indicated that CREB is required for reconsolidation of conditioned fear memory. We also observed protein synthesis-dependent reconsolidation of spatial memory using Morris Water maze. In this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of CREB in reconsolidation of spatial memory. Using conditional CREB mutant mice that enable to induce the inhibition of CREB activity in a Tamoxifen-dependent manner, we found that inhibition of CREB activity leads to disruption of spatial memory after the retrieval. This result indicates that CREB is required for reconsolidation of spatial memory.
Shunsuke Hasegawa, Hirosi Hosoda, Satoshi Kida Department of Bioscience, Tokyo university of Agriculture bHLH-PAS transcription factor BMAL1 ubiquitously expresses in brain. BMAL1 functions by forming a heterodimer with either CLOCK or NPAS2, which has been known to play important roles in control of circadian rhythm and memory formation, respectively. To understand roles of BMAL1 in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of BMAL1 function in a forebrain. Using a dominant negative mutant of BMAL1 (BMAL1 R91A) that forms a heterodimer with CLOCK but loses the binding activity with E-box (Hosoda et al., 2004), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. These mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tTA) under the control of alpha CaMKII promoter. We observed the expression of BMAL1 R91A in several double transgenic lines in a tTA-dependent manner. Behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of BMAL1 in learning and memory. Stress sometimes causes memory deficits. And chewing has been shown to reduce stress. However, the chewing-related mechanism in stress-induced memory deficits is unclear. We thus examined the effects of chewing on spatial memory using Morris water maze and Fos induction in the hippocampus and amygdala in stressed mice. When mice were exposed to restraint stress, reduction in learning ability and density of Fos-positive cells in the DG and the BLA was seen, but not in the mice chewing a thin wooden bar during stress exposure. The results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory.
Seiichiro Amemiya, Shinya Yanagita, Satoko Suzuki, Ichiro Kita Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan
We examined the effect of background noise (BGN) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-Fos immunostaining. Rats performed maze task under different intensity of BGN (0, 70, or 100 dB; intermittent white noise). 70 dB BGN induced significant decreases in number of error and time to goal in maze compared with 0 and 100 dB. Although BGN increased Fos positive acetylcholinergic neurons (Fos-ChAT) in mesopontine tegmentum (MT) regardless of the intensity, Fos-ChAT in basal forebrain (BF) increased intensity-dependently. In locus coeruleus (LC) and cortex, Fos positive cell increased intensitydependently. Furthermore, 100 dB BGN remarkably enhanced Fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. These results suggest that BGN improve spatial performance by enhancing arousal following activation of cholinergic neurons in MT and BF, and LC neurons. However, higher BGN intensity could evoke over-arousal and stress responses, thereby prevent the maze task.
Siriporn Chattipakorn 1,2 , Anucha Pongpanparadorn 2 , Wasana Pratchayasakul 2 , Anchalee Pongchaidacha 2 , Nipon Chattipakorn 2 1 Fac. Dent. CMU, Chiang Mai, Thailand; 2 CERT, CMU, Chiang Mai, Thailand
Current pharmacotherapy of AD is the use of AChE inhibitors. Previous in vitro study showed that TDE inhibited AChE activity. This is the first study investigating the effects of TDE on cortical AChE activity and neuronal activity in in vivo. We used Fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical AChE activity following the single injection of various TDE doses. Mean Fos-positive neurons in cortex were 101 ± 14, 124 ± 19 and 108 ± 22 in the groups administered 250, 500 and 1000 mg/kg TDE, respectively. Cortical Fos-positive neurons in all three TDE-treated groups were greater than those in the control group. Percent inhibition of cortical AChE activity was 17.4 ± 6.3, 22.7 ± 6.9 and 16.6 ± 5.0 for 250, 500 and 1000 mg/kg TDE, respectively. These AChE inhibitory effects were significantly different from the control. These findings suggest that TDE could be beneficial as a possible novel therapeutic agent for AD. We showed the effects of a 2-h TDE administration in animals on the inhibition of cortical AChE activity and the enhancement of cortical neuronal activity. AChE activity in circulation following a 2-h TDE administration was not different compared to the control. This study investigated that the effects of TDE on circulating AChE activity (cAChE) in animal models was time-dependent. We used the colorimetric method to investigate cAChE activity in rats following the single administration of TDE at various doses at different time courses (10, 60 and 120 min). Percentage inhibition of cAChE activity following a single TDE injection at doses 500 and 1000 mg/kg significantly decreased at 10 and 60 min after TDE injection, but not at 120 min. cAChE inhibitory effects among two doses of TDE administrated groups at various time courses were not significantly different. These findings suggest that TDE may be a short-acting AChE inhibitor. Donepezil, galanthamine and tacrine are acetylcholinesterase (AChE) inhibitors used for treatment of Alzheimer's disease. We examined the neuroprotective mechanisms of AChE inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. We show that they protect neurons through mechanisms other than AChE inhibition. The protective effects are mediated through nicotinic receptors (nAChRs). Donepezil and galanthamine protect neurons through ␣4and ␣7-nAChR and kinases involved in PI3K-Akt pathway, and increase the levels of phosphorylated Akt and Bcl-2. These results suggest that these AChE inhibitors express their neuroprotective effects against glutamate neurotoxicity through nAChRs and that donepezil and galanthamine protect neurons through PI3K-Akt pathway via ␣4and ␣7-nAChRs.
PS1A-I160 Arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats Yasuto Kashiyae 1 , Yoshiyuki Ishikura 2 , Shigeaki Fujikawa 2 , Yoshinobu Kiso 2 , Manabu Sakakibara 1 1 Lab. Neurobiol. Engr., Tokai Univ., Numazu, Japan; 2 Inst. Health Care Sci. Suntory, Shimamoto, Japan
Previous studies indicate that long-term dietary supplementation with arachidonic acid (AA) in 20-month-old rats (OA) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (YC). This study examined fluorescent recovery after photobleaching (FRAP) in YC, old control (OC), and OA neurons in hippocampal slice preparations. Three measures: mobile fraction (Mf), diffusion constant (D), and time constant (τ), were estimated among YC, OC, and OA. Each of these parameters was significantly different between OC and YC, suggesting that membrane fluidity is lower in OC than in YC. In contrast, D and τ were almost comparable in OA and YC, indicating that hippocampal neuronal membranes supplemented with AA were more fluid than those in OC, whereas the fraction of available molecules remained smaller than in YC. Long-term administration of AA to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity.
PS1A-I161 Thimet oligopeptidase co-exists in GFAP-and CD11b-positive glia in rat PC/RSC treated with MK-801
Takeshi Kato 1 , Mohammad Arif 1 , Michiyuki Yamada 2 , Toshiyuki Chikuma 3 , Md. Mahiuddin Ahmed 4 1 Lab. Natural Info. Sci., Grad. Sch. Integr. Sci., Yokohama City Univ., Yokohama, Japan; 2 Grad. Sch. Integr. Sci., Yokohama City Univ., Yokohama, Japan; 3 Dept. Hygien. Chem., Showa Pharmaceut. Univ., Machida, Japan; 4 Dept. R&D, Bioelectro. Anal. Sci., Japan
Thimet oligopeptidase (EP24.15) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. In the present immunohistochem study we found that MK-801 activated GFAPand CD11b-positive glia cells in rat posterior cingulate/retrosplenial cortex (PC/RSC) 3 day after the treatment. MK-801 also increased EP24.15 and prolyl oligopeptidase. Immunohistochem data showed that EP24.15 co-localized with GFAP and CD11b positive glial cells.
Since MK-801 causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. Clozapine co-administration suppressed the increased EP24.15 in the PC/RSC. These data suggest that EP24.15 in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms.
PS1A-I162 Effect of age and sex steroids on the expression of Alzheimer's disease presenilin (PS) 1 and 2 in the mouse brain
Soumi Ghosh, M.K. Thakur Banaras Hindu University, India
Alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. These functions are improved by supplementation of sex steroids. The genes causing lateonset of AD, presenilin (PS) 1 and 2, code for highly homologous integral membrane proteins. The proteolytic fragments of these proteins are main biological components. We have analysed the effect of age, sex and gonadal hormone supplementation on PS expression at protein level by western blotting. PS1 shows a significant decrease with aging in both males and females. However, there is no significant variation in expression of PS1 and PS2 with sex. Gonadectomy also lowers the level of presenilin proteins in old age. PS2 protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers PS1 level. These modulatory effects of age, sex and gonadal hormones on PS proteins may explain the therapeutic interventions of hormone replacement therapy.
Research funds: Ministry of Science and Technology, India PS1A-I163 Effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from SVZ
Dept. of Pharmacol., Seoul Natl. Univ., South Korea
The Subventricular zone is the largest neurogenic area of the adult brain. In this region, neural stem cells (NSC) serve to produce newly generated neurons and glia cells throughout adulthood. However, the common neurogenesis of NSC cannot replace neuronal loss in Alzheimerǐs disease (AD) induced by amyloid deposits composed mainly of amyloidproteins. In vitro, we examined the effects of various form of Apeptide on the proliferation and differentiation of NSC from SVZ of 10-week-old adult mice. In this study, A42 peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar A42 peptide. We found that treatment of NSC with oligomeric form of A42 peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. We also found that these neurogenesis was accompanied by morphological change of neuron. The number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric A42 peptide without shrinkage of axonal length. In Alzheimer's disease (AD) brain, the formation of senile plaque with accumulated microglia is observed. Although the role of microglia in AD is not clarified, their involvement in A clearance is noted. High mobility group box protein-1 (HMGB1) is a non-histone chromosomal protein.
Here, HMGB1 was associated with senile plaques and protein level was increased in AD brain. Diffuse HMGB1 immunoreactivity was observed around dying neurons in the kainic acid-and A1-42 (A42)-injected rat hippocampi. HMGB1 was not co-localized with A in transgenic mice which show massive A production without neuronal loss. Furthermore, co-injection of HMGB1 delayed the clearance of A and accelerated neurodegeneration in A42-injected rats. These results suggest that HMGB1 released from dying neurons may inhibit microglial A clearance and enhance the neurotoxicity of A. Perineuronal nets consisting of chondroitin sulfate proteoglycan (CSPG) and hyaluronic acid (HA) are associated with distinct populations in mammalian brain. In the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using Wisteria floribunda lectin, HA binding proteins, and CSPGspecific antibodies. This perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating GABAergic ones. It is well known that perineuornal nets-containing neurons are survive against Alzheimer disease in human. To elucidate significance of perineuronal nets in Alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. Perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. These results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid.
PS1A-I166 X11-like protein regulates metabolism of APP in the mouse brain Yoshitake Sano 1,2 , Tadashi Nakaya 2 , Shigeyoshi Itohara 1 , Toshiharu Suzuki 2 1 RIKEN BSI, Saitama, Japan; 2 Hokkaido University, Neuroscience, Sapporo, Japan
Abnormal metabolism of amyloid beta precursor protein (APP) results in the accumulation of beta amyloid (A) in the brain, and contributes to the pathogenesis of Alzheimer's disease. APP has a functional sequence in its cytoplasmic domain, the YENPTY motif, which is involved in trafficking, internalization, and metabolism of APP. X11-like protein (X11L) was identified as a molecule that interacts with the motif and regulates APP metabolism in cultured cells (1999 . J. Biol. Chem. 274, 2243 2003. J. Biol. Chem. 278, 49448) . There is no evidence, however, that endogenous X11L suppresses APP metabolism and A generation in vivo. To examine the physiologic role of X11L in APP metabolism in the brain, we generated X11L null mutant mice. The mutant mice developed normally without gross anatomic brain abnormalities. There were increased amounts of Cterminal fractions cleaved at the -site and A, but the amount of total APP was unaltered in the mutant mouse brain. These results suggest that X11L suppresses the production of A by inhibiting secretase-induced proteolysis of APP. It is still unknown how human's central nervous system (CNS) controls its body system to keep the body balanced. This study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a PID control model. In this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. Spectral response analysis showed the gain varied with input frequency and time lag. Peaks of the gain were intensively influenced by controller's parameters (KP, KD and KI). Parameters identification showed that KD is decreased in eye-closed. By simulation, the spectral responses of the PID model quite agreed with the experimental data. The results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in CNS can be modelled as a PID controller. Nuclear dysfunction is a critical element of the pathology of polyglutamine (polyQ) diseases. Proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin-1 protein by 2D-electrophoresis and TOF-MASS delineate that mutant AT1 and Htt proteins similarly reduce transcriptional factor X1 and X2. Immunoprecipitation and pull-down assays support interaction between polyQ and factor X1 and X2. Immunohistochemistry of Hela cells and primary neurons reveal sequestration of factor X1 and X2 into inclusion bodies and reduction of them in the nuclear matrix. Compensatory expression of factor X1 and X2 ameliorates poly-Q pathology in Htt-/AT1-expressing neurons and transgenic Drosophila. These results suggest that factor X1 and X2 are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics.
PS1A-J170 BA1-42 was reduced in rat brains fed with coconut juice N. Radenahmad, P. Subhadhirasakul PSU, Thailand
Young coconut juice (YCJ), Cocos nucifera (Arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. Sixty ovx rats were divided into six groups, 10 rats/group (g). Group 1 received E2 at 2.5 g/kg per day; groups 2 and 3 received YCJ at 20 mL, and 100 mL/kg day, respectively, once everyday. Group 4 received YCJ 100 mL/kg plus E2 at 2.5 g/kg day twice a week, all for 5 weeks. The other two were ovx and sham-operated controls. Using a chemiluminescent immunoassay, circulating E2 in group 3 was insignificantly different from the control groups. After rats were sacrificed, brains were removed, fixed and paraffin embedded for IHC staining. Using anti--amyloid 1-42 antibody, this Alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer 3 and layer 5 of cerebral cortex. It was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group 3 was lesser than ovx and control groups. Amyloid deposition was correlated with E2 serum at r = −0.4.
PS1A-J171 Correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. A voxel-based morphometry
Yasuyuki Taki 1 , Shigeo Kinomura 1 , Kazunori Sato 1 , Shinya Uchida 1 , Ryoi Goto 1 , Kentaro Inoue 1 , Ichiro Tsuji 2 , Hiroyuki Arai 2 , Ryuta Kawashima 3 , Hiroshi Fukuda 1 1 Department of Radiology and Nuclear Medicine, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan; 2 Tohoku Univ. Grad. School of Med., Sendai, Japan; 3 NICHe, Tohoku Univ., Sendai, Japan
The purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. We collected brain magnetic resonance images of 111 community-dwelling normal elderly subjects. We performed multiple regression analysis of raw score in the WAIS-R Information subtest, gender, and regional gray matter volume. The volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the Information subtest. Our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory.
Research funds: (H13-kenko-008), (H13-choju-007, H13-choju-023)
PS1A-J172 Effects of fluoxetine on the cognition of patients with mild cognitive impairments Arash Mowla, Azadeh Pani Shiraz University of Medical Sciences, Iran
Recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. In several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. We studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (MCI). Fifty-two non-depressed patients with MCI were randomly assigned to take fluoxetine or placebo. The patients were administered mini-mental status examination (MMSE) and Wechsler Memory Scale III (WMSIII) pre intervention. Twenty-six patients completed the 8 weeks trial. Treatment response was defined as a final MMSE and WMS-III scores. The patients in the fluoxetine group showed improvement in MMSE and immediate and delayed logical memory scores of WMS-III. The placebo group had not significant changes in the cognitive measurements. Fluoxetine enhanced memory and cognition in the patients. This was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. However, this conclusion should be tempered by the small sample size.
Lisa L. Cook 1 , D.G. Goodenowe 1 , Y. Yamazaki 1 , J. Flax 2 1 Phenomenome Discoveries Inc., Saskatoon, Canada; 2 PrecisionMed Inc., San Diego, CA, USA Dementia affects about 10% of the population over the age of 65 and can result from various neuropathological conditions. Currently, there is no way to differentiate specific forms of dementia (Alzheimer's disease (AD), vascular dementia, etc.) prior to autopsy. PDI has discovered an 8-metabolite biomarker panel within the serum of patients with AD, non-AD dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS). The accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. The 8-metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. The development of a high throughput assay for these markers will also be presented. Neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin-2 (ChR2). In this study, we quantitatively investigated the rapidness of the light-gated current of ChR2 expressed in PC12 cells using blue LED light. The light-gated current consists of two components, inactivating and noninactivating. The magnitude of inactivating component was almost linearly related to the light intensity. The non-inactivating component showed the tendency to saturate at high illumination. We also found that the activation rate is about 10-fold faster than the inactivating rate, but both are linearly dependent on the light intensity. Since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. It is suggested that the genetic expression of ChR2 is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system.
Yujiro Hattori 1 , Shigeki Ohta 1 , Kenji Hamada 2 , Naofumi Yamada-Okabe 2 , Yonehiro Kanemura 3 , Hideyuki Okano 4 , Yutaka Kawakami 5 , Masahiro Toda 1,6 1 Neuroimmnology Research Group, Keio Univ., Tokyo, Japan; 2 Chugai Co. Ltd., Kanagawa, Japan; 3 Inst. Cli. Res., ONH, Japan; 4 Physiology, Keio Univ., Tokyo, Japan; 5 Cellular Signaling, Institute for Advanced Medical Research, Keio Univ., Tokyo, Japan; 6 Neurosurgery, Keio Univ., Tokyo, Japan
To identify neuron specific genes, we performed two gene profiling techniques, DNA microarray and EST analysis. In this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human KIAA1110 gene which was a homologue of rat synArfGEF (Po). RT-PCR analysis revealed that the human KIAA1110 homologue was expressed only in adult brain tissue. Western blot and immunocytochemical analyses showed the KIAA1110 protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. In conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes.
Ritsuko The objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism.
Methods: Developing hypothalamic and cortical cells from fetuses on embryonic day 15 were dissociated. After the cells were treated with 100 nM estradiol-17beta (E2) or ethanol for 8 days, proteins were extracted and labeled with CyDyes. Two-dimensional difference gel electrophoresis (2D DIGE) was then performed. The differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
Results: More than 4000 spots were detected from 2D DIGE. Compared with ethanol treatment, E2 increased the expression of 501 spots in the hypothalamic cells and 169 spots in the cortical cells (p < 0.2, difference >1.5). Proteomics analysis showed different effects of E2 for hypothalamic and cortical cells. In order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. Genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. Here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (AlstR), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (AL). In rat barrel cortex neurons expressing AlstR, AL reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. Both inactivation and recovery were seen within several minutes. We also confirmed the effectiveness of the AL/AlstR system in ferret visual cortex, lateral geniculate nucleus (LGN), and monkey LGN. Therefore, the AL/AlstR system will be a powerful tool to investigate neuronal circuits and function. Prospective purification of neural stem cells (NSC) through the specific cell surface marker is crucial for functional recovery of the damaged brain. In the last meeting, we showed that living NSC were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (E-PHA), which binds to complex type Asparagine-linked oligosaccharide (N-glycans). In this study, by using FACS system, we found that high selective affinity of E-PHA binding to the brain cells; E-PHA negative cells were neurons, mid-positive cells were NSC, and highly positive cells were endothelial cells, respectively. Ligand blot analysis revealed the existence of the E-PHA binding proteins different from the known selective NSC markers in E14 days brain homogenate, suggesting that N-glycosylated proteins could be distinctive markers for NSC.
Masahiro Waza, Hiroaki Adachi, Masahisa Katsuno, Makoto Minamiyama, Fumiaki Tanaka, Manabu Doyu, Gen Sobue Department of Neurology, Nagoya University, Nagoya, Japan
The pathogenic gene product of spinal and bulbar muscular atrophy (SBMA) is polyglutamine (polyQ)-expanded androgen receptor (AR), which belongs to Hsp90 client protein family. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. 17-AAG is now in phase II as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. We examined the efficacy and safety of 17-AAG in a mouse model of SBMA. Administration of 17-AAG significantly ameliorated polyQ-mediated motor neuron degeneration by preferential proteasome degradation of mutant AR. The ability of 17-AAG to preferentially degrade mutant protein would be directly applicable to SBMA and other neurodegenerative diseases. Modulation of Hsp90 function by 17-AAG has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. The avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. In the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. The reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (MN) specific HB9 promoter. Tetracycline responsive elements were used for inducible GFP expression. After these constructs were introduced into neural tube by in ovo electroporation, GFP expression was induced in spinal MNs in the presence of doxycycline. Approximately 5-20% of MN express GFP very intensely whereas the remaining MNs never express GFP, suggesting that, although the transgene is induced in limited numbers of MNs, once activated, cells express a large amount of the protein product of the experimental gene. Thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo.
PS1A-K181 Selective collection of catecholaminergic (CA) neurons in the brain and its application to gene expression analyses
Hiroaki Nakamura 1 , Yoshiyuki Ishii 1 , Kazuto Kobayashi 2 , Yasufumi Sato 3 , Keiichi Itoi 1 1 Lab. Info. Biol., Grad. Sch. Info. Sci., Tohoku Univ., Sendai, Japan; 2 Dept. Mol. Genet., Fukushima Med. Univ., Japan; 3 Inst. Develop., Aging, Cancer, Tohoku Univ., Japan CA neurons are involved in a wide spectrum of physiological functions in the brain. Most CA neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (A1, A2 and A6) and dopaminergic (A9, A10 and A12) neurons predominate. In order to explore functional roles of these neurons, we collected CA neurons selectively using tyrosine-hydroxylase (TH)-green fluorescent protein (GFP) transgenic mice in which GFP was expressed under the control of TH gene promoter. In fetal mice, most GFP-positive neurons expressed TH-immunoreactivity in limited brain regions including the locus coeruleus (LC). Therefore, LC-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then GFP-positive cells were sorted out by flow-cytometry (FACS). RNA was extracted from the GFP-positive (TH) neurons, reverse-transcribed, and analyzed by PCR. Atg4B has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To understand the role of Atg4B in rat tissue cells, we prepared an antibody to Atg4B and PC12 cells in which Atg4B expression was knocked down by RNAi. In RNAitreated PC12 cells where Atg4B expression was 10% of that in the wild-type PC12 cells, the expression of cytosolic LC3-I was similar to that in wild-type cells. The knockdown cell lysates, however, suppressed cleavage of proLC3 to LC3-I. Moreover, the expression of Atg4B mRNA was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. Immunostaining for Atg4B was intense in neurons, especially in the cerebellum. These results suggest that Atg4B plays a major role in the processing of LC3, while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum.
PS1A-K183 Spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, HEV) in the rat central nervous system after its hind footpad inoculation Transneuronal propagation of HEV 67N strain into the central nervous system was examined after its subcutaneous inoculation (10 5 PFU) in the rat hind footpad. On day 3 post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. On day 4 p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. On day 5 p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. In this model the 4th day would be critical for the analysis of the long-distance connections. HEV can be used as a novel tracing probe, being equivalent to other reported virus probes.
Hiroyuki Hioki 1 , Hiroshi Kameda 1 , Hisashi Nakamura 1 , Taro Okunomiya 1 , Koji Ohira 1 , Kouichi Nakamura 1,2 , Takahiro Furuta 1 , Takeshi Kaneko 1,2 1 Dept. of Morphol. Brain Sci., Grad. Sch. of Med., Kyoto Univ., Kyoto, Japan; 2 CREST, Japan Vesicular stomatitis virus G-protein (VSV-G) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. VSV-G achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. Thus, VSV-G pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hCMV) promoter, infect and express transgenes in neuronal and glial cells. The purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. At first, we used five kinds of well-known neuron-specific promoters; hSYN1, rTa1, mCaMKII, rNSE and hPDGF promoters. Then, we developed new hybrid promoters by a combination sequence of hCMV enhancer and neuron-specific promoters listed above. All of the new hybrid promoters dramatically improved expression of reporter gene (GFP), but the specificity deteriorated in the rat striatum, thalamus and neocortex. Although green fluorescent protein (GFP) is a useful tool to label living neurons, neuronal processes are not completely labeled with GFP.
In the present study, we tried to develop dendritic membrane-targeted GFP using non-prolilferative lentivirus vector with human synapsin I promoter. Palmitoylation site of GAP43 N-terminal and myristoylation site of Fyn N-terminal were first tested for membrane targeting of GFP. Myristoylated GFP (myrGFP) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated GFP. Since myrGFP was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as C-terminals of telencephalin (TLC), Fc ␥ II  receptor (FcR), polymeric immunoglobulin receptor (PIgR), and low density lipoprotein receptor (LDLR), to C-terminal of myrGFP, and compared their efficiency on dendrite targeting. Recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. The vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. By using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. It was expected that we could detect and visualize the divergence or convergence of a neurocircuit. In the study, we could demonstrate the efficiency of double labeling in vivo. In the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. The experiments were performed in the hippocampus and the neighboring cortices of rats. We could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain.
Shuchen Lee, Lihao Ge Institute of Neurobiology, Institute of brain Science, Fudan University, Shanghai, China
Glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. Cone terminals were both gly␣1 and gly immunoreactive. In freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. The glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. These results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-Catenin has 10 Armadillo motifs and a carboxyl terminal type I PDZ ligand. In neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the NMDA and AMPA receptors. Electrophysiological recordings from CA1 hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the AMPA receptor-mediated EPSC but had no significant effect on the NMDA receptor-medicated EPSC. The effect of ␦-catenin on the AMPAR-EPSC was medicated by its PDZ ligand. In COS7 cells, co-transfection of ␦-catenin/GRIP showed that ␦-catenin regulated the membrane localization of GRIP through its PDZ ligand. Co-transfection of ␦-catenin/GRIP/GulR2 increased the surface expression of GluR2 in COS7 cells compared with GRIP/GluR2 or ␦-catenin/GluR2 transfection. This study points to ␦-catenin as a regulator of GluR2 receptor trafficking.
Inseon Song, Kunihiko Obata, Alexey Semyanov BSI, RIKEN, Japan GABA A receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. We measured density of picrotoxin (GABA A receptor antagonist) sensitive holding current (which reflects GABA A receptor mediated conductance) in interneurons of hippocampal CA1 area in wild type (WT) and GAD65 knockout (KO) mice. This parameter was twice lower in GAD65KO mice (WT: 2.0 ± 0.5 pA/pF, n = 7; GAD65KO: 0.9 ± 0.3 pA/pF, n = 8; p = 0.038). The total membrane conductance was similar in both types of animals suggesting adaptive compensation. Application of GABA (5 M) increased tonic current in both type of mice by the same amount. No significant difference in amplitude or frequency of spontaneous IPSCs was detected, although their decay time was shorter in GAD65KO animals (WT: 12.4 ± 0.7 ms, n = 14; GAD65KO: 10.8 ± 0.6 ms, n = 12; p = 0.047). The changes in inhibition which we have found may explain previously reported behavioral abnormalities in GAD65KO.
Research funds: BSI, RIKEN
Hiroki Mutoh, Thomas Knopfel Lab. for Neuronal Circuit Dynamics, BSI, RIKEN, Wako, Japan
Olfactory glomeruli constitute the first stage of central odor processing. Yet, their role in integration of odor information is only partially understood. We previously discovered that 5 Hz olfactory nerve (ON) stimulation induces long-term depression (LTD) in young (P5 to P8) mice. The present experiments were designed to understand in more detail the molecular mechanisms underlying ON LTD. Bath application of DHPG, a selective group I mGluR agonist, induced ON LTD and occluded subsequent 5 Hz stimulation-induced LTD. ON LTD was not induced by activation of group II or III mGluR agonists. The DHPG-induced ON LTD was mediated by mGluR1 but not by mGluR5 because it was antagonized by the mGluR1 antagonist LY367385 but not by the mGluR5 antagonist MPEP. Expression of DHPG-induced ON LTD was accompanied by an increase in paired-pulse ratio suggesting that ON LTD is caused by a decrease of release probability. We propose that mGluR1 is expressed at the ON. ON LTD may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants.
PS1P-A004 Involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway
Masatoshi Takita 1 , Michiko Ohtomi 2 1 Cognition and Action Group, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan; 2 Department of Biomolecular Science, Faculty of Science, Toho University, Chiba, Japan A mesocortical dopaminergic (DA) input to prefrontal cortex (PFC) with the D1 receptor is necessary for long-term potentiation (LTP) to occur at hippocampal-PFC synapses, which is involved by working memory (WM) in rats. Here the DA system was investigated in another WM-involved pathway from mediodorsal nucleus of the thalamus (MD) to PFC. Preliminarily, local perfusion of the D1 antagonist SCH23390 into PFC by using a microdialysis method impaired MD-PFC LTP but the D2 antagonist sulpiride did not. Extracellular DA levels in the PFC robustly increased after the tetanus of MD (by 110-120%). As a result both excitatory synaptic inputs to the PFC involved the WM-related DA profile, implying DA system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the PFC to selectively store intersectional information of multiple brain areas. Neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. In sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (RF) maps with defined RF size and shape. Evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive Hebbian plasticity. Dendritic excitability, mediated by voltage-gated Na + channels, is required for active backpropagation of axosomatic action potentials (APs) and initiation of dendritic spikes; backpropagating APs and dendritic spikes enable forms of synaptic Hebbian plasticity, such as spike-timing dependent plasticity (STDP). Here we examined the role of dendritic excitability in synaptic maturation of layer 2/3 pyramidal neurons in the rat somatosensory barrel cortex. In the present study we compared LTP induction in neocortex of Captreated and normal rats in present of GABA antagonist, Picrotoxin (PTX). The result of present experiment showed that PTX plays an important facilitatory role in the induction of LTP in both normal and Cap-treated group. In Cap-treated group, in present of PTX, the LTP responses significantly were higher than normal group. We conclude that the enhancement of LTP by PTX can be explained by product of competition between excitatory and inhibitory pathways or synapses. These results suggest that GABAergic system has an important role in synaptic plasticity. Also, these results indicated that GABAergic inhibition has been increased in Cap-treated group.
Tohru Kurotani, Komatsu Yukio Department of Visual Neuroscience, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan
We showed in previous study that somatic inhibitory synapses of neocortical layer 5 pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (SWS) and arousal states. In the present study, using a minimal stimulation method, we recorded somatic IPSCs from layer 5 pyramidal cells in visual cortical slices prepared from rats at SWS like state under urethane anesthesia and in those prepared from rats at arousal state. The average amplitude of somatic IPSCs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. The mean rise time, decay time constant of IPSCs and the mean input resistance of the cells were not significantly different between these two groups. The present results further confirmed that the somatic inhibition in neocortical layer 5 pyramidal neurons is bidirectionally modified in accordance with behavioral state. Corticothalamic fibers (CT), originated from cerebral layer 6 pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. Since these pyramidal cells abundantly express kainate receptors (KARs) mRNA, we studied the effect of kainate on the presynaptic function of the two CT synapses in mouse thalamic VB nucleus. Bath application of kainate (200 nM) depressed CT-EPSCs and increased the paired pulse ratio in relay neurons. In contrast, kainate at the same concentration facilitated CT-EPSCs and decreased the paired pulse ratio in reticular neurons. These results suggested that KARs differentially regulated release at the two CT synapses. Furthermore, high frequency stimulation of CT depressed relay cell synapses but facilitated reticular cell synapses. Blocking endogenous KARs abolished these effects. Because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous KARs presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. To examine the involvement of NTR1 in the regulatory mechanisms for LTP in the amygdala, we utilized NTR1-knockout (KO) mice. We performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (BLA), where DA-NT neurons project. We found that the BLA-LTP, induced by LA stimulation, was significantly greater in NTR1-KO mice than in wild-type mice. The BLA-LTP in NTR1-KO mice was attenuated by sulpiride, a D2 receptor antagonist. These results suggest that D2-NTR1 interaction regulates the extent of LTP in the mouse LA-BLA synapses.
PS1P-A010 Facilitation of axonal plasticity in recovery from traumatic brain injury and the role of TNF␣ in mouse model Recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. Apart from that, TNF␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. The present study evaluated motor function recovery after controlled cortical impact (CCI) brain injury, and also the facilitation of plasticity by biotin dextran amine (BDA) axonal tracing in TNF␣KO mice and wild type (wt) mice. Mice were subjected to left sided CCI or served as sham controls, and were evaluated by composite neuroscore and rotarod over 28-day period. BDA was injected in right cerebral cortex to observe new axonal connections. So far, we observed recovery of motor function in wt mice, whereas TNF␣KO mice showed continuous functional deficit. We also observed greater number of new axonal connections in wt mice. Our results suggest that TNF␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. Disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. Here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. Kinetics of short-term plasticity and unchanged apparent Ca 2+ sensitivity indicate that TTX-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the Ca 2+ at the final fusion step. In addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin-1, and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization".
PS1P-A012 Inhibitory modulation of the hippocampal CA3 transmission and plasticity by glucagon-like peptide-2
Jun-Ichiro Oka, Takashi Iwai Lab. Pharmacol., Fac. Pharm. Sci., Tokyo Univ. Sci., Japan
Glucagon-like peptode-2 (GLP-2) is a proglucagon-derived peptidehormone in the intestine and brain. We reported that GLP-2 (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. However, the mechanisms of GLP-2 effects on hippocampal neurons are unclear. In this study, we investigated the effects of GLP-2 on the synaptic function of neurons in the acute hippocampal slices. Hippocampal slices (400 m) were prepared from 7 to 35 days Wistar rats of both sexes. Patch-clamp recordings were made from pyramidal cells of the CA3 in the whole-cell mode using glass microelectrodes (resistance: 4-8 M ). In extracellular recordings, field excitatory postsynaptic potentials (fEPSP) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. GLP-2 (10 nM-1 M) inhibited spontaneous excitatory postsynaptic current. GLP-2 (10 nM) did not affect fEPSP amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. These results suggest that GLP-2 may play an inhibitory role in the DG-CA3 transmission.
PS1P-B013 Quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe Takuya Hkima, Rikita Araki, Toru Ishizuka, Hiromu Yawo Dept. of Dev. Biol. and Neurosci., Tohoku Univ. Grad. Sch. of Life Sci., Japan
Both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. However, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. It has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. To measure exocytosis directly, we applied the synaptopHluorin (SpH) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of VAMP-2 and superecliptic pHluorin selectively in the mossy fiber terminals. The SpH fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin A1, a vesicular H+-ATPase inhibitor. Therefore, the rising phase of SpH fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. This method would enable us to evaluate the presynaptic contribution to synaptic plasticity.
Jyoti Parkash, Gurcharan Kaur GNDU Amritsar, India
We have earlier reported that GnRH nerve terminals in the ME continue to express high levels of polysialylated form of neural cell adhesion molecule (PSA-NCAM) in a cyclic fashion and PSA-NCAM covers both the GnRH axon surfaces and the associated glial cells in the proestrous phase rats indicating that PSA plays important role in the neurosecretory activity in hypothalamus. To further establish the functional significance of PSA-NCAM molecule, we have studied the expression of PSA-NCAM on GnRH axon terminals and glial cells in the proestrous phase of cycling rats as well as GABA and PBZ treated proestrous rats by using dual immunohistofluorescent staining. Both GnRH and PSA-NCAM immunostaining was much higher in proestrous phase rats, whereas, GABA and PBZ treatments significantly reduced their expression. The expression of PST has been studied within GnRH cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in POA and ME-ARC regions of cycling female rats as well as in GABA and PBZ treated proestrous rats. Cortical plasticity has important roles in the development of neural circuits in sensory cortices. However, the roles and mechanisms for various types of LTP and LTD are not clear. We investigated supragranular LTP and two types of supragranular LTD in the slices obtained from the rat auditory cortex, and compared their properties. Frontal cortical slices were prepared from male Wister rats. Supragranular field potentials elicited by the stimulation applied to layer VI were recorded. LTP was induced by tetanic stimulation (TS, 100 Hz for 1 s) applied to layer VI. LTD was induced by low-frequency stimulation (LFS, 1 Hz for 900 s) applied to layer VI. LTD was also induced by TS applied to supragranular layers near the recording site. LFS-induced LTD and TS-induced LTD were completely abolished in the presence of 50 M APV, 3 M bicuculline, but not 500 M MCPG. LFS-induced LTD and TS-induced LTD occluded each other, suggesting that that both types of LTD share cellular and molecular mechanisms.
Kazuyoshi Kawa Department of Neurophysiology, Tohoku University, Graduate School of Medicine, Sendai, Japan
Using slice-patch techniques, synaptic transmission in neurons of the area postrema (AP) of the rat was studied. When 20 mM KCl was applied from a "Y tube" to AP neurons (whole-cell clamped at −10 mV), massive inhibitory postsynaptic currents (IPSCs) were induced. Most of the evoked IPSCs were blocked by bicuculline confirming GABAergic identity. When nicotine (5-100 M) or capsaicin (0.1-1 M) was applied to AP neurons, robust appearance of IPSCs with GABAergic identity was induced. After blocking action potential generation in the slice with tetrodotoxin (1 M), nicotine and capsaicin could still induce GABAergic IPSCs. Interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after 5 min of rigorous washout. It is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, TRPV1), are expressed at GABAergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions.
Research funds: Grant from the Smoking Research Foundation (Japan)
Takako Morimoto-Tanifuji 1 , Akira Komatu 2 , Akinao Nose 1 1 Dept. Phys., Univ. Tokyo, Tokyo, Japan; 2 Dept. Physiol., Sch. Med., Tokyo Women's Med. Univ., Tokyo, Japan
The molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. We investigated how the localization of glutamate receptors (GluRs) is regulated in neuromuscular junctions (NMJs) of Drosophila 3rd instar larvae. There are mainly two classes of GluRs, containing either GluRIIA or IIB. GluRIIA has a sequence predicted as Ca 2+ -permeable site. When CaMKII was inhibited by the expression of inhibitory peptide, Ala, the content of GluRIIA in synapses was dramatically increased and the mean amplitude of extrajunctional potential (EJP) was enhanced. The expression of constitutively active form of CaMKII (T287D) resulted in decreased GluRIIA content and enhanced GluRIIB content. Although miniature EJP amplitude was reduced, EJP amplitude was normal in T287D expressing larvae, suggesting the existence of some homeostatic mechanisms. Taken together, CaMKII regulates the localization of GluRs in a subunitspecific manner and modulates synaptic function in NMJs. ) . Notably, neuronal dNRs from dNR1* flies did not show Mg 2+ blockade, and dNR1* flies displayed significant impairment in transcription-dependent long-term memory (LTM) but not in transcription-independent acquisition and short-term memory. We identified salient increases in genes involved in L-LTP formation, e.g. homer, and activin, as well as the increase in genes involved in LTM, e.g. staufen, upon LTM formation. However, such increases were absent in dNR* flies. Transcription for LTM is mediated, at least, by transcription factors such as CREB, Adf-1, and Notch. We examined how Mg 2+ blockade of dNR links to these transcription factors.
Research funds: KAKENHI PS1P-B020 Response properties of wind-sensitive giant interneurons in the 4th-instar nymphs of the cricket Tetsuya Matsuura 1 , Masamichi Kanou 2 1 Dept. of Welfare Eng., Iwate Univ., Morioka, Japan; 2 Dept. of Biology, Ehime Univ., Matsuyama, Japan
The response properties of four wind-sensitive giant interneurons (GIs) 8-1, 9-1, 9-2 and 9-3 in the 4th-instar nymphs of the cricket Gryllus bimaculatus were investigated. Air current was presented to the animal from 12 different directions in the horizontal plane. The intensity-response curves showed that the response magnitudes of GI 8-1 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. The response magnitudes of GI 9-1 reached a plateau at a stimulus velocity of 30 mm/s in most stimulus directions. The response magnitudes of GIs 9-2 and 9-3 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. The directional sensitivity curves revealed that the preferential directions of the GIs in nymphs were the ipsilateral-side in GI 8-1, the ipsilateralfront and contralateral-rear in GI 9-1, the ipsilateral-rear in GI 9-2 and the ipsilateral-front in GI 9-3, designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults.
Yasuyuki Ishikawa, Sadao Shiosaka Division of Structural Cellular Biology, Nara Institute of Science and Technology, Nara, Japan
Long-term potentiation (LTP) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. LTP expression can be regulated by previous synaptic activity, a process known as "metaplasticity." We report a novel form of cellwide metaplasticity in hippocampal area CA1. Serine protease, Neuropsin, is involved in the regulation of synaptic plasticity. Neuropsin increased the stability of LTP induced later at the same inputs via L-type VDCC. Moreover, neuropsin-deficient mice impaired L-LTP induction by l-TBS. Our findings have revealed the effects of neuropsin on the conversion of E-LTP to L-LTP. PTP, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. Although it has been known that PTP is induced by the elevation of presynaptic Ca 2+ , the molecular mechanism of PTP is poorly understood. In order to elucidate the specific role of presynaptic TrkB receptors in PTP, we analyzed PTP using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkB gene is restricted to presynaptic sites in the CA1 region. We found that PTP induced by the 100-Hz tetanus was reduced in mutant mice, and that PTP in control mice was partially reduced by an N-type Ca 2+ channel blocker, while PTP in mutant mice was unaltered by the blocker. Thus, these data suggest that the N-type Ca 2+ channel-dependent component of PTP requires TrkB receptor activation.
Research funds: JSPS and MEXT of Japan
Kiyoshi Ohnuma 1 , Kunihiko Kaneko 1,2 , Makoto Asashima 1,2 1 Grad. Sch. of Arts & Sci., Univ. of Tokyo, Tokyo, Japan; 2 JST, Tokyo, Japan
Measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. We have used this approach to study intercellular interaction between neuronal cells.
Here we show that the shape of the population distribution of intracellular Ca 2+ concentration ([Ca 2+ ] i ) may change because of nonsynaptic communication. We loaded PC12 cells with a Ca 2+ indicator, Indo-1 AM, and the [Ca 2+ ] i of more than 10,000 cells was measured using flowcytometry. The [Ca 2+ ] i distribution of unstimulated single cells had a long right tail, suggesting that [Ca 2+ ] i is usually low but sometimes becomes high. On the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many Ca 2+ -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem.
Our results suggest that measuring the distributions is useful in researching intercellular interaction. Na x is a sodium channel involved in sensing the sodium level of the body fluid. Our recent studies showed that Na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the CNS organs involved in the sodium reception. However, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated.
To address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that Na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. These results suggest that Na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. The state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. We have isolated spinesin/TMPRSS5 from human and mouse CNS.
In mouse CNS, four isoforms (types 1-4) were expressed. Subcellular localization analysis revealed that type 4 (full length) spinesin was predominantly localized to the ER, Golgi apparatus and plasma membrane, whereas type1 variant was localized to the cytoplasm. Furthermore, we performed expression analysis of m-spinesin in some cell lines. NSC34 and NB2A derived from neuronal cell express only type 4, whereas OS3 and KT-5 derived from astrocyte express both type 4 and type 1. Interestingly, it was observed that the level of spinesin mRNA was increased by a dibutyryl cyclic AMP treatment only in OS3 and KT-5. We analyzed promoter region of m-spinesin gene, and identified that 5 -flanking region from −224 to −188 bp was essential for m-spinesin gene expression. However, this region did not involve cAMP-dependent regulation of m-spinesin expression.
These results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in CNS. It has recently been elucidated that l-serine (L-Ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme 3-phosphoglycerate dehydrogenase (Phgdh), which is the first committed enzyme of L-Ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. Since L-Ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of Phgdh in the mouse retina. Phgdh immunoreactivity was detected in Müller cell soma in internal nuclear layer, being close to internal plexiform layer. Immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. It was suggested that Müller cells contribute in L-Ser synthesis and its transportation to neurons in the retina. Astrocytes frequently show spontaneous intracellular Ca 2+ signals, such as intra-and intercellular Ca 2+ waves; however, their physiological roles remain elusive. The overexpression of an IP 3 -hydrolyzing enzyme, IP 3 5-phosphatase, suppressed the spontaneous Ca 2+ signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. Hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed.
The total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of Ca 2+ -signal-deficient astrocytes. Moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on Ca 2+ -signal-deficient astrocytes. These results indicate that spontaneous Ca 2+ signals in astrocytes are essential for glial cells to promote neurite outgrowth.
Katsuyasu Sakurai 1 , Noriko Osumi 1,2 1 Tohoku Univ. Sch. Med., Japan; 2 CREST, JST, Japan
Astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. We have previously reported that Pax6 transcription factor is expressed in GFAP positive cells in the rat hippocampus.
In the present study, we first investigated the expression patterns of Pax6 in postnatal mouse brain and found that Pax6 was expressed in almost all astrocytes in the cerebral cortex. To address the role of Pax6 in the astrocytes, we examined the morphology of the astrocytes in the wild type (WT) and Pax6 heterozygote mutant (Sey/+) mice at 8 weeks. Confocal imaging revealed that arborization and extension of the astrocytes were poor in Sey/+ mice as compared with the WT. In primary culture, the astrocytes isolated from Sey/Sey cortex showed no morphological difference. However, 6 and 24 h after dibutyryl-cAMP treatment, the majority of the WT astrocytes had undergone the conversion from a polygonal to stellate shape, while Sey/Sey astrocytes rarely showed this response. These results suggest that Pax6 regulates the morphology of astrocytes, thereby being involved in astroglial functions. We raised mouse monoclonal antibody (mAb) DIM21 to study its distribution and function in cell membrane and found not only its preferential reaction with PtdGlc on TLC, but also its labeling in rodent CNS (Yamazaki et al., 2006) . We previously reported a unique expression of DIM21 Ag in developing mouse brain, especially in cell membranes of embryonic radial glia (Kinoshita et al., 2005) . We show here that mAb DIM21 also recognizes adult neural stem cells and glial cells at postnatal period. DIM21, BrdU and GFAP co-expressed in cells of mouse neurogenic subventricular zone. We discuss a possibility that the DIM21 Ag may be expressed in the radial glia/astrocyte lineage cells. The bone morphogenetic protein (BMP) receptors are thought to have a role in neural patterning of early neuronal development. The BMP receptor is widely expressed throughout the central nervous system (CNS) including cerebellum. However, the physiological roles of BMP signaling in mature brain remains obscure. To understand BMP function in CNS, we generated a transgenic mouse line that conditionally overexpresses BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI) in a Purkinje cell-specific manner using a Cre-loxP system. We bred this mouse line with the Cre transgenic mouse line of which expression was driven by L7 promoter. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following a constitutively active Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 (P-smads) in Purkinje cells. We will discuss functional changes of the purkinje cells which receive excess amount of BMP signaling through ALK2. Lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. Glial scar being considered as an impediment for axonal growth, it had been proposed in 1950s and 1960s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. We tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from Salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. Therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury.
Kavita Seth, R.K. Chaturvedi, S. Shukla, A.K. Agrawal Dev. Tox. Div., Industrial Toxicology Reserch Center, Lucknow, India
Crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as Parkinson's disease has gained attention of more than supportive interaction. Here contribution of glial cells in 6-OHDA induced degeneration of dopaminergic neurons was investigated. Glial cultures showed significant loss in cell viability after 48 h (34 and 19%) and 72 h (56 and 33%) exposure to 10 −4 and 10 −5 M 6-OHDA respectively. It was accompanied by morphological changes and induction of GFAP, S-100 and OX42. 6-OHDA (10 −6 M, 72 h) was found to cause a significant impairment in 3 H glutamic acid uptake (31%) and GSH levels (27%). Further neurons (in coculture with 6-OHDA pre exposed glial cells) on exposure to 6-OHDA (10 −6 M), showed loss of TH expression and significant neuronal cell death (34%). The results of the present study suggest that 6-OHDA may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. Nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. We studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (OE). The OE presented a near-complete destruction at 1 day after injection, then started to repair at 3 days and returned to the normal levels at 6 weeks. Nestin was expressed in olfactory ensheathing cells (OECs) of the olfactory mucosa at 3∼7 days, but not in those of the olfactory bulb (OB). Simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. The reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (NG2) is currently considered a marker of multipotent progenitor cells in the brain. In the present study, most Iba1+ cells accumulated in stab wounds and ischemic lesions were found to express NG2, of which molecular weight of its core protein was higher by 10 kDa than that of NG2 expressed in contralateral brain region. This was due to the lack of shedding of NG2 in the brain lesions. We found that Iba1+ cells accumulated in stab wounds and ischemic core lesion, most of which were NG2+/PDGFRa+. Furthermore, some of these cells expressed GFAP, nestin, CD163 and von Willebrand factor. NG2+ MG isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. These variety of antigens expressed by NG2+ MG in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue.
Saroj Sharma, L.K. Singh, B. Ray, T.S. Roy 1 All India Institute of Medical Sciences, India
Oculomotor nerve (ON) supplies most of the extra-and intraocular muscles. It shows changes with normal ageing, metabolic and degenerative diseases. Though there are various studies on the ON, no definitive data regarding the morphometry and the fine structure is available. So, in the present study, neural and the connective tissue organization of the extradural part of the ON from 20 cadavers were studied. Light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. Small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. Using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from <1 to 40 m 2 . Most of the fibers were myelinated and counts produced a mean of 16,891 (12,000-21,500). Ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. The cell junctions of the perineurial cells and the endoneurial capillaries were observed. Myelin thickness ranged from 0.29 to 3.16 m (from fetal age to 60 years age). During the development of the Drosophila visual system, retinal axons project to the optic lobe through the optic stalk. The optic stalk is composed of glial cells and adopts tube-like structure. FAK is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. In Drosophila FAK (Dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. CdGAPr encodes one of GAPs that regulate Rho-family GTPases. Putative CdGAPr mutants showed Dfak-like phenotype.
Since Dfak and CdGAPr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via Rho-GTPases. Tissue specific rescue experiment showed that Dfak autonomously acts in the glial cells. Our results suggest that Dfak and CdGAPr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. Astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. Spontaneous GABAergic postsynaptic activity is reported to be decreased in small neurons of the caudal NTS at the end of the first postnatal week. To investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of GFAP, an astrocytic marker. GFAP began to be immunohistochemically expressed in the caudal NTS at P5-10. Costaining with calbindin, a marker for a certain type of small neurons, showed that GFAP positive processes were thereafter closely apposed to soma of small calbindin neurons. Electron microscopy showed that some astrocytic processes were interposed between orphan GABAergic varicosities and soma of small neurons at the specific developmental stages. These findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal NTS.
Hitoshi Ozawa, Naoyuki Yamamoto, Nobuhiko Sawai, Hao-Gang Xue Department of Anatomy and Neurobiology, Nippon Medical School, Tokyo, Japan
It is well known that the hypothalamo-pituitary-adrenal (HPA) axis is an important system for responding and mediating the stress. In addition, hippocampus is also an important area for the stress response.
In the hippocampus, the expression of glucocorticoid receptor (GR) has been reported in the CA1, CA2 pyramidal neurons and the dentate gyrus neurons. On the other hand, while astroglia around the hippocampus also expresses GR, the morphological and functional changes under different corticosteroid condition have not been well elucidated. In the present study, we investigated morphological changes of astroglia around pyramidal neurons. Under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. These suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons.
PS1P-D042 Impact of S100B on hippocampal spontaneous activities in anesthetized and epileptic conditions Seiichi Sakatani 1 , Akiko Seto-Ohshima 2 , Shigeyoshi Itohara 2 , Hajime Hirase 1 1 Neuronal Circuit Mechanisms Research Group, Japan; 2 Lab. for Behavioral Genetics, BSI, RIKEN, Wako, Japan S100B is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. In order to assess the physiological roles of S100B, we have recorded hippocampal spontaneous activities from urethane anesthetized S100B KO and WT mice. Typical EEG patterns including theta (3-8 Hz) and sharp wave associated fast ripple (120-180 Hz) oscillations were observed in both populations and these patterns were indistinguishable between the WT and KO. When epileptic activity was induced by kainic acid (i.p.), a difference appeared in CA1 radiatum, where ictal event was characterized by hyper-synchronous gamma band (30-80 Hz) activity. While both populations developed ictal event within 40 min, mean power during the development was significantly smaller in KO mice. Our results suggest that deficiency of S100B does not have a profound impact on neural activity in normal conditions. However, when neural activity was raised, activation of S100B related pathways could potentially be activated.
Yoshiko Takagishi 1 , Erina Okabe 2 , Xiaoyang Sun 1 , Sen-ichi Oda 2 , Yoshiharu Murata 1 1 RIEM, Nagoya Univ, Nagoya, Japan; 2 Grad Sch Bio-Agricult Sci, Nagoya Univ, Nagoya, Japan Shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. We have recently identified the shm gene that encodes Caspr, which constitutes paranodal junction of myelinated nerves. To determine whether the mutation alters the node of Ranvier, we performed morphological analysis of myelinated nerves in Shambling mice. By electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. Immunohistochemistry revealed that Caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. Contactin, a component of the paranodal junction, was distributed similar pattern to that of Capsr. Further, K + channels were mislocalized to the paranode, while Na + channels were normally restricted to the node. These findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in Shambling mice.
PS1P-D044 Regulation of hippocampal neurocircuit activity by glutamate transporter GLT-1
Noriko Koganezawa, Shinsuke Muraoka, Ken-Ichiro Tsutsui, Toshio Iijima Div. Systems Neurosci. Tohoku Univ. Grad. School of Life Science, Japan
Glial cells are now recognized as an essential functional element in synapses. In order to investigate their function, we focused on the activity of GLT-1, the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations.
Response to an electrical stimulation of the Schaffer collaterals was recorded using the optical imaging technique. By combining the application of the glutamate receptor blockers (NBQX, AP5) and the GLT-1 blocker (DHK) with the signal subtraction, we could visualize the activity of GLT-1 as a slow, tonic rise of the optic signal following electrical stimulation. Then we evaluated the function of GLT-1 by applying its blocker DHK. An obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of DHK. Furthermore, the blocking of GLT-1 function in the CA3 region was elicited by much lower concentration of DHK than that in the CA1 region.
PS1P-D045 Monoclonal antibody rip specifically recognizes 2 , 3 -cyclic nucleotide 3 -phosphodiesterase in oligodendrocytes The antigen recognized with monoclonal antibody (mAb)-Rip has been used as marker for oligodendrocytes and myelin sheaths. However, the Rip-antigen has been unknown yet. To identify the Rip-antigen, we performed immunopurification with mAb-Rip using the differentiated CG-4 cells lysate. MALDI-QIT/TOF MS n analyses revealed that one of molecules was 2 ,3 -cyclic nucleotide 3phosphodiesterase (CNP). Immunocytochemical and immunohistochemical studies showed that Rip-antigen colocalized with CNP in rat cerebellum, cultured rat oligodendrocytes and CG-4 cells. Moreover, the same localization was also observed in rat Cnp1 transfected HEK293T cells. Overall we first demonstrated that the antigen labeled with mAb-Rip is CNP in oligodendrocytes. The expression of BDNF gene is regulated by four promoters (pI-pIV), and is under activity-dependent control. Until now, it has been established that BDNF pI is activated by Ca 2+ signal via CRE. On the other hand, neuron-restrictive silencer cis-element (NRSE), located in BDNF pII, represses BDNF gene expression through binding NRSF and recruiting HDAC in non-neural cells. Here, we found that NRSE repressed the activity of BDNF pI in neuron. Using RT-PCR and ChIP assay, the BDNF exon I expression and the histone acetylation of BDNF pI were increased by the administration of Ca 2+ signals or HDAC inhibitor. In addition, NRSF bound to BDNF pII in neurons but was detached by Ca 2+ signals. These results suggest that BDNF pI activity is regulated by CREB and NRSF through an alteration of chromatin structure. Since CREB and NRSF are playing an important role in neuronal differentiation, it is considered that the BDNF pI is deeply involved in the regulation of neurogenesis.
Singo Suzuki 1,2 , Hisatsugu Koshimizu 1 , megumi Kashihara 1 , Tomoko Hara 1 , Masami Kojima 1,2 1 Research Institute for Cell Engineering; 2 SORST, JST, Japan
Brain-derived neurotrophic factor (BDNF) plays a crucial role in synapse development, especially, in the central nervous system (CNS) . Although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. Here we show that 3-day treatment with BDNF leads to a significant increase in cholesterol content in primary neuron. This change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. To understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and SV proteins in lipid raft fractions prepared from cultured cortical neurons. The results indicated that BDNF treatment increased the amount of cholesterol and SV proteins in lipid rafts, but not in non-rafts fraction. These data suggested a possibility that BDNF regulated synapse development by increasing the amount of cholesterol and SV proteins in synaptic rafts. (p38) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. In this study, we found that p38 expressed in the neural progenitor cells at embryonic 14 days (E14) mice brain. To ascertain the function of p38 on these cells, we treated the cultured E14 cells with the selective chemical inhibitor for p38 (SB203580) for 7 days, and determined the number of neural progenitor cells. The inhibitor specifically enhanced the number of neural progenitors compared to the control cells. This result suggests the involvement of p38 in the proliferation and/or survival of neural progenitor cells in developing mouse brain.
Hemragul Sabit 1 , Takashi Yamazaki 1,2 , Takeshi Oya 1 , Yoko Ishii 1 , Masakiyo Sasahara 1 1 Pathology II, University of Toyama, Toyama, Japan; 2 Oral and Maxillofacial Surgery, University of Toyama, Toyama, Japan
Purpose: We had reported the increase of PDGF-B and active Src in rat peripheral nerve regeneration. Here examined activation of PDGF receptors (PDGFRs) and signals in the peripheral nerve regeneration. Method and result: Crushed sciatic nerve was removed on 3 to 28 days after injury, and activation of PDGFRs, MAPKs, Akt and p38 were examined by Phosphoprotein Purification kit (QIAGEN) and Western blot. Expression of PDGFRs increased from 3 to 21 days after injury. P-Tyr was highly detected from 3 to 21 days after injury, and activation of PDGFRs also increased during this period. Activation of ERK and JNK increased up to 7 days after injury and then gradually decreased. Activation of Akt and p38 continuously increased from 3 to 28 day after injury.
Conclusion: PDGFRs and their signals were activated in rat peripheral nerve regeneration. Autocrine signal of PDGF may contribute to the regenerative processes, such as proliferation and differentiation of Schwann cells and axonal extension. Cbln1 is a cerebellum-specific protein structurally related to the C1q and Tumor Necrosis Factor families. Recently, we have shown that Cbln1 is secreted from cerebellar granule cells (GC) and controls synaptic structure and plasticity of GC-Purkinje cell (PC) synapses. However, because Cbln1 was previously shown to serve as a precursor of a PC-specific peptide cerebellin, it remains unclear whether Cbln1 needs to be processed before it trans-synaptically activates signaling pathways in PC. Here, we show that purified recombinant Cbln1 proteins, which formed a hexamer, preferentially bound to spines on PC dendrites. Furthermore, Cbln1 mutants that did not form a hexamer lost the binding affinity to PC spines. Although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that Cbln1 released from GC directly bind to postsynaptic PC as a hexamer and activates signaling pathways in PC. Activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. Recently, we found that specific kinds of mRNA can be stabilized in an activity-dependent manner. To elucidate the mechanisms for activity-dependent mRNA stabilization, we have focused on BDNF, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. We constructed firefly luciferase gene fused to 3 -untranslated region (UTR) of BDNF mRNA to investigate the effect of the 3 UTR on the calcium signal-mediated mRNA stabilization. In cultured neurons, we found that the degradation of firefly luciferase-BDNF3 UTR mRNA induced by the treatment with actinomycin D was prevented by calcium signals evoked via L-type voltage-dependent calcium channels (L-VDCCs) and NMDA receptors. We are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of BDNF mRNA using a series of mutant BDNF3 UTR. Recently, it has been established that BDNF and PACAP regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. In this study, we found that the treatment of cultured rat cortical neurons with BDNF or PACAP acutely induced the mRNA expression of the activityregulated cytoskeleton-associated protein (Arc) and homer1a, whose products are necessary for the synaptic plasticity. BDNF induced Arc mRNA expression through the activation of TrkB-ERK/MAPK pathway, whereas PACAP induced it partly through the activation of NMDA-receptors. Using Affymetrix GeneChips, we are now investigating a comprehensive profile of gene expression controlled by BDNF or PACAP in cultured rat cortical neurons.
PS1P-E056 BMP4 expression in the adult rat brain Bone morphogenetic protein-4 (BMP4) is a member of the transforming growth factor  (TGF-) superfamily and plays important roles in multiple biological event. Although BMP4 expression has been well described in the early development of central nervous system (CNS), little information is available for its expression in the adult CNS. We, thus, investigated BMP4 expression in the adult rat CNS using immunohistochemistry. BMP4 is intensely expressed in most neurons and their dendrites. In addition, intense BMP4 expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes also express BMP4 protein. These data indicate that BMP4 is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that BMP4 plays pivotal roles also in the adult brain.
PS1P-E057 HGF as a target-derived trophic factor for rat nigro-striatal dopaminergic (DA) system during post natal development
Wakana Ooya, Hiroshi Funakoshi, Toshikazu Nakamura Div. Molecular Regenerative Medicine, Osaka Univ. Grad. Sch. Med., Osaka, Japan HGF is a novel neurotrophic factor in vitro on DA neurons. However, little is known about expression and biological activities of HGF in nigral-striatal system in vivo. Here we show that HGF is a targetderived trophic factor for rat DA system. Real-time RT-PCR revealed that c-met mRNA was expressed in substantia nigra (SN) and striatum (STR), while HGF mRNA was expressed in STR but not in SN in programmed cell death period. HGF, c-Met, phospho-c-Met, TH, DA transporter immunostaining revealed the presence of concentration gradient of HGF from SN to STR and c-Met was phosphorylated in DA nerve end during early postnatal development. Phospho-c-Metpositive DA neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. HGF application into STR increased DA neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for HGF. Therefore, HGF may be a critical trophic factor for nigro-striatal DA system development. The neuroprotective effects of G-CSF were reported in neurological disease models. In the present study, we examined whether G-CSF can protect dopaminergic neurons from MPTP-induced cell death in a PD. The mice were intraperitoneal injected with MPTP for five consecutive days, G-CSF is intraperitoneal administered two days and one day before first MPTP injection, and 30 min before each MPTP injection. In our results, G-CSF significantly prevented MPTPinduced loss of TH-positive neurons, and increased Bcl-2 protein, decreased Bax protein expression. These findings suggest that G-CSF has therapeutic potentiality to protect MPTP-induced cell death through increasing the level of bcl-2 expression, decreasing the level of Bax expression in C57BL/6 mice.
Kazue Takahata
has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase 1. Here, we evaluated the effects of (−)-BPAP on the phosphorylation of mitogen-activated protein kinase (MAPK) and Akt in slice cultures as well as in an in vivo model. (−)-BPAP significantly increased phosphorylation levels of MAPK, but not those of Akt. (−)-BPAP attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-treated mice. (−)-BPAP may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in Parkinsonian substantia nigra.
Shyuichi Maeda 1 , Yoko Tohyama 1 , Shinichi Kohsaka 2 , Tadashi Kurihara 1 , Kazuyuki Nakajima 1,2 1 Soka University, Hachioji, Tokyo, Japan; 2 Dept. of Neurochem., National Institute of Neuroscience, Kodaira, Tokyo, Japan Astrocytes (Ast) are a cell type that supports CNS not only nutritionally but also neurotrophically, by supplying neurotrophic factors (NTF) required in the neuronal survival, maturation and protection. However, the ability of Ast to produce/secrete NTF has not been accurately known. Thus, in the present study, we investigate the capacity of Ast to produce/secrete various NTF in vitro. Ast were prepared from the mother culture of neonatal rat brain. The NTF in the conditioned medium (CM) were detected by immunoblotting. The analysis of neurotrophins in the CM revealed that Ast produce/secrete NGF, BDNF and NT-3, and promote the production of them by stimulation with lipopolysaccharide (LPS). Furthermore, TGF2 among TGF family was detected in the CM of Ast, and the production was enhanced by stimulation with LPS. These profiles of Ast were different from those of microglia, suggesting the differential regulation of NTF by glial cells.
Ritsuko Katoh-Semba 1 , Chiaki Nakagawa 1 , Masako Tsuzuki 1 , Motoko Matsuda 1 , Satoshi Ichisaka 2 , Yoshio Hata 3 1 Inst. Dev. Res., Aichi Human Ser. Ctr., Kasugai, Japan; 2 Div. Neurobiol., Tottori Univ., Sch. Med., Yonago, Japan; 3 Div. Integrative Biosci., Tottori Univ. Grad. Sch. Med., Yonago, Japan
The sleep-awake rhythm is formed during the early period after birth. The rhythm is very important for the functional development of brain. When the rhythm formation is disturbed, autism-like behaviors are often observed. Brain-derived neurotrophic factor (BDNF) is known to be one of the factors forming circadian rhythms. We have found increases in levels of BDNF in the entorhinal cortex as well as the visual cortex from adult male rats 12 h after beginning an 8-h phase advance of the light-dark cycle. Here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. We first examined circadian changes in the concentrations of BDNF and neurotrophin-3 (NT-3) in selected brain regions from 14-day-old male rats and compared to those from adults. The changes in levels of BDNF and NT-3 were observed in the neocortex and hippocampus. Objective: This study was aimed to investigate the possible beneficial effects of Granulocyte colony stimulating factor (G-CSF) compared to methylprednisolone (MP) in experimental spinal cord injury (SCI).
Materials and methods: (in vivo) Adult female Sprague-Dawley rats had moderate SCI (200 kdyne, IH injury device) at T8/9 and were assigned to three groups; A (placebo), B (MP treated; 30 mg/kg i.v. immediately after injury), C (G-CSF treated; 15 g/kg i.v. for 5 days after injury). Animals were assessed with the BBB locomotor rating scale for 6 W post injury and then killed for assessment of tissue sparing around the lesion.
Result: The behavioural recovery rates of the group C was as good as group B and significantly better than that of group A. Morphological assessment showed better tissue sparing in group B and C compared to group A.
These results suggest that G-CSF is a possible neuroprotective agent in SCI.
Research funds: KAKENHI (16390427) PS1P-E064 Glutamate signals enhance the expression of RNase-L in primary cultured cortical neurons of mice Chie Sugiyama, Kiyokazu Ogita Dept. Pharmacol., Setsunan Univ., Osaka, Japan
Mitochondrial dysfunction results from a decline in the mitochondrial RNA (mtRNA) transcripts and mitochondrial enzyme activity, as well as from mitochondrial DNA (mtDNA) damage. To evaluate involvement of mtDNA expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtRNA level in cultured cortical neurons of mice. Cultured neurons (12 DIV) exposed to glutamate for 15 min at 100 M. Glutamate exposure led to a decrease in mRNA of ND1 and ND6, which are subunits of NADHubiquinone oxidoreductase, before cell death. Since mtRNAs level is regulated at least in part by RNA degradation mediated by RNase-L, we next examined the effect of glutamate on expression of RNase-L. RT-PCR analysis revealed that glutamate was effective in increasing the level of RNase-L mRNA at least 2-12 h after treatment. The increase in the expression of RNase-L was abolished by the NMDA receptor antagonist MK-801. These results suggest that the activation of NMDA receptor by glutamate reduces mtRNA level probably through enhanced expression of RNase-L in cultured cortical neurons.
Yuka Gotoh, Kiyokazu Ogita Dept. Pharmacol, Setsunan Univ, Osaka, Japan
Expression of DJ-1 is enhanced by oxidative stresses. Although exact functional significance of DJ-1 has still unknown, it is thus proposed that DJ-1 is protective against neural damage under oxidative stresses.
In this study, we tested expression of DJ-1 in the hippocampus damaged by trimethyltin (TMT) treatment in mice. TMT was systemically injected into mice to cause neural damage in the dentate gyrus selectively. Immunohistochemical analysis indicated that DJ-1 was markedly increased in the molecular layer of the dentate gyrus on days 4 and 14 post TMT injection. On day 14 post TMT injection, enhanced expression of DJ-1 was observed in the stratum lucidum of the CA3. In glutathione-depleted mice, TMT was more effective in enhancing expression of DJ-1, compared with that in untreated mice. Furthermore, double staining of DJ-1 and GFAP demonstrated that most of cells highly immunoreactive to DJ-1 were co-localized with GFAP in the dentate gyrus of TMT-treated animals, but not of untreated animals. These results suggest that DJ-1 is enhanced in the dentate astrocytes activated by TMT treatment.
Kei Higuchi, Kiyokazu Ogita Dept. Pharmacol, Setsunan Univ., Osaka, Japan
The systemic administration of trimethyltin (TMT) is known to induce granule cell death in the dentate gyrus of mice. We have previously shown that an injection of TMT (2.8 mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus 2 days later, with visually apparent recovery of the granule cell layer 14 days afterward. In this study, we examined the effects of glucocorticoids on TMT-induced damage in the dentate granule neurons of mice. TMTinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded DNA. The systemic injection of dexamethasone (0.5-5 mg/kg) led to a significant reduction in neuronal damage induced by TMT in the dentate gyrus. The neuronal damage induced by TMT at the dose of 2.0 mg/kg was enhanced by adrenalectomy. Dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. Taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against TMT-induced dentate granule cell death in mice.
Masami Ishido National Institute for Environmental Studies, Tsukuba, Japan
Melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. Furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. In this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. Neurotoxicity of maneb (1 g/ml) on the PC12 cells was elicited through apoptotic cell death. Activation of caspase-3/7 was associated with this process. A fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. However, melatonin (1 nM) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. Thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in PC12 cells.
PS1P-E068 Kinesin superfamily protein 4 (KIF4) regulates activity-dependent neuronal survival by suppressing PARP-1 enzymatic activity
Ryosuke Midorikawa, Yosuke Takei, Nobutaka Hirokawa Department of Cell Biology and Anatomy, University of Tokyo, Tokyo, Japan In brain development, apoptosis is a physiological process that controls the final numbers of neurons. Here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein 4 (KIF4), a microtubule-based molecular motor. The C-terminal domain of KIF4 is a module that suppresses the activity of poly (ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme known to maintain cell homeostasis by repairing DNA and serving as a transcriptional regulator. When neurons are stimulated by membrane depolarization, calcium signaling mediated by CaMKII induces dissociation of KIF4 from PARP-1, resulting in upregulation of PARP-1 activity, which supports neuron survival. After dissociation from PARP-1, KIF4 enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. We suggested that KIF4 controls the activity-dependent survival of postmitotic neurons by regulating PARP-1 activity in brain development.
Research funds: 1684304
PS1P-E069 Expression of HSP105, APG-1, and APG-2 in the hippocampal neural cells by trimethyltin
Masanari Orita, Kiyokazu Ogita Dept. Pharmacol, Setsunan Univ, Osaka., Japan
We tested changes in expression of high-molecular-weight heat shock proteins (HSPs) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (TMT) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. TMT (2.8 mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against HSPs, NeuN, and GFAP in coronal sections of hippocampus. TMT was effective in enhancing the expression of HSP105, APG-1, and APG-2 in the granule cell layer of the dentate gyrus, but not in CA1-CA3 pyramidal cell layer, 16 h to 2 days later. Double staining of NeuN and these HSPs revealed that these HSPs expressed by TMT almost co-localized with NeuN in granule cells of the dentate gyrus. Whereas HSP105 highly expressed in survival neurons in the culture, APG-1 and APG-2 highly expressed in damaged neurons with nuclear condensation. Taken together, the high-molecular-weight HSPs may be involved in neuronal survival and damage caused by TMT. Brain irradiation is often performed in patients with brain tumors. However, little has been known about radiosensitivity of neurons, especially in the developmental stages. In this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. Primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day 18. Thirty gray of X-irradiations were performed on the cultured cells at 7 or 21 days in vitro (DIV). Then the cells were fixed at 24 h after the irradiation with DAPI. At 7-DIV, irradiation significantly increased the number of nuclear pyknosis of neurons. In contrast, radiation did not induce any nuclear pyknosis of neurons at 21-DIV. This indicates that the radiosensitivity of 7-DIV immature neurons is higher than that of 21-DIV mature neurons. Glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. Ataxic mice Lurcher (Lc) are caused by a mutation in the ␦2 glutamate receptor (GluR␦2), which shows constitutive channel activities in Purkinje cells and leads to the cell death. Thus, Lc is the first example of neurodegeneration caused by chronic excitotoxicty. Interestingly, GluR␦2 is also suggested to regulate autophagy via its association with Beclin. However, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. Here, using heterologous cells in vitro, we show that continuous influx of Na + , but not Ca 2+ , was necessary and sufficient to induce autophagic cell death.
In addition, we found that intracellular ATP levels and subsequent activation of MAP kinase are involved in this process.
Junko Taniguchi A major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (NIIs) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. But, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. Here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the NF-kB-dependent transcriptional activity. Expression of expanded polyglutamine proteins increases the stability and the levels of IkB-a and its phosphorylated form. We have also found that various NF-kB subunits and IkB-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. Finally, we have shown several NF-kB-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. Molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. By microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin-1 and found a novel gene down-regulated in response to mutant ataxin-1 in cerebellar granular neurons. We named the novel gene Maxcell (mutant ataxin-affected gene in the cerebellum). Nothern blot shows that Maxcell mRNA in human brain is expressed in cerebellum and cerebral cortex. Immunohistochemistry with anti-Maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. Confocal microscopy shows that Maxell-EGFP is colocalized with ribosomal protein s6 as a ribosomal marker. We are analyzing the function of Maxcell protein that might relate to SCA1 molecular pathology.
PS1P-F080 Permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity The purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to Ca 2+ -induced permeability transition (mPT). The spinal cord is more traumatized than the brain during the extraction because it takes more time. In order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. Sensitivity to Ca 2+ -induced mPT was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mPT inhibitor, cyclosporin A (CsA). The present findings imply that the general features of mPT are similar in brain and spinal cord mitochondria and that mPT may be an important pharmacological target in disorders affecting the spinal cord. The role of cyclophosphamide monohydrate (CP), which is known as an immunosuppression drug, in the central nervous system (CNS) has not been elucidated. In the present study, we found that treatment with CP prevented the cultured cortical neurons from cell death induced by serum deprivation. Furthermore, CP exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways. Interestingly the up-regulation of Bcl-2, a survival promoting molecule was observed after CP treatment. These observations suggest that CP protects CNS neurons from neuronal damage through intracellular signaling pathways. The research of cell death mechanisms has rapidly progressed. However, cell death is an inherently difficult process to measure. To investigate the roles of cell death in vivo, we introduced SCAT3 probe. SCAT3 is an indicator protein for caspase-3 activation that uses FRET between two types of fluorescent protein, ECFP and Venus, linked by a peptide containing the caspase-3 cleavage sequence. Using this probe, we could monitor the activation of caspase-3 at the singleneuron level in culture. We will discuss about neural cell death through the detection of caspase activity.
Keiichi Seko, Koichi Kawada, Chie Sugiyama, Masanori Yoneyama, Kiyokazu Ogita Dept. Pharmacol., Setsunan Univ., Osaka, Japan
Trimethyltin chloride (TMT) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. In this study, we examined TMT-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. In vivo analysis using mice revealed that injection of TMT (2.8 mg/kg, i.p.) led to an increase in single-stranded DNA-positive cells, as well as in DNase II-positive cells, in the frontal cortex 2 days later. In cortical neurons, TMT exposure for 48 h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and LDH released. TMT exposure was effective in activating DNase II in the nucleus. In addition, caspases 3 and 8, but not caspase 9, were significantly activated by TMT treatment. Cytochrome c release was not affected by TMT treatment. The caspase inhibitor ZVAD-FMK completely prevented TMT-induced neuronal death. These results suggest that TMT-induced neuronal death is involved in caspases and DNase II activated by mitochondria-independent pathway in cortical neurons. We investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (TMT) chloride (2.5 mg/kg, i.p.) in 3-(3W) and 7-week-old (7W) ICR mice. In addition, we measured the brain tin level following TMT injection. TMT induced marked, localized cell death in granule neurons of the dentate gyrus in 3W mice. By contrast, slight, diffuse neuronal damage was found in the CA1 and CA3 subfields and dentate gyrus of 7W mice. The hippocampal putrescine level was elevated markedly in 3W mice on TMT administration, whereas a minor putrescine increase was detected in 7W mice. There was no difference in the brain tin level between these two age groups. These results revealed the age-dependent vulnerability of mice hippocampal neurons to TMT administration, and suggest that massive activation of polyamine metabolism is associated with TMT-induced neurodegeneration.
Withdrawn PS1P-F086 Establishment of memory guided actions of taking food with tweezers in monkeys Naoki Hirai 1 , Toshinori Hongo 2 , Kimisato Naito 2 , Shigeto Sasaki 2 1 Department of Physiology, Kyorin University, School of Medicine, Tokyo, Japan; 2 Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan
Monkeys learned a task of taking food with tweezers (Twz) under the visual guidance. The task consisted of sequential actions of looking at Twz, grasping it by hand simultaneously shifting gaze to food, bringing Twz to food, and picking it up with Twz. Brief interruption of vision for 0.3-1.0 s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. This contrasted with the task of taking directly with hand, which was done without vision. With repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. They directed their gaze to invisible food in advance, and when vision of 0.1 s became available, they grasped Twz, brought the Twz to memorized location of food and grasped the food without vision. These results show that they acquired food taking actions using Twz based on memory and somatosensory cues, the latter allowing monkeys to use Twz as an extension of the hand.
Masahiko Nishimura, Yoshihiko Yoshii University of Ryukyus, Okinawa, Japan
We have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. Lt. parietal lobe and IFG are commonly recognized tools-semantic neuro-system. However, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. We examined an fMRI to evaluate the brain activation of tools-manipulation in 17 volunteers. Experiment was performed by three tasks, control task is a forearm rotation, Task1 is simulation of tools-manipulation, Task2 is execution of tools-manipulation. We found different brain regions by this experiment. Task2 To investigate the role of synchronous firing in the prefrontal cortex (PFC), we performed cross-corelational analysis of the PFC neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. First, we analyzed synchrony among PFC neurons during the execution period in comparison with that during the preparatory period. We found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. Next, we examined relationship between neuronal synchrony and task-related activities. We found that the relationship between neuronal synchrony and response selectivity of PFC neurons was more distinct during the preparatory period than during the execution period. We would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. The functional significance of primate medial prefrontal cortex in the selection of action has been unclear. We studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. The location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. We found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-SMA. The activity of neurons in this newly identified area was dependent on conflict. Intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the SEF. We found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand.
Masaki Maruyama, Peter Fenwick, Andreas A. Ioannides Laboratory for Human Brain Dynamics, RIKEN-Brain Science Institute, Saitama, Japan
We used infrared corneal reflection, sampling at 1 KHz, to record simultaneously and independently the 12 • horizontal saccades of each eye for 12 subjects. Two paradigms were used, in Go-Only sessions saccade direction with the cue to move, and in Go/No Go sessions, saccade execution, direction and move. Mutual information (MI) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. MI showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. Surprisingly leftward movement latency was longer to peak-velocity and showed more complex MI interactions. Comparing Go-Only to Go/No Go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. A recent MEG study using this protocol, found just before and during the saccade the additional Go/No Go difficulty led to more interaction between left and right brainstem and cerebellum. These could be related to eye velocity changes with the higher cognitive loading. Wriggle Mouse Sagami (WMS) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. By extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. In the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. On the other hand, the discharge rate of cerebellar Purkinje cells in WMS was markedly decreased. These results suggest that the decreased activity of Purkinje cells may be responsible for movement disorders in WMS.
Hiromi Hirata Division of Biological Science, Nagoya University, Nagoya, Japan
Wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at 1 day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with GlyR antibody failed to show clusters in beo neurons. These data suggest that clustering defect of GlyRs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. Indeed, mutations in the glycine receptor beta subunit were identified in beo. This is the first direct demonstration that GlyR is essential for physiologically relevant clustering of GlyRs in vivo. Since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. Medium-sized spiny projection neurons in the striatum receive inputs from GABAergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. In the present study, the effect of GABAergic modulation on striatal projection neuron activity was investigated by infusion of the GABA A receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. The gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. These results suggest that local GABAergic input may play an important role in fine tuning of striatal projection neuron activity.
Research funds: KAKENHI (17022042) PS1P-F094 Dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs Katsunori Kitano, Fumito Kosuga Department of Human and Computer Intelligence, Ritsumeikan University, Japan
The subthalamic nucleus (STN) and the external segment of global pallidus (GPe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. The evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as Parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. Among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of STN neurons is likely to be one of the most crucial origins. To clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. We apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. In particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. During phasic voluntary movement, electromagnetic oscillatory activities of ∼20 Hz around the central sulcus show decrement and increment (ERD/ERS, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. We investigated the correlation between personality and these oscillatory changes. From 35 healthy subjects, high and low scorers (n = 12 each) of novelty seeking dimension on the psychometry were selected. Magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band (18-24 Hz). High NS group showed less amount of ERD in the left hemisphere, smaller magnitude, larger latency of ERS in the right hemisphere and smaller amount of baseline activity in both hemispheres than low NS group. It was suggested that individuals with high NS trait may have less inhibition after the movement and higher readiness during resting state. Despite the emerging methodology of combined fMRI and TMS, the quantitative relationship between TMS intensity and BOLD signals is poorly understood. Eight healthy subjects were scanned on a 3-T scanner, with an MRI-compatible figure-of-eight TMS coil attached for eliciting right hand movement. BOLD measurement was performed with the stepping stone sequence (TR = 2.7 s) with online monitoring of MEPs. The intensity of TMS pulses was varied from 30% to 95% at a 5% step (frequency at ∼0.15 Hz). BOLD signal changes were assessed in the primary motor cortex. A sharp increase in BOLD signals was observed above 80% stimulation. BOLD signals were weakly but significantly correlated with TMS intensity adjusted by the resting motor threshold (R = 0.46, P = 0.001). This finding gives a theoretical background for the application of fMRI with TMS to cognitive brain regions.
PS1P-F097 Shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: An NIRS study
Kotaro Takeda 1,2 , Yukihiro Gomi 1 , Itsuki Imai 3 , Nobuaki Shimoda 1,2 , Hiroyuki Kato 1,2 1 International University of Health and Welfare, Ohtawara, Japan; 2 CREST, JST, Kawaguchi, Japan; 3 Nasu Neurosurgical Center, Nasushiobara, Japan
We investigated the cerebral hemoglobin (Hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (NIRS). Fortyfour channels (22 channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral Hb changes were observed during four to six cycles of 15 s task and 30 s resting periods while sitting on a chair. The amounts of Oxy-Hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. This result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex.
Research funds: CREST, JST
Todd Pataky 1,2 , Rieko Osu 2 , Hiroshi Imamizu 2 , Mitsuo Kawato 1,2 1 Computational Brain Project, ICORP, JST, Japan; 2 ATR CNS Laboratories, Japan
Movement direction encoding in primate single cortical cells has been widely documented. This study was designed to test whether this directional tuning is observable at the voxel level in human fMRI. Three subjects performed 1 Hz isometric force pulses to seven targets separated by 30 • in the shoulder/elbow flexion-extension plane. While in MRI, online force feedback was provided by a 2D strain gauge. Twenty repetitions of each condition were performed in 12 s blocks (TR = 2 s). All subjects showed broad activation over the contralateral motor area, and from functional ROIs an average of 661 voxel time series were extracted. Many voxels exhibited continuous cosine-like tuning with movement direction. Decoding using linear SVM revealed that while correct classification rate was only 31.3% (chance: 14.3%), errors were distributed normally about the target such that 80.4% (chance: 59.2%) of the data was classified correctly to within 60 • . These data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. The behavioral thermoregulation is important for living in various temperature environments. However, the neural mechanism of behavioral thermoregulation is poorly understood. In this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. We investigated whether zebrafish perform behavioral thermoregulation against heating. When water temperature was changed from 28 • C, fish showed repetition of short time swimming in the range 32.5-40 • C. The frequency of the heat induced escape behavior was increased with temperature dependant. These results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. The heat induced escape behavior was observed stably in 3-5 days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in 3 days. In conclusion, we established an effective new model to analyze behavioral thermoregulation.
PS1P-F100 To learn with one limb or two: Limited transfer between unimanual and bimanual skills Recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. Here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. First, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. Second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. Rather, some learning remained intact (but invisible) until the original context was again performed. Lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. These results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning.
Research funds: JSPS and NSERC
Rieko Osu 1 , Ken-ich Morishige 2 , Jun Nakanishi 1,3 , Hiroyuki Miyamoto 2 , Mitsuo Kawato 1 1 ATR Computational Neuroscience Labs, Kyoto, Japan; 2 Kyushu Institute of Technology, Japan; 3 JST-ICORP, Japan
Human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. Recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. It, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. On the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. Considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. Here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. The purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (PM) and primary motor area (MI). The activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. Electromyograms (EMGs) and kinematics were also measured. We reconstructed the EMGs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed EMGs with an artificial neural network model and proportional derivative controller. As a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. The advantage of our BMI system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. We have recently reported essential role of the tongue in breastfeeding in the hypoglossal (XII) nerve-injured newborn rats. Of particular interest were the findings that the rates of the amounts of milk intake in the unilateral XII nerve-injured P1 pups of the surviving cases increased greatly between P4 (30% of the control value) and P7 (52%), suggesting adaptive tongue movement during development. This study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. After resection of the ipsilateral XII nerve on P1, DiI, a postmortem neuronal tracer, was applied to the contralateral uninjured XII nerve of P4 and P7 pups. DiI-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the XII nerve-injured side with gradual increase from P4 to P7. We show evidence for functional neural plasticity that allows effective suckling in the XII nerve-injured newborns with suckling disturbance. Previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (ARR) could be evoked purposefully (180 • turn around the body axis) in decerebrated ones. Thus, the basal ganglia might send interference signals to the ARR center via the midbrain tegmentum. To clarify its functional roles in ARR control, we examined ARR movements in rats with the midbrain lesioned. Wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. ARR movements were examined post-operative 1, 4, and 7 days. In rats with the superior colliculus lesioned extensively on both sides, ARR onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. Furthermore, coordination between the body and tail rotations was lost in many cases. The ablated region may relay cortical signals that give a top priority to the ARR center.
PS1P-G110 Role of plateau potentials in feeding system of Aplysia kurodai
Aiko Kinugawa 1 , Tatsumi Nagahama 2 1 Dept. of Life Sci., Grad. Sch. of Sci. & Technol., Kobe Univ., Kobe, Japan; 2 Fac. Phar. Sci., Toho Univ., Funabashi, Japan
Rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (CPG). In the feeding system of Aplysia kurodai, the LE neuron we identified produces the long-lasting plateau potentials and may be a CPG element. During the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the LE. In this study we found that the LE plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. And various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the LE can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. The LE may be an important CPG element to determine the size and duration of the basic depolarization of many buccal neurons.
Withdrawn PS1P-G112 Neural organizations for vocal control in a social rodent, the deguNeural organizations for vocal control in a social rodent, the degu Naoko Tokimoto 1 , Sayaka Hihara 1 , Kazuo Okanoya 2 , Atsushi Iriki 1 1 Lab for Symbolic Cognitive Development, BSI, RIKEN, Japan; 2 Lab for Biolinguisutics, BSI, RIKEN, Japan
Vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the PAG. On the other hand, a few animals with the Cortico-Medullary Projection Path can learn a new sound. In this research, we investigated about vocal control in PAG of social rodent, the Degu (Octodon degu). It is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. We verified the hypothesis by electrical stimulation of the PAG that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. Guinea pig is near relation with Degu. As a result, in guinea pigs, each sound is controlled in different area of the PAG. In degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. The sound with the time-series specific to the spontaneous vocalization of degu was not emitted. The effects of a heat-and steam-generating sheet (HSG sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, The HSG sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about 38.5 • C. Application of the HSG sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. As for changes in R-R, the HF increased after application, suggesting that the parasympathetic nerve system had become dominant. Bowel movement assessed by electrogastrography increased in amplitude. Based on the above findings, we concluded that the application of an HSG sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility.
PS1P-G115 Prostaglandin E 2 -induced thermogenesis involves a GABA-receptive mechanism in the preoptic area Toshimasa Osaka National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku,162-8636, Japan
Unilateral microinjection of PGE 2 into the region around the rostroventral wall of the third ventricle (AV3V) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. The magnitude of these responses increased dose-dependently in a range of 20-1000 pg, except for the vasoconstrictive response. Next, the effects of pretreatment with a GABA A receptor antagonist, bicuculline methiodide (0.5 mM, 100 nl), microinjected into the preoptic area (POA) ipsilateral or contralateral to the PGE 2 injection site was examined. This treatment alone had no effect on the O 2 consumption rate and temperatures of colon and skin but elicited a bradycardic response. However, all PGE 2 -induced responses were blocked 10 min after the pretreatment with bicuculline, and recovered at ∼70 min. Pretreatment with vehicle saline had no effect on the PGE 2 -induced responses. These results suggest that the GABA-receptive mechanism in the POA is required for the PGE 2 -induced thermogenesis.
Tetsufumi Ito 1 , Hiroyuki Hioki 2 , Kouichi Nakamura 2 , Takeshi Kaneko 2 , Yoshiaki Nojyo 1 1 Dept. Anat., Univ. of Fukui, Fukui, Japan; 2 Dept. of Morphological Brain Sci., Kyoto Univ., Kyoto, Japan
Although GABA-immunoreactive (ir) fibers in the rat superior cervical ganglion (SCG) were thought to originate in small cells located in the cervical sympathetic trunk (CST), almost all GABA-ir axon terminals showed markers for sympathetic preganglionic neurons (SPNs) in our recent report. In this study, we performed series of experiments to confirm the origin of GABA-containing fibers. GAD67-ir fibers were not found in dorsal roots (DRs), but in ventral roots (VRs), stellate ganglion, CST, and SCG. GAD67-positive somata were not found in DR ganglia and CST, but in intermediolateral (IML) nucleus of thoracic spinal cord (TSC). After intraperitoneal injection of fluorogold (FG), to label the entire SPNs, some FG-ir neurons were also positive for GAD67. We injected Sindbis virus, an anterograde tracer, in IML, and some labeled terminals in SCG showed GAD67-ir. After cutting T1-T4 VRs and DRs, almost all GAD67-ir fibers were abolished in SCG.
These results indicate that GABA-containing fibers in SCG originate from SPNs in IML of TSC.
Masato Nagahama 1 , Ning Ma 1 , Reiji Semba 2 , Satoru Naruse 3 1 Dept. of Anat. II, Mie Univ. School of Med., Tsu, Japan; 2 Inst. for Developmental Research, Kasugai, Japan; 3 Dept. of Int. Med., Nagoya Univ. Graduate School of Med., Nagoya, Japan Aquaporin 1 (AQP1) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. In the present study, we have demonstrated the presence of AQP1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ENS) of the rat ileum. AQP1-immunoreactive (IR) neurons simultaneously expressed a neuronal marker HuC/D. Moderate numbers of AQP1-IR neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. AQP1-IR neurons can be classified as Dogiel type I cells, which have several short processes and a single long process. Many AQP1-IR fibers were found both in the myenteric and submucosal plexi. Many AQP1-IR varicose fibers were closely associated with neuronal somata in the ganglia, whereas other AQP1-IR fibers penetrated into the muscle layers. These results suggest that AQP1-IR neurons probably play a significant role within the ENS to control gut functions.
Research funds: KAKENHI (13137204) PS1P-G118 Abdominal expiratory nerve activity in the decerebrate neonatal rat
Center for Medical Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan
The abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days 1-4. The increase in the volume and frequency setting of the artificial ventilator (FIO2 = 50%, FICO2 = 0%) failed to make the rat apnea. Under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. Although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. When FICO2 was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. In many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. The preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. Since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. We investigated the role of GABAergic neurons in the rostral ventrolateral medulla (RVM) in central respiratory control. We used GAD67-GFP knock-in mice in which we could identify GABAergic (i.e., GFP-positive) neurons in a living condition. We recorded GABAergic neuron activities (n = 40) in medullary transverse slices. About 20% of GABAergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. About 50% of GABAergic neurons recorded in the superficial RVM were CO 2 inhibitory, and all of the remaining neurons were CO 2 insensitive. We suggest that GABAergic inhibition in the RVM respiratory neuron network is mediated mainly by inspiratory neurons. GABAergic neurons are also involved in central chemosensitivity. We investigated two groups of people with a different initial level of an emotional tension before Intellectual Loading (IL). First group had the initially increased emotional level, Stressed Group (SG), and the second group had not, Calm Group (CG). Reaction time (RT) of simple visual sensorymotor reaction and asymmetry of Skin Potential Level (SPLa) in two facial zones: forehead and nasal were measured. From Research groups, subgroups with 0 and 10 mV SPLa were extracted. Thus the distinction in the greater RT gain after IL in subgroups with 10 mV forehead SPLa, in comparison to subgroups 0 mV forehead SPLa. Such law was common for both for SG and for CG. In two subgroups of 0 and 5 mV nasal zone SPLa in SG the greater RT gain after IL was in 0 mV nasal SPLa subgroup, and for CG in 5 mV subgroup. It was shown, that individual features of IL performance are related to SPL lateralization. But the low of the relationships of SPL asymmetry in nasal zones depends on the level of emotional tension of investigated groups.
Mitsuko Kanamaru, Ikuo Homma Department of Physiology, Showa University School of Medicine, Tokyo, Japan
We have reported that serotonin (5HT) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via 5HT2 receptors. Peripheral administration of p-chlorophenylalanine (PCPA) reduces the whole brain 5HT level. The present study examined whether peripheral PCPA pretreatment affects hypercapnic ventilatory responses in mice. Adult male mice (C57BL/6N) were pretreated with PCPA (0.1 g/10 ml/kg body weight) or saline intraperitoneally for consecutive 4 days. On the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. One hundred percent O 2 inhalation was changed to stepwise 5, 7 and 9% CO 2 in O 2 inhalation every 5 min. Hypercapniainduced increases in tidal volume and minute ventilation during 9% CO 2 inhalation were reduced by PCPA pretreatment; these results suggest that 5HT may increase tidal volume in hypercapnic ventilation.
Saori Nishijima, Kimio Sugaya, Minoru Miyazato Division of Urology, Department of Organ-oriented Medicine, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
We investigated the effect of Gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. Forty-two female rats were divided into a control diet group and a 1.08% Gosha-jinki-gan diet group. After 4 weeks, continuous cystometry with physiological saline or 0.1% acetic acid solution and biochemical analysis were done. The amplitude of bladder contraction with physiological saline was lower in the Gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the Gosha-jinki-gan diet group. When cystometry was done with 0.1% acetic acid, the interval between bladder contractions was shortened in the both groups. However, the interval and duration of bladder contractions were longer in the Gosha-jinki-gan diet group than in the control diet group. Therefore, it is suggested that Gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level.
Satoko Suzuki, Shinya Yanagita, Seiichiro Amemiya, Ichiro Kita Graduate School of Science, Tokyo Metropolitan University, Japan
We examined the effects of negative air ions (NAI) on physiological responses and neuronal activity with c-Fos immunohistochemistry.
In addition, we investigated the effect of vagotomy to reveal afferent pathways of NAI stimulation. We analyzed neuronal activity of the paraventricular nucleus of hypothalamus (PVN), the locus ceruleus (LC), the nucleus ambiggus (NA), and the nucleus of solitary tract (NTS). NAI significantly decreased blood pressure, heart rate, and respiratory rate, and increased HF component which is an index of parasympathetic nervous activity. NAI decreased c-Fos expression in the PVN and LC, and enhanced in NA significantly. After vagotomy, the physiological responses and changes of c-Fos expression in PVN, LC, and NA was disappeared. Furthermore, increase of c-Fos expression in NTS induced by NAI was also disappeared. These results suggest that effect of NAI on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the PVN and LC, whereas enhancing the NA activity, and that these effects of NAI was caused through vagus nerve.
Yusuf O. Cakmak, Umit Suleyman Sehirli School of Medicine, University of Marmara, Turkey
Previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. We challenged this consensus by using neuronal tracer Fluorogold in rats. Fluorogold dye solutions was injected unilaterally under the capsule of rat testis. Rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. Brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. In the main group the fluoroscent-labelling were dense in area postrema. Dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. These preliminary data provide an evidence of testicular innervation by vagus nerve. Taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. The results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia.
Research funds: Scientific Researches Comittee of Medical School of Marmara University PS1P-G127 Differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats
Yoshimi Tahara, Misa Yoshimoto, Keiko Nagata, Kenju Miki Integrative Physiol. Grad. Sch. Humanities and Sci. Nara-Women's Univ., Nara, Japan
The present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. Wistar male rats were instrumented with electrodes for measurements of renal (RSNA) and lumbar (LSNA) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. Rats were exposed to 90 dB white-noise for 10 min. 90 dB noise exposure resulted in an immediate and significant increase in RSNA while LSNA did not change significantly during the exposure in sham-operated (SO) rats. There was a significant difference in the response between RSNA and LSNA during the 90 dB noise exposure in SO rats. Sinoaortic denervation attenuated the magnitude of the increase in RSNA while it had no influence on the changes in LSNA observed in SO rats. These data suggest that arterial baroreceptor significantly contribute to the differential control of RSNA and LSNA during freezing behaviour in conscious rats. Here, we first demonstrated that in both the Kolliker-Fuse nucleus (KF) and the rostral ventral respiratory group (rVRG) region, phrenic nucleus (PhN)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlNST) and that the vlNST axon terminals made synaptic contacts with somata and dendrites of the PhNprojecting neurons, using a combined anterograde and retrograde tracing technique. Secondly, we indicated that some of the vlNST neurons innervate both the KF and the rVRG by way of axon collaterals, using the double-labeling method. Using retrograde tracing combined with in situ hybridization for mRNA encoding glutamic acid decarboxylase 67 (GAD67), we finally showed that most of the KF/rVRG-projecting vlNST neurons expressed GAD67 mRNA. These results suggest that vlNST neurons may exert inhibitory influences upon the PhN-projecting KF/rVRG neurons for inspiratory control. We have examined whether the neurons of the DMV have direct synaptic contacts on the myenteric ganglia using WGA-HRP. The myenteric ganglia of the stomach were composed of four types of neurons. The average numbers of axosomatic terminals per profile were 2.0 on the small neurons, 3.1 on the medium-sized neurons, 1.2 on the large neurons, and 4.2 on the elongated neuron. Most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. About 80% of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. When WGA-HRP was injected into the DMV, many anterogradely labeled terminals were found around the myenteric neurons. The labeled terminals were large (3.16 m), and contacted exclusively the somata. Most of them contained round vesicles and formed asymmetric synaptic contacts. Serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the DMV.
PS1P-H131 Neuronal mechanisms of respiratory rhythm modulation induced by external K + concentration change in the newborn rat brainstem-spinal cord preparation
Hiroshi Onimaru, Ikuo Homma Dept. Physiol., Showa Univ. School of Med., Tokyo, Japan
It has been suggested that two distinct rhythm generators (pFRG-Pre-I and pre-Bötzinger Insp) for respiration in the medulla possess different sensitivity to various neuromodulators. We hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. To verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [K + ] change. We recorded membrane potential of Pre-I neurons, C4 nerve and facial nerve activities. Addition of 4 mM K + to the standard superfusate decreased burst rate of C4 activity. Addition of 6 or 8 mM K + caused initial inhibition of C4 burst and subsequent high frequency C4 burst. The facial nerve burst was depressed. Pre-I neuron was depolarized strongly by application of high K + , and the burst activity was disturbed and action potentials were inactivated. Results suggest that pFRG-Pre-I or pre-Bötzinger Insp rhythm generator is dominant in low or high back ground stimulation level, respectively.
Research funds: KAKENHI (16500208) PS1P-H132 Regulation of synaptic transmission in the reticular formation of medulla oblongata by substance P We have examined the response of neurons in the reticular formation near the nucleus ambiguous (NA) to the administration of substance P (SP). Whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. Bath application of SP (1 M) increased or decreased the frequency of spontaneous activities. Several neurons were clamped at −60 mV and recorded EPSCs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. In several neurons, evoked inward EPSCs were augmented by SP perfusion. I-V curve suggested that voltage dependent current was both augmented and not changed by SP. Our previous studies have shown that administration of neurokinin 1 receptor (NK1R) antagonist near the NA inhibited gastric and respiratory movement in anesthetized rat. These results indicated that SP affect to both post and presynaptic NK1R and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions.
PS1P-H133 Effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room To investigate effects of local warming of the body on physiological functions as well as subjective feeling, EEGs, ECG, respirometer, BIS (Bispectrum) index, blood pressure (BP), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for 1 h in the cold room. In the control experiment without the heat pack, LF/HF of HR variability (LF/HF-HR) and LF of BP variability (LF-BP) increased, while HF of HR variability (HF-HR) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. In the warming experiment with the heat pack, an increase in LF/HF-HR and/or LF-BP was suppressed and HF-HR increased. We will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, EEG and BIS data.
Junichi Arai, Yasuhisa Endo, Ryouichi Yoshimura, Huan Wang Kyoto Institute of Technology, Japan
In the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. We conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and MIN-6 cells (a cell line of pancreatic B cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. These effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. We discuss the possible mechanism of their intracellular signal transduction.
Research funds: KAKENHI To study the correlation between the trans-cranial oxy-and deoxyhemoglobin (Hb) dynamics and sBP, we measured Hb dynamics (f-NIRS ® , OMM-3000, SHIMADZU Corp. Japan) over the frontal area and sBP (Finapres ® , BP monitor, Ohmeda 2300, USA) at the right middle finger from 12 volunteers (22.8 ± 2.1 years). Mild thermal stimuli (17 ± 1 • or 40 ± 1 • ) were administered every 2 min alternatively to the left hand. Some area showed positive correlation between the oxy-Hb and sBP, the other showed negative correlation between them. Hb dynamics over the frontal area have any correlation to sBP to some extent. So, trans-cranial NIRS should be discussed carefully for neural activation.
We thank SHIMADZU Corp. for the use of OMM-3000. We examined the effects of color environments on cognitive function in 12 healthy subjects and 12 patients after traumatic head injury using P300 components and LORETA analyses. The examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. The P300 latency in the red environment was significantly shorter in controls than in patients. The P300 amplitude in the red environment was significantly larger in controls than in patients. LORETA analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and Brodmann area 10 in the red environment were significantly higher in controls than in patients.
Hironori Nakatani, Cees van Leeuwen RIKEN Brain Science Institute, Saitama, Japan
Some figures, such as Rubin's vase/face and the Necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. When an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. As this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. We analyzed eye-movement and EEG during perceptual switching in the Necker cube. Blink probability showed a peak about 800 ms before the button press responses. We found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of EEG. Our results indicate that some, but not all, blinks play an active role in perceptual switching processes.
PS1P-H139 Neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain Tsuneyuki Kobayashi 1,2 , Mikinobu Takeuchi 1,2 , Takahiro Omote 3 , Naoyuki Yosimura 3 , Etsuro Hori 1,2 , Kazuo Sasaki 3 , Taketoshi Ono 2,4 , Hisao NIshijo 1,2 1 System Emotional Science, Univ. Toyama, Toyama, Japan; 2 CREST, Japan Science and Technology Agency, Japan; 3 Bio-information Engineering, Univ. Toyama, Toyama, Japan; 4 Molcul. & Integ. Emotional Neurosci., Univ. Toyama, Toyama, Japan
Neural basis of social cognition was investigated by functional near infrared spectroscopy (fNIRS) and electroencephalograms (EEGs). A head cap for recording fNIRS and EEGs was set on heads of subjects. The probes of the fNIRS imaging systems (103 channels) and/or electrodes of the EEG system were attached on the heads of the subjects. The subjects were required to perform social cognition tasks to discriminate (1) human facial stimuli with different gaze directions and (2) simple animation videos representing social interaction. Whole brain hemodynamic images were superimposed on the 3D reconstructed MRI images of the brains. Now we are analyzing hemodynamic images and EEG data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition.
Hiroshige Takeichi 1 , Sachiko Koyama 2 , Ayumu Matani 3 , Andrzej Cichocki 1 1 RIKEN, Wako, Japan; 2 Hokkaido University, Sapporo, Japan; 3 University of Tokyo, Kashiwa, Japan
To evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (EEG) were recorded from five Japanese adults, while they were listening to fifty-second spoken sentences. Natural Japanese (native) and Spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at 40 Hz, and played twice: forward and backward. Evoked responses to the modulation were analyzed as follows: (1) Circular cross correlation functions were calculated between the EEG data and the m-sequence for each subject.
(2) The functions were averaged across subjects.
(3) Independent component analysis (ICA) was applied to the averaged functions and independent EEG components were estimated for each stimulus for each subject. (4) Phase-locked component responses to the modulation were inspected. As a result, two components showed differential responses to the comprehensible forward Japanese and the other incomprehensible stimuli.
Research funds: JST and KAKENHI (17022004) Perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. In this study, we carried on MEG recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. We investigated power and phase synchronization analyses of MEG signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. As results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. These results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry.
PS1P-H142 The reflection of category perception of sound in the auditory evoked N1m Magnetic responses to periodic complex sounds with equivalent acoustic parameters except for 2 different fundamental frequencies (F0) and 12 different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. Responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. N1m source strength for nonlinear sounds was significantly larger than that for linear sounds. N1m peak latency only for vocal sounds was not affected by F0. These results suggest that perceptual categorization was reflected in N1m source strength and location (linear or nonlinear), and in N1m latency (vocal or nonvocal).
Sunao Iwaki 1 , Hiroko Kou 1 , Kouichi Sutani 1 , Mitsuo Tonoike 2 1 National Institute of Advanced Industrial Science and Technology, Osaka, Japan; 2 Chiba Univ., Chiba, Japan
Interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using MEG and the causal modeling. MEG signals were measured during subjects performing a visual infrequent target detection task. Distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (SEM) was then used to compare two possible causal models underlying the generation of major event-related components, namely P3, related to the target detection. We used Akaike information criteria (AIC) and goodness-of-fit index (GFI) as measures of the goodness of the models. The results of the comparison of two possible SEM models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of P3 components, suggested the involvement of frontal and anterior cingulate cortex in the early P3 component (P3a) and the contribution of the parietal and temporal regions to the later component (P3b In our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (L1 and L2, respectively). We compared the brain activity of 11 Chinese learners of Japanese as L2 with that of 28 Japanese natives studied in our previous study. We obtained written informed consent from each subjects. In data analysis, we used SPM2. While natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. These results indicate that there are distinct neural substrates for word recognition of L1 and L2. Neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (MEG) and functional magnetic resonance imaging (fMRI) on ten right-handed subjects, and their time courses were analyzed with an fMRI-constrained MEG-multi-dipole method. The average activations rose at latencies around 100 ms in the occipital gyrus or cuneus (OG/Cuneus) and ventral occipito-temporal areas (vOT), and at latencies around 200 ms in the posterior superior temporal and inferior parietal areas (pST/iPL), anterior temporal area (aT), and posterior inferior frontal gyrus (pIFG). The differences in activation between tasks are considered to reflect visual-form process in the OG/Cuneus and R.vOT, phonological process in the L.pST/iPL and L.pIFG, and semantic process in the L.aT. The decay of activation for these areas was found to be well fitted to exponential functions with time constants around 500 ms. The effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a 1.5 T fMRI. Healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. After habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. The two blocks were repeated alternately for 6 times. Comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. The results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. The issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. The present fMRI study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. Fifteen native Japanese volunteers participated. Stimuli were pairs of definitions and their target words, including correct words and foils. The subjects were asked to decide the correct target words of definitions. The results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. Orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. These findings reflected the roles of both orthography and phonology in kanji reading. Moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. This might suggest the role of this area in character-to-sound conversion of kanji words.
Chieko Takamiya 1 , Mie Matsui 2,3 , Tsuneyuki Kobayashi 2,4 , Hisao Nishijo 2,4 , Michio Suzuki 2,5 , Yasuhiro Kawasaki 2,5 , Masayoshi Kurachi 2,5 , Jun Nakazawa 6 , Kyo Noguchi 7 , Hikaru Seto 7 1 Neuropsychiatry, Univ. Toyama, Toyama, Japan; 2 CREST, Japan Science and Technology Corporation, Japan; 3 Psychology, Univ. Toyama, Toyama, Japan; 4 System Emotional Science, Univ. Toyama, Toyama, Japan; 5 Neuropsychiatry, Univ. Toyama, Toyama, Japan; 6 Developmental Psychology, Univ. Chiba, Chiba, Japan; 7 Radiology, Univ. Toyama, Toyama, Japan
An individual has a theory of mind (ToM) if he imputes mental states to himself and others. This ability is necessary for our well-rounded social communication. We used functional magnetic resonance imaging (fMRI) in ten healthy subjects to study the neural mechanisms underlying ToM. We adopted the picture sequencing tasks which demanded inferring mental states to self and others as ToM task. As a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. These activations coverged with a part of results in previous neuro-imaging studies on ToM and social cognitive functions. Objective: The purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fMRI).
Methods: Participants underwent fMRI scanning during which they performed a task evaluating facial expression of human (happy or sad). The task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. Pictures were chosen from ATR facial expression image database.
Results: Subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression.
The present results suggest that these area may be involved in evaluation of ambiguously expressed emotions.
Motoaki Sugiura 1 , Atsushi Sekiguchi 2 , Keisuke Wakusawa 2,3 , Yuko Sassa 2,4 , Hyeonjeong Jeong 2,5 , Kaoru Horie 5,6 , Shigeru Sato 5,6 , Ryuta Kawashima 2,6 1 Miyagi University of Education, Sendai, Japan; 2 NICHe, Tohoku Univ., Japan; 3 Dep. Pediatrics, Tohoku Univ. School of Medicine, Japan; 4 RISTEX, JST, Japan; 5 GSICS, Tohoku Univ., Japan; 6 LBCRC, Tohoku Univ., Japan
Using an fMRI, we examined the cortical mechanisms for risk perception during observation of risky tool usage. Normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. After the fMRI, each subject self-evaluated the degree of risk in each picture. Main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. Significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. In this fMRI study, we identified cortical areas where activation during experience of risky situation is correlated with the Harm Avoidance (HA) scores, subscale of Temperament and Character Inventory (TCI). Forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fMRI. Each situation was arranged for subjects to gain 10, 50, 100 points or lose 100, 50, 10 points, respectively. Cortical activation induced by experience of risky situation was estimated. A significant positive correlation with the HA scores, was observed in activated areas in the right anterior insula in Risky versus Safe comparison. The results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. This finding indicates that the right insula underlies individual difference in response to risky situation.
PS1P-H152 Brain activation related to the evaluation of absolute and relative value of outcome Juri Fujiwara 1 , Masato Taira 2,3 , Toshio Iijima 1 , Ken-Ichiro Tsutsui 1 1 Div. Sys. Neurosci., Tohoku Univ. Grad. Sch. Life Sci., Sendai, Japan; 2 ARISH, Nihon University, Tokyo, Japan; 3 Appl. Sys. Neurosci., Nihon Univ. Grad. Sch. Med. Sci., Tokyo, Japan
One way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). Here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fMRI. Subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either 0 yen or a gain or loss of 200, 400, or 800 yen. In each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. We analyzed the brain activity during the presentation of each outcome. The activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively.
PS1P-I153 Neural activation during experience-based reasoning
Chisato Suzuki 1,2 , Takashi Tsukiura 1 , Hiroko Mochizuki-Kawai 1 , Yayoi Shigemune 1,2 , Toshio Iijima 2 1 Neurosci. Res. Inst., AIST, Japan; 2 Div. Systems Neurosci., Tohoku Univ., Japan
The aim of this study is to investigate neural activations when reasoning future events based on experienced events. Before fMRI, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. After encoding, subjects performed three tasks during fMRI. In the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (Memory-based Reasoning: MR), whereas in the second task, subjects recognized a last scene encoded in the complete version (Memory: M). In the third task, subjects chose a last scene appropriate to the first scene in the new comics (Reasoning: R). Activations specific to MR was found in a relatively anterior part of the left PFC and right PFC. The common activations between MR and M were identified in the right MTL, whereas a relatively posterior part of the left PFC was activated commonly between MR and R. The findings suggest that the network including bilateral PFC and right MTL may contribute to the experience-based reasoning of future events. To assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fMRI study in 16 healthy subjects who participated in the Chicken Game. Statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (PCC) and the posterior superior temporal sulcus (STS). When we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior STS showed that the counterpart had a reliable effect regardless of risky or safe decisions. In contrast, a significant opponent x selection interaction was revealed in the anterior PCC. It could be inferred that the posterior STS and the anterior PCC play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. Creativity is the ability to generate new and original ideas. The most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. We used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (OS: originality score) of the products. Using fMRI, we observed BOLD signal change during designing task in art students (trained) and non-art students (untrained). We observed clear difference between two groups; in the trained highly creative group, the OS is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance.
In the untrained group we saw no such correlation. Thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity.
PS1P-I156 The difference of brain activity elicited by different styles of art
Hiromi Yamamura 1 , Yasuyuki Kowatari 1,2 , Shigeru Yamane 2 , Miyuki Yamamoto 1,2 1 Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan; 2 System Brain Science Division, AIST, Tsukuba, Japan
Artworks are categorized according to time and place where they were produced (cultural effects). Surrealistic art is one of those categories and it gives uneasy impression to our mind. We investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fMRI). Works of several artists who are well-known as representatives of Renaissance, Impressionism and Surrealism were used as stimuli and results were analyzed by SPM2. While Renaissance arts or Impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, Surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (BA9, BA46). These results suggest that a particular style of artwork may have commonly activated brain regions.
Research funds: COE(J-3)
PS1P-I157 Effects of chewing on the activity of the prefrontal cortex in working memory processing: An fMRI study In general, it has been proposed that chewing produces holding or enhancing effect on attention. Furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. We therefore examined the influence of chewing on brain activities using fMRI. The subjects used were 20-30 aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. Gum without odors and taste component was used to remove effects other than chewing. The results indicated that chewing tended to increase the BOLD signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. This suggests the possibility that chewing may accelerate the process of working memory.
Research funds: KAKENHI 17,6577
PS1P-I158 The tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names Hirohito M. Kondo 1 , Michio Nomura 2 , Jun Kawaguchi 3 1 NTT Commun. Sci. Labs., NTT Corp., Atsugi, Japan; 2 Dept. Psychol., Tokai Women's Univ., Kakamigahara, Japan; 3 Dept. Psychol., Grad. Sch. Environ. Studies, Nagoya Univ., Nagoya, Japan
The tip-of-the-tongue (TOT) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. The TOT state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. Participants were instructed to recall the full names of celebrities when their faces were presented. Event-related fMRI analysis demonstrated that the anterior cingulate cortex (ACC), anterior insular cortex (AIC), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the TOT state with frustration. Activity of the ACC and right AIC was positively correlated with the degree of frustration in unsuccessful retrieval. ROI analysis indicated that the ACC and right AIC were sensitive to retrieval demands and awareness of frustration, respectively. We suggest that the cinguloinsular circuit regulates the self-monitoring processes during the TOT state.
Noriko Kudo 1,2,3 , Yulri Nonaka 1 , Katsumi Mizuno 4 , Kazuo Okanoya 1,5 1 RIKEN, BSI, Biolinguistics, Saitama, Japan; 2 Chiba University, Chiba, Japan; 3 JSPS, Japan; 4 Department of Pediatrics, Showa University, Tokyo, Japan; 5 PRESTO, JST, Japan
Segmentation of speech stream is a prerequisite for language acquisition. Language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. We consider that the ability for statistical learning is not specific to language, but more general cognitive competence. And we ask whether this ability could be considered as innate. In this study, we measured ERPs for 18 neonates within 3 days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. Four three-tonal-words were presented in random order without intervals during recording of the EEG. As a result, only the first tone of each word evoked a significant positive component in the frontal area. Since this potential is not evident during the first session, this is likely to be due to statistical learning. These results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. Using near-infrared spectroscopy (NIRS), changes in concentration of oxygenated hemoglobin (oxy-Hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. In this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. Subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. It was shown that the peak of averaged oxy-Hb change was higher while subjects appreciated abstract paintings. Average differentiation for each oxy-Hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. These results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. We used MEG to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. Eleven healthy subjects have participated in the study. Three conditions were considered: EASY: Add three (3) to a two-digits number without carry-over; DIFFICULT: Stimuli were the same as EASY, but with carry-over; NOCALC: Add zero to the two digits number. Probe stimuli were presented 1 s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. Root-mean-square values for 12 different MEG sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. Increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around 700-900 ms. The activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in DIFFICULT condition compared to EASY condition. We investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the Lombard effect. Ten children with autism (9:4-14:11) and 18 agematched typically developing children (9:5-15:2) were instructed to name 50 pictures of objects aloud in Control and Masking conditions. In Masking, weighted-white noise was continuously delivered through a headset. The subjects' speech responses were recorded from a microphone. In typically developed children, the enhancement (Masking/Control) in Masking was significantly greater (duration = 1.2 ± 0.2, loudness = 1.4 ± 0.3) than in the children with autism (duration = 1.1 ± 0.2, loudness = 1.2 ± 0.3) (p < 0.001). The present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. Since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (Xenopus laevis) gastrocnemius or not. Muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at 0.5/s chronologically attenuated and showed a simple fatigue curve. Direct irradiation of laser (532 nm, 180 mW) to the muscle surface (28.3 mm 2 ) significantly delayed its attenuation (p < 0.05). When the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for 2 min decreased according to the session sequence. However, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < 0.05). It is suggested that laser irradiation has a potential to more activate ATP synthesis during as well as after muscle contraction.
PS1P-I165 NEDL1, a novel E3 ubiquitin ligase for dishevelled-1, targets mutant superoxide dismutase-1 and interacts with p53
Yuanyuan Li 1,2,3 , Kou Miyazaki 1 , Toshinori Ozaki 1 , Akira Nakagawara 1 1 Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba, Japan; 2 Production Technology Development Center, The Furukawa Electric Co., LTD., Ichihara, Japan; 3 Hisamitsu Pharmaceutical Co., LTD., Tokyo, Japan
We have cloned a novel HECT-type E3 ubiquitin ligase gene termed NEDL1. Previous study has shown that NEDL1 is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. Dishevelled-1, a regulatory molecule in the Wnt signaling pathway, was identified as the physiological target of NEDL1 for uniquitination and proteasome-mediated degradation. On the other hand, NEDL1 bound and ubiquitinated mutant (but not wild-type) SOD1 in a mutant SOD1 type-dependent manner, which is proportionally related with the FALS severity. In the present study, we show that NEDL1 physically bound p53, and induced apoptosis in a p53-dpendent manner. Taken together, our results suggest that NEDL1 may play a critical role in neuronal cell death occurring in FALS through interacting with mutant SOD1 and p53. Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (AR). CHIP (carboxyl terminus of Hsc70interacting protein), U-box type E3 ubiquitin ligase, has been shown to interact with Hsp90 or Hsp70 and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. We demonstrated in a neuronal cell model that transient over-expression of CHIP reduced the monomeric mutant AR more than the wild-type, suggesting that the mutant AR is more sensitive to CHIP than is the wild-type. We also demonstrated high expression of CHIP ameliorated motor impairments in the SBMA transgenic mouse model. These findings suggest that CHIP over-expression ameliorates SBMA phenotypes in mice by reducing nuclear-localized mutant AR, which probably due to enhanced mutant AR degradation. We performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with Guillain-Barré syndrome (GBS) serum, as well as purified IgG, from selected patients with GBS. Using a whole-cell recording technique, we then investigated the effects of serum and purified IgG from patients with GBS on voltage-dependent calcium currents (VDCC) in NGF-differentiated PC12 cells and cerebellar Purkinje cells. Serum from selected patients with GBS and purified IgG from some serum of patients with GBS inhibited Ca 2+ current in both cells. These results suggest that muscle weakness in some patients with GBS might be induced by changes in P/Q-type calcium channel function within motor nerve terminals. The aim of the present study was to explore the possible role of COX-2 inhibitor, rofecoxib in pentylenetetrazol (PTZ, 40 mg/kg, i.p.)induced kindling. Rofecoxib was administered orally daily 45 min before either PTZ or vehicle. Seizure severity was measured according to a prevalidated scoring scale. Biochemical estimations were performed on the 16th day of PTZ treatment. Chronic treatment with rofecoxib (2.0 and 5.0 mg/kg, p.o.) for 15 days showed significant decrease in PTZ-induced kindling score. Chronic treatment with PTZ significantly increased lipid peroxidation, nitrite levels (NO levels), and myeloperoxidase levels and decreased the reduced glutathione (GSH) levels in brain homogenate, which was reversed with rofecoxib treatment.
Research funds: University Supportted study
Ashish Dhir, Shrinivas Kulkarni UIPS, Panjab University, Chandigarh, India
The objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (PTZ)-induced (80 mg/kg) convulsions in mice with possible mechanism of action. Various COX-inhibitors were administered 45 min prior to the PTZ administration. Onset, duration of clonic convulsions and percentage mortality/recovery were recorded. Pretreatment with COX-inhibitors aspirin (10 and 20 mg/kg, p.o.), naproxen (7 and 14 mg/kg, p.o.), nimesulide (1-5 mg/kg, p.o.) or rofecoxib (1-4 mg/kg, p.o.) dose dependently showed protection against PTZ-induced convulsions. Rofecoxib (1 mg/kg) or nimesulide (1 mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing GABAergic modulation of COX-2 inhibitors. COX-2 inhibitors also antagonized the effect of flumazenil (4 mg/kg) against PTZ-induced convulsions further confirming the GABAergic mechanism.
PS1P-J171 Cell proliferation after domoic acid-induced neuronal damage in adult rats Domoic acid (DA) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. DA-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. In this study we examined the neuronal damage after intraperitoneal DA administration and cell proliferation in the adult rat brain. The most extensive neuronal cell damage was observed in CA3 subfield as evaluated by HE staining, while TUNEL positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (DG) of the hippocampus. To elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (BrdU) labeled cells. BrdU labeled cells were detected in DG and the granular cells of cerebellum. The cell proliferation was not associated with damage.
PS1P-J172 A-type potassium channel truncation mutation in temporal lobe epilepsy The role of voltage dependent calcium channels on the pentylenetetrazol (PTZ) kindling induced learning deficits was investigated in rats.
In this study animals were divided into three groups. In the test group Verapamile were injected in the hippocampus (4 mg/4 min).
After 20 min Kindling was established in rats with PTZ. The control animals were the same age and undergone the same treatment in term of ACSF injections and post-kindling waiting time as the kindled animals. And in sham group the animals received saline. One month after induction of kindling spatial learning and memory was tested by morris water maze.
Results showed that intra-hippocampal injection of Verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact.
The results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. We showed that 8-oxoguanine (8-oxoG) in mitochondrial (mt) DNA and cellular RNA increased significantly in the CA3 subregion of the mouse hippocampus after kainate administration. Laser scanning confocal microscopy revealed that 8-oxoG accumulated greatly in mtDNA of the CA3 microglia. Wild-type and MTH1-null mice, the latter lacking an ability to hydrolyze 8-oxo-dGTP and 8-oxo-GTP to the monophosphates to avoid their misincorporation into DNA or RNA, exhibited similar degree of the CA3 neuron loss after kainate administration, however, levels of 8-oxoG accumulated in mtDNA and cellular RNA in the CA3 microglia were significantly increased in MTH1null mice in comparison to wild-type mice. We thus demonstrated that MTH1 efficiently suppresses the accumulation of 8-oxoG in both cellular DNA and RNA in the hippocampus, especially in microglia, caused by excitotoxicity.
PS1P-J176 Transcription factor Nrf2 regulates brain response to kainate-induced excitotoxicity Yukihiko Dan 1 , Kosuke Kajitani 1 , Noriko Yutsudo 1 , Ken Itoh 2 , Masayuki Yamamoto 2 , Yusaku Nakabeppu 1 1 Kyushu Univ., Med. Inst. Bioreg., Div. Neurofunc. Genomics, Japan; 2 Univ. Tsukuba, Grad. Sch. Comp. Hum. Sci., Japan NF-E2 related factor 2 (Nrf2) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (ARE), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. Since loss of Nrf2 has been reported to increase neuronal death under increased oxidative stress, Nrf2 seems to play a role for neuroprotection. Administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the CA3 subregion of hippocampus. To unveil the functional significance of Nrf2 in the brain, we compared seizure responses between wild-type and Nrf2-null mice after systemic kainate administration. We found that Nrf2-null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation.
PS1P-J177 Synaptic plasticity and 4-aminopyridineinduced epileptic discharges in rat hippocampal slices
Makoto Otani, Tetsuo Furukawa, Kiyohisa Natsume Department of Brain Science and Engineering, Kyushu Institute of Technology, Kitakyushu, Japan
Four-aminopyridine (4-AP) at the concentration below 0.1 mM suppresses K D channel and induces the epileptic discharges in rat hippocampal slices. In the present study, the involvement of the activation of NMDA receptor on the ictogenesis of the 4-AP induced discharges in CA3 region was studied. Ten M 4-AP induced the epileptic discharges with the frequency of 0.33 ± 0.04 Hz (mean ± S.E.M.; n = 3) and the amplitude of 1.23 ± 0.86 mV. When AP-5, an NMDA receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. On the other hand, when AP-5 was applied from the ictogenesis period of the discharges, the discharges did not appear. These results suggest that the NMDA receptor-dependent synaptic plasticity involves in the ictogenesis of 4-AP-induced epileptic discharges. Chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. Using primary astrocyte cultures, we observed that under thyroid hormone (TH) deficient conditions, morphine significantly decreased cell viability. Further studies showed that the loss of cell viability was due to apoptosis of the cells. The effect is attenuated by TH supplementation to the culture medium. The observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. 7NI, a specific inhibitor of nNOS, completely blocked loss of cell viability suggesting that morphine induced intracellular NO production, leads to cell death. Studies suggested that NO acts through a cGMP independent pathway. The involvement of NO induced cGMP independent pathway in morphineinduced apoptosis during TH deficiency has been investigated. Collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium.
PS2A-A001 Influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons
Takashi Tateno 1 , Hugh P.C. Robinson 2 1 Engineering Science, Osaka University, Osaka, Japan; 2 University of Cambridge, UK
In the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. A quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. Neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. We have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and Wiener-kernel estimation to calculate the average conductance input trajectory (ACIT) preceding spikes. The time course of the ACIT provides a general description of a neuron's response to dynamic conductance stimuli. Our analysis showed that the ACIT reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. Our study has investigated the effects of ALS-CSF on voltage-gated calcium P/Q-type channel (␣1A) expression in pre synaptic terminals of rat spinal motor neurons. CSF from ALS and non-ALS (neurological patients) was injected into the 3-day-old rat pup spinal subarachnoid space at the rate of 1 l/2.5 min. The rats were sacrificed 48 h after CSF injection and spinal cord sections were processed for immunocytochemistry with P/Q-type channel ␣1A antibody and also for cytochrome oxidase labeling. ALS-CSF significantly increased P/Qtype channel expression compared to CSF from Non ALS patients. ALS-CSF significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. It is probable that, toxic factors present in the ALS patients CSF might induce the expression of P/Q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons.
PS2A-A003 On the membrane potential profile of CA1 pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices
Takashi Tominaga 1,2 , Yoko Tominaga 1 1 Dept. Neurophysiol., Kagawa Sch. Pharmaceutical Sci., Tokushima-Bunri Univ., Kagawa, Japan; 2 Lab. for Dynamics of Emergent Intelligence, RIKEN BSI, Hirosawa 2-1, Wako, Saitama, Japan
Integration of membrane potential response in a single neuron is a basis of neuronal calculation. We have been aiming to visualize this with voltage sensitive dye (VSD). Hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. But, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. To solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. A steep peak in optical signal was seen along stratum pyramidale. An application of TTX diminished this peak, and made the optical signal profile flat along the cell. Thus, we concluded that the specificity of the VSD is small. With "NEURON", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated.
PS2A-A004 Overexpression of inwardly rectifying K + channel 2.1 in hippocampal slice culture
Masayoshi Okada, Hiroko Matsuda Department of Physiology, Kansai Medical University, Japan
The expressions of mRNAs for the inwardly rectifying K + channel (Kir) 2.1 have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. In our rat hippocampal slice cultures, the endogenous Kir current was hardly detected with whole cell recordings in the CA1 pyramidal neurons. Then, EGFP and Kir2.1 expressing virus vectors were constructed, and infected to the neurons in the slices. The vectors succeed to express the Kir current, and the translocation of the fusion protein to the plasma membrane was also observed. Furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that Kir plays a role of noise-filter for synaptic input in central neurons.
Takeshi Otsuka, Mieko Morishima, Yasuo Kawaguchi Div. Cerebral Circuitry & Structure, NIPS, Okazaki, Japan
Layer 5 pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. Although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. Here, we obtained whole cell recordings from rat frontal layer 5 pyramidal cells that project to the striatum (CCS) or pontine nucleus (CPn), identified by injection of fluorescent retrograde tracer to these regions. Firing properties of pyramidal cells had similarity depending on the projection regions. CCS cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. However, CPn cells exhibited very little spike frequency adaptation during current injection. We also examined synaptic inputs from layer 2/3 neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. Our results suggest that physiological properties of layer 5 pyramidal cells are correlated with their subcortical target. This study aimed to clarify expressional changes in types 1 and 2 of ryanodine receptors (RyR1 and RyR2) in the cerebellum of a Ca 2+ channel ␣ 1A subunit mutant, rolling mouse Nagoya. Semi-quantitative RT-PCR revealed altered mRNA signal levels of RyR1 but not RyR2 in the RMN cerebellum: a less RyR1 mRNA signals than in the control cerebellum. Well consistent with the semi-quantitative RT-PCR results, RyR1 immunostaining in soma and primary dendrites of Purkinje cells was less intense in RMN than in control mice. In contrast, RyR2 immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between RMN and controls. The present study suggests that somatodendritic RyR1 expression in Purkinje cells was decreased in the cerebellum of RMN. This may suffer RyR1-mediated Ca 2+ release, contributing altered Ca 2+ homeostasis in the RMN Purkinje cells.
PS2A-A007 Dopamine-based modulation of lateral amygdala neuron excitability: A possible involvement of potassium current Ryo Yamamoto, Yoshifumi Ueta, Noubuo Kato Integrative Brain Sci. Med., Kyoto Univ., Kyoto, Japan
The amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. In the present slice experiments, we investigated the effects of Dopamine (DA) on lateral amygdala (LA) neurons by whole cell recordings. Application of DA depolarized LA neurons, reduced the action potential threshold, and induced slow afterdepolarization (sADP). This sADP was induced voltage dependently, and lasted for more than 5 s. D1 receptor agonists induced the same sADP. Previous reports have repeatedly suggested that sADP is triggered by calcium influx. Consistently, calcium channel blockers or chelating intracellular calcium inhibited the present DA-induced sADP. A membrane conductance decreased at the peak of sADP current (I sADP ). Also, I sADP was suppressed by including cesium in the pipette solution. These results suggest that the present DA-induced modulation of LA neuron excitability may depend on a potassium current that can be masked by calcium influx.
Toru Aonishi 1,2 , Hiroyoshi Miyakawa 3 , Masashi Inoue 3 , Masato Okada 2,4 1 Tokyo Institute of Technology, Japan; 2 Brain Science Institute, RIKEN, Japan; 3 Tokyo University of Pharmacy and Life Science, Japan; 4 The University of Tokyo, Japan
It has been reported that amplification of AP paired with EPSP boosts the induction of LTP. There are two alternative hypotheses of such amplification mechanisms; one is activation of the Na channel and other is inactivation of the A-type K channel. Which is essential? In this talk, by mathematical analyses and the NEURON simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the A-type K channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of AP paired with EPSP. The balance of Na and A currents is essential for controlling dendritic integration manners. We also show that the down-regulation of the A-type K channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying AP mode to shunting EPSP mode.
Miharu Komai 1 , Maya Yamazaki 1,2 , Mika Tsujita 1 , Manabu Abe 1 , Rie Natsume 1,2 , Kenji Sakimura 1,2 1 Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan; 2 SORST/JST, Saitama, Japan
We previously reported that Stargazin family (␥2, ␥3, ␥4, and ␥8) not only promoted AMPA receptor surface expression but also modulated receptor activity and channel property (Yamazaki et al., 2004) . Therefore, we assumed these family proteins were auxiliary subunits of AMPA receptors. To prove this hypothesis, we generated ␥4 subunit knockout (KO) mice using the Cre/loxP recombination system and analyzed their phenotypes. The ␥4 subunit KO mice were viable, fertile, and displayed no overt phenotype. On the other hand, on Western blot analysis, protein expression levels of AMPA receptor subunits were reduced in KO mice compared with those in wild-type at postnatal day 11, while the reduction was not so significant in adult brain. These results suggested that ␥4 might regulate dynamics of AMPA receptor subunits during early development. In the CNS, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the pH environments. Ambient pH plays as a significant signal for neural functions. Microglia (brain phagocytes) express abundant voltagegated proton (Hv) channels which have extremely high selectivity for H + and potent H + efflux ability. Exposure to Na-lactate (pH 6.8) induced cell acidosis and activation of the Hv channels. The channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. Consequently, the Hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis.
Mari Sasaki, Masahiro Takagi, Yasushi Okamura Okazaki Institute for Integrative Bioscience, Aichi, Japan
Here we report a novel four transmembrane protein similar to the voltage sensor domain (VSD) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. Voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. However, the molecular basis underlying this channel has been elusive. Here we identify a novel cDNA clone named as mouse voltage-sensor domain only protein (mVSOP1). Cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. Imaging analysis demonstrated that pHin recovers rapidly after an acid load in mVSOP1-transfected cells. mVSOP1induced currents exhibited two key features of native voltage-gated proton channels: pH-dependent gating and Zn 2+ sensitivity. Neutralization of a positive charge in the S4-like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. Oscillatory extracellular electric fields have been observed in mammalian brains. The electric fields modulate neuronal excitability and synaptic events. To investigate the effect of the oscillatory electric fields on the CA1 pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (RH414). Application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the CA1 region. In the pyramidal layer, the amplitudes of the responses to the 1-Hz field were the largest. The amplitudes were decreased monotonically when the frequency of the fields became higher. However, in the stratum radiatum, the amplitudes of the responses to the 3-10-Hz fields were larger than those to the other frequencies. The frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. Acid sensing ion channels 2 (ASIC2) have proposed to constitute mechanoreceptors and nociceptors. We examined the localization and characterization of ASIC2-expressing cells in rat central nervous system (E17-P14) using immunohistochemical techniques. ASIC2positive fiber first appeared in brain stem and spinal cord at E17-18 stage. ASIC2-expresseing cells appeared in white matter of brain stem and spinal cord at E20 stage. In early postnatal stages ASIC2expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at P4 stage. These cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. These results are suggested the hypothesis that the function of ASIC2 mediate the myelin formation in the developmental stages of central and peripheral nervous system.
Masato Shino, Seiji Ozawa, Yasuhiko Saito Department of Neurophysiology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan
Nucleus prepositus hypoglossi (NPH) is involved in horizontal eye movement. Previously, we found NPH neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (FIL neurons). FIL neurons exhibited a spike train with a long first interspike interval (1st ISI) that is attributed to a large, slow hyperpolarization (AHP) after the first spike. In this study, we investigated ionic conductances underlying the long 1st ISI by whole-cell recordings in rat slices. Application of 100 M apamin, an SK-type Ca 2+ -activated K + (Kca) channel blocker, shortened the 1st ISI and decreased the amplitude of the slow AHP. The shortening of the 1st ISI was observed when membrane potentials were depolarized. Moreover, application of 3 M mibefradil, a T-type Ca 2+ channel blocker, shortened the 1st ISI. These suggest that the firing pattern of FIL neurons arises from activation of SK-type Kca channels induced by Ca 2+ influx through T-type Ca 2+ channels.
Research funds: KAKENHI (C) (17500270)
Jafar Vatanparast 1,2 , Mahyar Janahmadi 1 , Houri Sepehri 2 , Ali Haeri-Rohani 2 , Ali Reza Asgari 1 1 Neuroscience Research Center, Shaheed Beheshti Medical Sciences University, Tehran, Iran; 2 Dept. of Biology, University of Tehran, Tehran, Iran
The roles of the ionic channels and muscarinic receptors in paraoxon (PX) induced burst firing in snail neurons were studied using current clamp method. PX (0.6 M, within 5 min) increased the frequency of spikes and shortened AHP. Slow waves of depolarization with superimposed bursts were recorded within 20 min. Atropine blocked the depolarization shift but not the other effects of PX. PX was able to reversibly decrease the duration of calcium spikes elicited in a Na + free Ringer. This effect observed in the presence of atropine and was along with a decrease in the duration of Ca 2+ spike AHP and an increase in the spike frequency. The PX blockade of Ca + channels may decrease the activation of Ca 2+ dependent K + channels that underlies AHP. Blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by PX underlies the depolarization shift with associated bursts. Dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. However their distribution is still unclear. We estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal CA1 slices to perturbation, such as propagating EPSP induced by synaptic inputs and biphasic response to extracellular electric field. By numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. This implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal CA1 pyramidal neuron. It is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved.
In this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (TRPM8). Repeated menthol application induced TRPM8 desensitization. This desensitization was depended on extracellular Ca 2+ , indicating that involvement with Ca 2+ -dependent kinase. PKC activator (PMA) desensitized TRPM8 and Go6976 (PKC inhibitor) abolished PMA-induced TRPM8 desensitization. PMA similarly desensitized wild type TRPM8 and mutant TRPM8, in which serine or threonine residues in some putative PKC phosphorylation sites were replaced by alanine. PMA treatment did not induce internalization of TRPM8. As the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated TRPM8 causes PKC to desensitize TRPM8 itself.
Yosuke Sawada 1 , Hiroshi Hosokawa 1 , Kiyoshi Matsumura 2 , Shigeo Kobayashi 1 1 Dept. of Int. Sci. and Tech., Grad. Sch. of Info., Kyoto Univ., Kyoto, Japan; 2 Dept. of Info. Sci. and Tech., Osaka Institute of Technology, Osaka, Japan
Cooling below 17 • C evokes cold pain sensation. However, the molecular basis of the cold pain sensation is still unknown. TRPA1 is activated by pungent compounds stimuli. If TRPA1 responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. However, whether TRPA1 is activated by cooling or not is still controversial. Here, we investigated that TRPA1-expressing HEK293 cells responded to noxious cold stimuli. Whole-cell recording demonstrated that cooling below threshold evoked inward current. Threshold temperature was 17.5 ± 2.7 • C. In inside-out singlechannel recording, cooling activated TRPA1 directly. Single channel conductance was 74.1 ± 18.8 pS. Single channel currents showed inword rectification. In conclusion, TRPA1 is the cooling activated cation channel.
Yoshiki Matsuda, Foong Yen Ang, Jinsun Yoon, Noriko Ebisu, Satoshi Takahashi, Shinichi Kogure Dept. Bioengin., Soka University, Tokyo, Japan
Hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (HCN1-4) have been demonstrated in the CNS. Since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the PNS using the frog (Xenopus laevis) sciatic nerves. Western blot analyses for HCN1-4 demonstrated that samples from the nerve and the heart showed an HCN2 band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for HCN1, HCN3 and HCN4 could not be found in those samples. When an HCN channel blocker, ZD7288 was applied on the stimulus portion of sciatic nerve and the nerve was elicited at 0.5/s by a duration of 10 ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < 0.01). It is suggested that HCN2 channels exist in the PNS and they contribute to the burst or recurrent discharges. Ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ 1 adrenergic, N-methyl-d-aspartate, serotonin and receptors. However, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. Here, we show that ifenprodil inhibited G protein-activated inwardly rectifying K + (GIRK; Kir3) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in Xenopus oocytes. In contrast, Kir1.1 and Kir2.1 channels in other Kir channel subfamilies were insensitive to ifenprodil. The GIRK currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. The inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. Our results suggest that inhibition of GIRK channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects.
PS2A-B022 Proliferation of rat C6 glioma cells is controlled by the concentration-sensitive Na + channel (Na C ) Shigeru Yoshida, Hiroyuki Yamaguchi, Takashi Takeuchi, Hokuto Tanaka, Yoshiyuki Morimoto, Teruki Hagiwara Department of Life Science, Kinki University, Higashi-Osaka, Japan
The role of Na + as a regulator of cell growth was studied using the tumor cell line (C6), which has a large quantity of concentrationsensitive Na + channels (Na C ; c = concentration). Cell proliferation was suppressed when [Na + ] o was raised from control (140 mM) to 190 or 240 mM. An increase in [Na + ] i was revealed by an image processor in C6 cells loaded with a Na + indicator (SBFI), under high [Na + ] o conditions. [Na + ] i elevation was augmented by ouabain or by bumetanide (Na + /K + /Cl − cotransporter blocker), while it was decreased when Na C expression was inhibited by RNAi techniques. The real-time PCR method revealed that the expression level of the immediate early gene egr-1, which is involved in cell growth, was concomitantly reduced. It is to be noted that similar alterations in cell growth, egr-1 level and [Na + ] i were induced by a Na + ionophore (monensin) without raising [Na + ] o . These data indicate that Na + enters through Na C upon [Na + ] o increase, and [Na + ] i elevation itself is responsible for these phenomena.
Hiroshi Kuba, Takahiro Ishii, Harunori Ohmori Dept. Physiol., Univ. Kyoto, Kyoto, Japan Na + channels are concentrated in the axon to generate action potentials. However, little is known about how distribution of Na + channels contributes to the activity and function of single neurons. In avian nucleus laminaris (NL), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (CF) and interaural time difference (ITD) of sounds. We show here in the chick that NL neurons have distinct distribution of Na + channels along the axon and optimize the ITD sensitivity depending on their CF. Neurons of high and middle CF (higher than 1 kHz) had small action potentials, and had no Na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma (20-50 m). While, neurons of low CF generated large overshooting spikes, and Na + channels were clustered closer to the soma (5 m) in the axon. Thus, NL neurons had a spike generator on the axon, at a greater distance from the soma with the increase of CF. By computer simulation, these unique distributions of Na + channels were found essential to enhance the coincidence detection.
Research funds: KAKENHI (17021021)
Il-Sung Jang 1 , In-Sun Choi 1 , Eun-Ju Park 1 , Jin-Wha Cho 1 , Man-Gee Lee 2 , Byung-Ju Choi 1 1 Kyungpook National University, School of Dentistry, South Korea; 2 Kyungpook National University, School of Medicine, South Korea Bisphenol A (BPA), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. Here we report the effect of BPA on GABA A receptors using a conventional whole-cell patch clamp technique from acutely isolated rat CA3 pyramidal neurons. BPA itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. BPA increased postsynaptic currents induced by GABA at lower concentrations (<10 M), but decreased those induced by GABA at higher concentrations (>100 M). In addition, BPA decreased both the current amplitude and decay time constant of GABAergic mIPSCs. Finally, mechanisms underlying BPA-induced modulation of GABA A receptors will be discussed. We recently generated Nav1.1-deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. We have now used these Nav1.1-deficient mice as negative controls to examine Nav1.1 distribution in the mouse brain using RNA in situ hybridization histochemistry and immunohistochemistry. At low magnification, Nav1.1 expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. Contrary to previous studies indicating a somato-dendritic Nav1.1 distribution, in the present study, higher magnification analysis revealed that Nav1.1 is predominantly distributed to axons in some brain parts. This apparent discrepancy may reflect the lack of specificity of anti-Nav1.1 antibodies used in these previous studies, none of which utilized Nav1.1-deficient mice. Based on our findings, we propose that Nav1.1 might be involved in propagating action potential to presynapses.
Keiji Miura 1,2 , Masato Okada 2,3,4 , Shun-ichi Amari 4 1 Department of Physics, Kyoto University, Kyoto, Japan; 2 "Intelligent Cooperation and Control", PRESTO, JST, Japan; 3 Department of Complexity Science and Engineering, University of Tokyo, Chiba, Japan; 4 Brain Science Institute, RIKEN, Saitama, Japan
We considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. A gamma distribution is a natural extension of Poisson process and it can generate spike trains with various irregularities. The model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. Because the environment changes over time, the firing rate varies for each interspike interval. We used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. Our estimator is simple and easily applicable to experimental data. The estimator is useful for characterizing spiking irregularity which varies among neuron types. It may be possible to classify neurons into functional groups according to their spiking irregularities.
Research funds: Grant-in-Aid for Scientific Research (Nos. 14084212 and 16500093)
Mitsuyo Watanabe, Yuko Ishimaru, Taketo Nakadai, Tomoyuki Kanamatsu Graduate School of Bioengineering, Soka University, Tokyo, Japan
We examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. The rats were injected with [1-13 C] glucose intravenously (1 g/kg) 24 or 48 h after the intraventricular injection of colchicine (75 g/10 l) and the amino acid fractions were extracted from the brains at 10, 20 or 40 min after the glucose injection. The amount of [1-13 C] glucose in the cerebra was increased, however, the 13 C incorporation into glutamine, glutamate, GABA and Aspartate from [1-13 C] glucose were decreased. Only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. The microdialysis analysis showed that the amount of Gln in the dialysate was increased by three times in the colchicine group compared with the control group. These data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. These results indicate that A  alters the bHLH gene expression in neural stem cells toward cell death.
Ken Kojima, Akiko Nishida, Shinji Takebayashi, Jyuichi Ito Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Japan
Basic helix-loop-helix (bHLH) transcription factors play crucial roles in development of the central and peripheral nervous systems. To visualize expression of Hes1 or Hes5 gene, pHes1-and pHes5-EGFP transgenic (tg) mice were generated (Ohtsuka et al., 2006) . In each transgenic mouse, a promoter of Hes1 or Hes5 gene drives enhanced green fluorescent protein (EGFP) gene. In the inner ear, it is suggested that Hes1 or Hes5 regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. By use of immunohistochemical technique, we examined distribution of GFP expressing cells in the inner ear of the transgenic mice from embryonic day 10 (E10) to postnatal day 60 (P60). In the pHes1-EGFP tg mouse inner ear, GFP immunoreactive (GFP-IR) cells were detected from E10 to P60. In the pHes5-EGFP tg mouse inner ear, GFP-IR cells were observed from E16.5 to P15. GFP-IR cells in pHes1-GFP tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear.
Ohtsuka et al., 2006. Mol. Cell Neurosci.
PS2A-C033 Expression of zfh-5 in the developing mouse brain: mRNA, antisense RNA and protein expression Yuriko Komine 1 , Kenji Nakamura 2 , Motoya Katsuki 1 , Tetsuo Yamamori 1 1 National Institute for Basic Biology, Okazaki, Japan; 2 Mitsubishi Kagaku Institute of Life Science, Machida, Japan ZFH-5 is a transcription factor containing three homeodomains and 18 Zn fingers and expressed in differentiating neurons. We have reported that the level of zfh-5 mRNA is negatively regulated by antisense transcripts of the zfh-5 gene. In several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh-5 antisense RNA is expressed prior to the mRNA; as the level of the antisense RNA gradually decreases, zfh-5 mRNA starts to be expressed. Recently, we have raised an antibody against mouse ZFH-5 and examined the expression profile of the ZFH-5 protein. In the most regions of the brain, the protein expression pattern consisted with that of mRNA. However, in the several types neurons mentioned above, ZFH-5 protein was not detected even when the zfh-5 mRNA was already expressed. This observation together with other data suggested that the ZFH-5 protein level is regulated by several mechanisms including suppression by the antisense RNA and translational control.
Takashi Inoue 1 , Maya Ota 1 , Katsuhiko Mikoshiba 2 , Jun Aruga 1 1 Laboratory for Comparative Neurogenesis, RIKEN BSI, Saitama, Japan; 2 Laboratory for Developmental Neurobiology, RIKEN BSI, Saitama, Japan
Zic family zinc-finger proteins play various roles in animal development. In mice, five Zic genes (Zic1-5) have been reported. Despite their partially overlapping expression profiles, mouse mutants for each Zic gene show distinct phenotypes, suggesting the functional redundancy of Zic proteins. It is expected that the common and specific roles of mouse Zic proteins can be clarified by studying compound mutant mice. In the present study, we characterized Zic1/Zic3 compound mutant mice. Mice carrying homozygous Zic1 mutant allele together with Zic3 null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. Especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either Zic1-or Zic3-single mutant. These observations indicate that Zic1, in cooperation with Zic3, have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon.
Chiaki Maruyama, Haruo Okado Department of Molecular Physiology, Tokyo Metropolitan Institute for Neuroscience, Japan RP58, a novel zinc finger protein containing a POZ domain, functions as a sequence specific transcriptional repressor. RP58 gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that RP58 has a crucial role in cerebral development. To understand the role of this protein in brain development, we examined RP58 gene expression in mouse embryo and adult brain by in situ hybridization. As a result, we found that RP58 transcripts are first detected at embryonic day 10 in the neuroepithelium of the spinal cord and telencephalic vesicle. In the day 12-13 embryos, RP58 transcripts are predominantly observed in the preplate region but not in outside the nervous system. At E15, RP58 transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. In the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. In adult mice, RP58 is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex
Toshiki Kameyama, Fumio Matsushita, Yuzo Kadokawa, Tohru Marunouchi Division of Cell Biology, Fujita Health University, Toyoake, Japan
Neural zinc finger (NZF) proteins are transcription factors with DNAbinding domains of C2HC-type zinc finger motifs. Using P19 cells, we demonstrated that NZFs were expressed transiently during neuronal differentiation, and forced expression of NZF cDNAs resulted in neuronal differentiation. These results suggest that NZF family have a function regulate neuronal differentiation. To elucidate in vivo functions of NZF family in detail, we generate knockout mice of NZF-2 and NZF-3 respectively. NZF-2 null mice are born alive, but die within 10 min after birth with cyanosis. On the other hand, NZF-3 null mice are viable, fertile and appear normal. These mice look normal morphologically. Then we generate double knockout mice of NZF-2 and NZF-3 by intercrossing. Double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. And we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , 2006) . In the present study, to examine the role of Runx3 in the development of DRG in more detail, we examined the development of DRG neurons in Runx3-deficient mice from the early embryonic stages to birth, using various markers for subpopulation of DRG neurons. In newborn Runx3−/− mice, parvalbumin-positive DRG neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. Similar decreases were observed in embryonic days 14.5 and 15.5.
Shin Hisahara 1,2 , Susumu Chiba 2 , Hiroyuki Matsumoto 2 , Yoshiyuki Horio 1 1 Department of Pharmacology, Sapporo Medical University, Sapporo, Japan; 2 Department of Neurology, Sapporo Medical University, Sapporo, Japan
In mammalian CNS, the function of histone deacetylase SIRT1 is still unclear. Recent studies indicated that SIRT1 interacts with nuclear receptor co-repressor (N-CoR) and N-CoR represses intracellular domain of Notch-ICD activation of the Hes1 promoter. We performed overexpression of SIRT1 and N-CoR in neurosphere by Nucleofection, then induced differentiation. We found remarkable promotion for neural differentiation by overexpression of SIRT1 and N-CoR in the SIRT1 with N-CoR. SIRT1 and N-CoR suppressed Hes1 transcription by Notch-ICD in the luciferase assay. Hes1 transcription was suppressed in overexpression of SIRT1 and N-CoR, suggesting that interaction between SIRT1 and N-CoR represses Hes1 transcription. Consistent with this, ChIP assays revealed that not only N-CoR but also SIRT1 bind to the promoter of Hes1 gene. Taken together, these results indicate that SIRT1 and N-CoR accelerate neural differentiation of the undifferentiated cells via binding Hes1 promoter site and repressing Hes1 transcription.
Yasushi Maruyama 1 , Mitsuhiko Kurusu 2 , Masataka Okabe 3 , Katsuo Furukubo-Tokunaga 1 1 Grad. School Life and Envir. Sci., Univ. Tsukuba, Japan; 2 Natl. Inst. Genetics, Mishima, Japan; 3 Inst. DNA Medicine, Jikei Univ. School of Medicine, Japan During brain development, a large number of neurons are generated by proliferation of neural stem cells. With a characteristic proliferation mode that persists through development, the neuroblasts of Drosophila mushroom bodies (MB) provide an attractive model system to study mechanisms of neural stem cell proliferation. Here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the MB neuroblasts. Mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the MB neuroblast cell cycles. Moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. We propose that tll plays pivotal roles in proliferation of the MB neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/Tlx homologs in both Drosophila and vertebrate brains.
Kouji Senzaki, Masaaki Yoshikawa, Shigeru Ozaki, Takashi Shiga Graduate School of Comprehensive Human Science, University of Tsukuba, Ibaraki, Japan RUNX family transcription factor is an important component of TGF- and BMP signaling. We reported previously that Runx3 mRNA is expressed in the dorsal root ganglion (DRG) from the early developmental stages, and that RUNX3 regulates axonal projection of TrkCexpressing proprioceptive DRG neurons (Inoue et al., 2002) . Furthermore, we announced previously that Runx3 mRNA is expressed in cranial ganglia of V, VII, VIII, IX and X in mouse developmental stages. The expression was restricted to subset of neurons in each ganglion.
To examine the influence of Runx3 on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of Runx3 knockout mice using immunohisitochemical staining. We found the decrease of TrkC-expressing neurons in trigeminal ganglia of neonatal Runx3 knockout mice, however, we observe little change in the proportions of NueN-expressing neurons.
Kouko Tatsumi 1 , Hirohide Takebayashi 2 , Takayuki Manabe 1 , Kazuhiro Ikenaka 2 , Akio Wanaka 1 1 Dept. Anatomy, Nara Med. Univ., Kashihara, Nara, Japan; 2 Division of Neurobiology and Bioinformatics, NIPS, NINS, Okazaki, Aichi, Japan
Our previous study with double labeling of BrdU and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (OPCs)-like cells in the injured adult brain. And we found out that these OPCs expressed NG2proteoglycan and Olig2 at early phase after injury. To directly trace the lineages of these OPCs, we employed double transgenic mice that express tamoxifen-sensitive CreER under the control of the Olig2 promoter together with ROSA-EGFP reporter. The GFP positive cells were detected around the injured region, and the almost all of these cells co-expressed GFAP at late phase after injury. Furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. Our results clarified that dormant OPCs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. Olig2 is a bHLH transcription factor, essential for oligodendrocytes (OLs) and motoneurons differentiation in the spinal cord. However, differentiation of Olig2 lineage cells in the forebrain is largely unknown. Here we examined fates of Olig2 expressing cells in the fetal forebrain by tamoxifen (TM)-inducible Cre-loxP system. Olig2-CreER knockin mice were mated with reporter mice, and TM was injected at embryonic day (E) 12.5 or 14.5, when most of Olig2+ cells are observed in the basal forebrain. The Olig2+ cells at E12.5 gave rise to more neuron than glia that included both OLs and astrocytes. Majority of neuronal Olig2 lineage cells differentiated into GABAergic neurons, and a lesser number, into cholinergic neurons. The Olig2+ cells at E14.5 generated more glial cells than neurons. These results indicate that Olig2 lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain.
Mana Igarashi 1,2 , Masato Yano 1,2 , Satoru Hayashi 1,2 , Hirotaka J. Okano 1,2 , Hideyuki Okano 1,2 1 Dept. Physiol., Keio Univ. Sch. Med, Tokyo, Japan; 2 SORST JST, Japan
The mammalian neuronal Hu RNA binding protein family is homolog of Drosophila ELAV protein which is essential for differentiation and maintenance of the nervous system. In mammals, neuronal Hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the UTRs of specific target mRNAs. To understand the molecular mechanism of Hu induced neuronal differentiation, we purified HuB associated complexes. Among them, NF90 family, a double strand RNA binding protein which is one of Hu associated proteins, is known to bind to UTRs of p21, p27 and tau mRNA known as Hu targets. We generated rabbit polyclonal antibodies against NF90 and NF45, binding partner of NF90, respectively. In mouse embryonic brains, we found that NF45/90 expressed highly in postmitotic neurons where neuronal Hu proteins are highly distributed. Moreover, we found that Hu and NF45/90 formed mRNP complexes in mouse brain extracts. We will discuss the role of Hu binding partners in neuronal differentiation through post-transcriptional regulation.
Sachiko The pallium is specified as a homologous field in the vertebrate telencephalon. However, little is known about how species-specific pallial structures are generated during embryogenesis. To address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. Cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. Next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er81 and Brn2) in the cerebral cortex. In situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. Our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species.
Research funds: KAKENHI (16027202) PS2A-D047 Protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures We established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. Protein phenotype of the cells was analyzed using immunocytochemical technique. Antinestin immunoreactivity was observed in cells with short processes 2 days in the re-cultivation. The anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-III tubulin immunoreactivity increased through the re-cultivation for 1-4 weeks. Presence of neurons, astrocytes and oligodenderocytes was examined using anti-III tubulin, anti-glial acidic fibrillary protein and RIP antibody, respectively. Apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. These results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. We have shown that perineuronal NG2 + cells are major populations of proliferating cells in the cerebral cortex of rats. In the adult cortex, NG2 is known as a marker for oligodendrocyte progenitor cells (OPCs) that retain ability to proliferate and differentiate into new oligodendrocytes. However, it is still unclear whether all NG2 + cells in the neocortex are the OPCs. We investigated about subtypes of NG2 + cells found in the perineuronal regions of the cerebral cortex using cell markers. Two subtypes of perineuronal NG2 + cells could be distinguished by the subcellular localization of GST-protein. One is nuclear type, the other is cytoplasmic type. Only the nuclear GST-+ cells have the proliferative activity. These data suggest that the nuclear GST-+ /NG2 + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells.
Muguruma Keiko, Su Hong-Lin, Matsuo-Takasaki Mami, Watanabe Kiichi, Sasai Yoshiki Neurogenesis and Organogenesis Group, RIKEN Center for Developmental Biology, Kobe, Japan
In this study, we report in vitro generation of Math1 + cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1 + . The induced Math1 + cells mitotically active and express markers characteristic of granule cells precursors (Pax6, Zic1, and Zipro 1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1 + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7 + /Calbindin-D28K + neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of cerebellar neurons by using the ES cell system.
Sachiyo Misumi, KIM Hye-Jung, Hideki Hida, Hitoo Nishino Department of Neurophysiology and Brain Science, Nagoya City University Graduate School of Medical Science, Nagoya, Japan Regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. We have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (NPCs) without influencing apoptosis after differentiation. In this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. In RT-PCR, the expression of p21 cip1 , p27 kip1 and p57 kip2 mRNAs were elevated during differentiation to neuron from NPCs. Especially, prolonged enhancement of p27 kip1 mRNA was shown. Transfection of p27 kip1 into NPCs induced activation of neuroD promoter and increase of number of tublin III-positive cells. Treatment with deferoxamine to NPCs from E12.5 rat midbrain and HB1.F3 cell line did not activate Erk and Akt phosphorylation during the treatment. Date suggest that prolonged p27 kip1 elevation is related to enhanced production of neuron from NPCs, and that cell cycle regulation in G1/S phase did not activate MAPK and PI3-K signaling.
Yuichi Tanaka 1 , Yusuke Tozuka 1 , Dai Muramatsu 1 , Kin-ichi Nakashima 2 , Tatsuhiro Hisatsune 1 1 Departement of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan; 2 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan
We previously reported no definite evidence for in vivo neurogenesis in adult neocortex. However, we also confirmed dividing cells in this area. In this study, we analyzed the characteristics of adult cortical nestin+ cells. In vivo, they belonged to NG2+ and Olig2+ cells, showed slowly proliferating ability compared to those in adult dentate gyrus. For in vitro analysis, we precisely isolated progenitor cells by Percoll gradient procedure. They differentiated into Tuj-1+ or MAP-2+ neuronal cells by adding retinoic acid or BDNF. More than 90% of newborn neurons expressed GABAergic neuronal markers, GABA, GAD or calretinin. We also purified nestin-GFP+ cells from Nestin-GFP transgenic mice using the FACS system, and confirmed their neuronal potential. Moreover, integration of a neural bHLH transcription factor NeuroD1 significantly promoted this neurogenesis. We demonstrated neurogenic potential of adult cortical nestin+ cells.
Mie Gangi 1 , Michiko Imanishi 2 , Teiko Kuroda 2 , Masao Tachibana 1 , Masahiko Takada 2 1 Department of Psychology, Graduate School of Humanities & Sociology, University of Tokyo, Tokyo, Japan; 2 Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, Tokyo, Japan A Kv3 subfamily of voltage-gated K + channels is thought to play an important role in high-frequency repetitive firings. It is unknown which subtype of Kv3 channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. We found immunohistochemically that Kv3.1b and Kv3.3 were expressed both in the mouse and frog retinas. However, a laminar pattern with two bands in the inner plexiform layer was displayed by Kv3.3 in the frog retina and by Kv3.1b in the mouse retina. It has been shown that mammalian cholinergic amacrine cells express Kv3.1b. Thus, the differential expression of Kv3 channels may reflect their functional diversity between the frog and mouse retinas.
Hiroshi Jouhou 1,2 , Kazunori Yamamoto 1 , Masayuki Hara 1 , Akinori Homma 1 , Akimichi Kaneko 3 , Masahiro Yamada 1 1 Tokyo Metropolitan Univ., Hino, Tokyo, Japan; 2 Astellas Pharma. Inc., Osaka, Japan; 3 Sch. Rehabili., Seijoh Univ., Aichi, Japan
In order to interpret the formation of receptive field surrounds in the retinal neurons, Hirasawa and Kaneko (2003) proposed a pHmediated mechanism to substitute for the GABA-mediated feedback hypothesis from horizontal cells (HCs) to cone photoreceptors. To verify the idea that the depolarized HCs release protons we measured, by a fluorescent ratio imaging technique, the pH of the immediate external surface (pH o ) of HCs isolated from carp or goldfish retina. When HCs stained by 5-hexadecanoylaminofluorescein, a pHsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular K + , pH o acidified. The amount of pH o acidification was monotonically dependent on the amount of depolarization, as much as 0.21 ± 0.05 pH unit by 100 mM K + . Acidification of pHo was suppressed by 0.4 M bafilomycin A1, a specific inhibitor of V-ATPase, suggesting that the HC depolarization enhanced an outward proton movement by the outward electrogenic H + pump.
PS2A-E055 Analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system Penphimon Phongphanphanee, Katsuyuki Kaneda, Tadashi Isa National Institute for Physiological Sciences, Japan
To study how the visual signal is processed in the local circuit of superior colliculus (SC) from the superficial layers (sSC) to the deeper layers (dSC), we analyzed the propagation of excitation following the electrical stimulation of the sSC by using a planar 64-channel field potential recording system in slice preparations obtained from 16 to 24 days old mice. Stimulation at sSC induced negative field potential with short latency and short duration (100-200 ms) at the recording site in sSC adjacent to the stimulating site. After application of bath containing 10 M bicuculline, the same stimulus induced a large negative field response with long duration (200-400 ms) that spreads laterally in sSC and ventrally to dSC. These responses disappeared after application of 50 M APV, when only short latency response remained. The results suggest that when GABA A receptormediated inhibition is reduced, visual signal in the sSC propagates to the dSC as large response with long duration and NMDA receptors contribute to propagation of the response.
Osamu Hosoya 1 , Ken Tsutsui 2 , Kimiko Tsutsui 1 1 Dept. of Neurobiol. and Neuroanat., Okayama Univ. Grad. Sch. of Med., Dent., and Pharm. Sci., Japan; 2 Dept. of Genomics and Proteomics, Okayama Univ. Grad. Sch. of Med., Dent., and Pharm. Sci., Japan Amphiphysin Ir (amph Ir) is alternatively spliced variants of amphiphysin I which is specifically expressed in retina. Amph Ir is composed of conserved domains including the N-terminal BAR, the central clathrin/AP-2 binding, and the C-terminal SH3 domains and the variant specific two novel insertions (A and B). Insert A may be a determinant for the retina-specific expression. Insert B has no significant homology to known proteins and two shorter transcripts with 3 -truncations in the insert were also expressed. Recently, we found that a human retinal pigment epithelia cell line, ARPE-19, also expresses amph Ir. ARPE-19 thus can be a useful tool for investigating the cellular function of amph Ir in retina. Immunofluorescence analyses with ARPE-19 cells revealed that amph Ir occasionally colocalized with mitochondria, raising the possibility that amph Ir may participate in structural or functional organization of mitochondria. Further characterization of the variant is under investigation.
Hironori Takamura 1 , Satoshi Ichisaka 2 , Chihiro Hayashi 2 , Hirotoshi Maki 2 , Yoshio Hata 1 1 Div. Integrative Biosci., Tottori Univ. Grad. Sch. Med. Sci., Yonago, Japan; 2 Div. Neurobiol., Sch. Life Sci., Fac. Med., Tottori Univ., Yonago, Japan Monocular deprivation (MD) induces significant plasticity in the primary visual cortex (V1) during critical period. It was reported that inhibition of extracellular signal-regulated kinase (ERK) activity in the visual cortex suppressed the ocular dominance plasticity. If ERK is involved in the mechanism of this plasticity, visual deprivation would change the activity of ERK in V1 and such change might be induced only in the critical period. To test this possibility, we examined effects of MD on the amount of phosphorylated (activated) ERK (pERK) in the rat visual cortex. By MD, we found a significant decrease in the amount of pERK in V1 receiving deprived eye inputs in both young and adult rats. As to the subcellular localization of ERK, we found a significant increase of the nuclear pERK only after MD in young rats. These results suggest that ERK signaling might be regulated by different mechanism between young and adult rats.
Research funds: KAKENHI (176959) PS2A-E058 Rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex Nobuko Mataga, Yoko Mizuguchi, Takao Hensch Neuronal Circuit Development, RIKEN Brain Science Institute, Saitama, Japan
In the binocular zone (BZ) of mouse visual cortex, critical period (CP) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. To explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the BZ by vesicular glutamate transporters (vGlut)1 and vGlut2, respectively. A complementary distribution of vGlut1 and vGlut2 was established by postnatal day (P)18 and both signal intensities increased further by P29-30 (peak CP). The immunoreactivity for vGlut2 decreased around layer IV after brief monocular deprivation (4dMD) during the CP. Interestingly, both signals in all layers were lower in the BZ contralateral to an eye injected with TTX than in the ipsilateral BZ, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to 4dMD. These results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition.
Reiko Meguro, Masao Norita Department of Sensory and Integrative Medicine, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan
We investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. Using anterograde/retrograde tracer, biotinylated dextran amine (BDA), we made a small injection into the dorsal lateral geniculate nucleus (dLGN) or the lateral posterior nucleus (LP) of the degenerated side of the monocular deprived rat. The geniculate projection terminated mainly in layer IV of area 17, with a small projection to layer VI of areas 17 and 18a. Cells in layer VI of area 17 projected to dLGN. In addition, cells in layer V of area 17 projected to dLGN, which is not observed in normal rats. In area 18a, cells in layers V and VI projected to dLGN. The projection from LP terminated mainly in layer IV of 18a. Cells in layers V and VI of area 18a projected to LP. Smaller number of cells in layer V of area 17 also projected to LP. These findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems.
PS2A-E060 Activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus Miho Yoshida 1 , Takemasa Satoh 2 , Yoshio Hata 1 1 Div. Integrative Biosci., Tottori Univ. Grad. Sch. Med. Sci., Yonago, Japan; 2 Div. Neurobiol., Sch. Life Sci., Fac. Med., Tottori Univ., Yonago, Japan
The projection from the lateral geniculate nucleus (LGN) to the primary visual cortex (V1) shows significant morphological plasticity responding to visual experiences in early life. Such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. It is not clear whether similar plasticity operates in other neural connections in the visual system. Therefore, we tried to investigate the plasticity of feedback projection from V1 to the LGN. We focused on the density of type 1 metabotropic glutamate receptor ␣ (mGluR1␣) in the LGN which locate postsynaptically at synapses of feedback projection. Immunohistochemical signal for mGluR1␣ in the LGN decreased after elimination of V1, showing that this signal reflects density of functional feedback synapses. To explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mGluR1␣ signal in the LGN of young rats.
Yu Morishima 1 , Hiroshi Sakamoto 2 , Takahumi Akasaki 1 , Yoshio Hata 1 1 Div. Integrative Biosci., Tottori Univ. Grad. Sch. Med. Sci., Japan; 2 Div. Neurobiol., Sch. Life Sci., Fac. Med., Tottori Univ., Japan Monocular deprivation (MD) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. However, when MD is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. To clarify mechanisms underlying the two forms of ocular dominance (OD) plasticity in different direction, we examined other characteristics of them, (1) how rapidly the reverse OD shift proceeds and (2) whether the shift is induced only in young animals. We infused the cortex with muscimol in 4-week-old kittens and in adults. The reverse OD shift was observed after 6 days MD, but not significant after 3 days MD. In adults, OD distribution remained unchanged. Morphological change of individual input axons was also examined after 6 days MD. The reverse OD shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal OD shift.
PS2A-E062 Experience-dependent plasticity in the absence of AMPA receptor subunits in mouse visual cortex Youichi Iwai 1 , Nafiseh Atapour 1 , John Renger 1 , John Roder 2 , Peter Seeburg 3 , Takao Hensch 1 1 Neuronal Circuit Dev., RIKEN, BSI, Wako, Japan; 2 Mount Sinai Hospital, Toronto, Canada; 3 Max Planck Inst. Med. Res., Heidelberg, Germany Two AMPA receptor subunits (GluR1, 2) play prominent roles in hippocampal models of homosynaptic plasticity (LTP/LTD). Brief monocular deprivation (1d MD) rapidly alters both the phosphorylation state and surface expression of GluR1 in visual cortex. Here, we addressed whether these coincidental events are essential for subsequent ocular dominance (OD) plasticity. Mice lacking GluR1 (KO) displayed little LTD in visual cortical slices, while baseline transmission was normal. They also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after 4d MD during a typical critical period. The rate of plasticity appeared somewhat slowed, as 20d MD eventually led to full OD shifts. In GluR2 KO mice, even 4d MD robustly activated OD plasticity. Thus, experience-dependent modification of AMPA receptors is not essential for plasticity in vivo, although GluR1 may contribute to the very earliest stages.
Shigeyoshi Higo, Nobuaki Tamamaki Department of morphological neural science, Kumamoto University, Kumamoto, Japan Virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. However, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. Since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which GFP expression is driven by Dlx2 promotor and Dlx5/6 enhancer. This virus labels GABAergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace GFP-labeled axons of GABAergic neurons in serial brain sections. We used this virus to investigate GABAergic neurons with long projecting axon branches beyond a functional area. The virus was injected into the stratum oriens of the mouse hippocampal field CA1 and revealed a nonpyramidal neuron projecting to CA3 and fimbria. Further we shall introduce this virus to the cat brain and investigate axon branches of GABAergic projection neurons in the neocortex.
Akiko Yamashita 1 , Takao Oishi 2 , Motoharu Hayashi 2 1 Div. Appl. System Neurosci., Nihon Univ. Grad. Sch. Med. Sci., Tokyo, Japan; 2 Dept. Cell. and Mol. Biol., Primate Res. Inst., Kyoto Univ., Inuyama, Japan GABAergic cells in the cerebral cortex are divided into subgroups: parvalbumin (PV)-, somatostatin (SOM)-, calretinin (CR)-, and calbindin-containing types. To clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within GABAergic cells in the various cortical areas. PV, SOM or CR did not coexist with each other in primates as observed in rodents. More than 60% of GABAergic cells contained PV; showing that PV cells are more abundant in primates than in rodents. Proportion of SOM cells in GABAergic cells was smaller in the primary visual area (5.3%) than in other areas, such as the prefrontal (10.8%), primary motor (9.8%), somatosensory (10.0%) and secondary visual areas (8.5%), indicating cortical differentiation in GABAergic system of the primate cerebrum. Our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by GABAergic projection neurons. In order to address the importance of the GABAergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the GABAergic projection axons. Since more than 80% of the GABAergic projection neurons showed NPY immunoreactivity, we used NPY-Cre transgenic mouse that express Cre in NPY neurons and adenovirus that encodes GFP in the downstream of floxed stop to label the GABEergic projection axons. After injection of the adenovirus into deep layers of the NPY-Cre mouse neocortex and immunoperoxidase staining of GFP in the brain section, we could reveal GABAergic neurons in a Golgi-like image with their axons. Also this method seemed to allow us to label GABAergic projection neurons retrogradely.
Koji Ikezoe 1 , Guy N. Elston 2,3 , Tomofumi Oga 1 , Hiroshi Tamura 1,3 , Ichiro Fujita 1,3 1 Osaka Univ., Japan; 2 Univ. Queensland, Australia; 3 CREST, JST, Japan Layer III pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. Basal dendrites of cells in visual association cortex such as inferior temporal area TE spread more extensively and are more branched than those in the primary visual cortex (V1). Pyramidal cells in prefrontal cortex, such as area 12, have even more dendritic branches than those in area TE. Here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. We stained individual layer III pyramidal cells in V1 (n = 52), area TE (n = 46), and area 12 (n = 43) of a 3-week old monkey (Macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. The number of branches and the tangential extent of dendrites was greatest in area 12, followed by area TE, and V1. Thus, considerable heterogeneity in pyramidal cell structure already exists 3-weeks after birth.
Hiroaki Matsushita 1 , Mahito Ohkuma 1 , Masami Watanabe 2 , Ei-ichi Miyachi 1 1 Department of Physiology, Fujita Health University, Aichi, Japan; 2 Department of Perinatology, Institute Developmental Research, Aichi, Japan Acetylcholine (ACh) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. We performed the patch-clamp recording and fura-2 based calcium imaging in cat retinal ganglion cells (RGCs). Under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated RGCs. However, these currents and spikes were not observed in the 50% of axotomized RGCs. Bath application of 100 M carbachol, an ACh receptor agonist, rose [Ca 2+ ] i in 22% of normal RGCs. Although the 85% of RGCs responded to carbachol at 3 days after axotomy, no responsive RGCs appeared during 7-15 days. ACh responsiveness recovered in axonal regenerated RGCs (17%). Since pycnotic cells were observed few days after axotomy, ACh may modulate neurotrophic effect in survived RGCs. These results suggest that ACh is an important marker for neuronal degeneration and regeneration in cat RGCs.
Research funds: KAKENHI (17500217 to EM, 16591780 to MW)
Kenichiro Miura, Masakatsu Taki, Hiromitsu Tabata, Kenji Kawano Dept. Integrative Brain Sci., Grad. Schl. of Med., Kyoto Univ., Kyoto, Japan
The initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (Hashimoto et al., 2004) . To study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. The pursuit target, presented on a CRT monitor (75 Hz), was a Gaussian patch of texture displayed on a neutral gray background. The Gaussian envelope moved at 30 deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). The number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames (107 ms) or only one frame (13 ms). The initial tracking responses were larger when the target became visible eight frames before the motion onset. The result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target.
PS2A-E069 Motion picture effects on eye movements and blood flow in the frontal area Atsuhiko Iijima 1,3 , Tohru Kiryu 2 , Kazuhiko Ukai 3 , Takeshiko Bando 1 1 Div. Integrative Physiol., Grad. Sch. Med., Niigata Univ., Niigata, Japan; 2 Div. Inform. Sci., Grad. Sch. & Tech., Niigata Univ., Niigata, Japan; 3 Dept. Appl. Phys., Sch. Sci. & Tech., Waseda Univ., Tokyo, Japan Motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. 20 subjects watched the two-dimensional motion pictures in random order. Eye movements were measured by a binocular video oculography (Newopto), and head movements were measured by a magnetic motion sensor (Polhemus). Blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (Hamamatsu). The patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. Possible mechanisms of the differences will be discussed.
Kiyoto Matsuura 1 , Kenichiro Miura 1 , Masakatsu Taki 1 , Hiromitsu Tabata 1 , Naoko Inaba 1 , Kenji Kawano 1 , Frederick A. Miles 2 1 Grad. Schl. Med., Kyoto Univ., Kyoto, Japan; 2 Lab. Sensorimotor Res., NEI, NIH, Bethesda, MD, USA Human OFRs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (Sheliga et al., SFN 2005) . We recorded the OFRs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency 3f and 5f, which created a repeating pattern with beat frequency, f, in two monkeys. Motion consisted of successive steps (100 Hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the 5f forwards and the 3f backwards. The contrast of the 5f was fixed at 4, 8, or 16%, while the contrast of the 3f was varied from one-fourth to four times the contrast of the 5f. When the contrast of the 3f (5f) was less than about half that of the 5f (3f), the 5f (3f) dominated initial OFR: winner-take-all. Thus, the motion processing underlying the OFR in monkeys, like that in humans, includes nonlinear interactions.
Masazumi Katayama, Takahiro Fujita Department of Human and Artificial Intelligence Systems, Faculty of Engineering, University of Fukui, Fukuki, Japan When executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. While, Goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. From the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. In this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. As a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < 0.05). We concluded that grasping positions of visual estimation and motor execution are independent and these results support the Goodale's hypothesis.
Ryuichi Hishida, Masaharu Kudoh, Katsuei Shibuki Dept. Neurophysiol., Brain Res. Inst. Niigata Univ., Niigata, Japan Cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. Recently, Wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. Multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. In this study, we tested the third possibility. We prepared the mouse cortical slices including the visual and auditory cortices. The horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. These results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons.
Jumpei Naito 1 , Yaoxing Chen 2 , Yukiko Tanada 1 1 Dept. Animal Sci., Teikyo Univ. Sci. & Tech., Uenohara, Japan; 2 China Agricul. Univ., Beijing, China Twenty white-leghorn chicks (P0-8) were perfused with 1% paraformaldehyde through the heart under deep anesthesia of Nembutal (32 mg/kg BW). Two to three small crystals of DiI were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. A total of 225 RGCs were classified into six groups according to the somal area and dendritic field (Naito and Chen, 2004). Group Ic projected dominantly to the tectum. Group Is and IIIs showed high hypothalamic-and thalamic-dominance, respectively. Group IIc was non-specific in the central projections. Group IVc was tectal-dominant. Patterns of the dendritic stratification were counted to 32 in 121 tec-RGCs, 28 in 77 tha-RGCs, and 16 in 38 hyo-RGCs. Of these stratification patterns, many patterns were common among tec-, tha-, and hyp-RGCs. In contrast, the RGCs that showed a same dendritic pattern were consisted of a single RGC in most of the non-common RGCs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers 1-3).
Yasuro Atoji, Shouichiro Saito Laboratory of Veterinary Anatomy, Gifu University, Gifu, Japan
The present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (NCI) in the pigeon by a tract-tracing method. In the present study we define NCI an area which is located lateral to the Field L complex and ventral to NCL. Following a CTB injection into NCI, a large number of neurons was labeled in NCI, the mesopallium, and intermediate arcopallium (AI) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. Contralateral AI contained a small number of labeled neurons. A few labeled neurons were found in LSt. Few labeled cells were found in NCL, Field L, piriform cortex, or hippocampal formation. Following a BDA injection into NCI, a large number of labeled fibers extended in NCI, mesopallium, and AI. LSt contained a small number of labeled fibers. Few labeled fibers were located in NCV and limbic regions. The diencephalon contained very few labeled fibers. In summary, NCI has strong reciprocal connections within NCI itself and with the mesopallium and AI, and little connections with the limbic system.
Hidenori Horie 1 , kenji Yuda 3 , Eiichi Okawa 4 , Katsuyoshi Maruyama 4 , Hiroshi Uozato 5 , Hiroko Horie 1 , Satomi Nakajima 1 , Kenkichi Tanioka 6 , Yuji Ohkawa 6 , Tomoki Matsubara 6 , Wolfram Tetzlaff 7 1 Advanced Res. Centr. Biological Sci., Waseda Univ., Tokyo, Japan; 2 TechnoMaster Co. Ltd., Yokohama, Japan; 3 Kikuna Yuda Eye Clinic, Yokohama, Japan; 4 Healthcare Business Co., Matsushita Electric Ind. Co. Ltd., Yokohama, Japan; 5 Dept. Ophthalmol. & Visual Sci., Kitasato Univ., Kanagawa, Japan; 6 NHK Sci. Technical Res. Labo., Tokyo, Japan; 7 ICORD, Univ. British Columbia, Vancouver, Canada
We describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. In our experiments on human subjects, we used an LCD screen that was rhythmically moved between 25 and 70 cm toward and away in a high speed (top speed: 1000 mm/s) from the subjects. The device significantly improved visual performances in over 60% of the school-aged children with myopia and 80% of adults with presbyopia.
Hiroyuki Miyamoto 1 , Toral S. Surti 2 , Takao K Hensch 1 1 Laboratory for Neuronal Circuit Development, RIKEN Brain Science Institute, Wako, Japan; 2 San Francisco, USA Competitive plasticity of binocular response following monocular deprivation (MD) is prominent in the primary visual cortex (V1) during an early critical period. Recently, MD has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of V1. Here, we addressed to what extent these two types of plasticity induced by the same MD share common mechanisms. Adult mice lacking a GABA-synthetic enzyme (GAD65 KO), which do not exhibit ocular dominance (OD) plasticity by brief MD during the critical period, showed normal optomotor acuity and enhancement with 4 day MD. OD shifts did not correlate with optomotor enhancement in these mice. Finally, early MD spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. These observations support the view that adult perceptual learning and classical OD plasticity are independent.
Junya Hirokawa 1 , Miquel Bosch 2 , Shuzo Sakata 3 , Yoshio Sakurai 4 , Tetsuo Yamamori 1 1 Division of brain biology, National Institute for Basic Biology, Okazaki, Japan; 2 MIT, MA, USA; 3 Rutgers University, NJ, USA; 4 Department of Psychology, Kyoto University, Japan The brain is able to integrate information from different sensory sources to enhance behavioral responses. To identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (Sakata, et al., 2004) by the expression of c-Fos. A pronounced c-Fos upregulation was found in superior colliculus and in layer IV and deep layer of latero-medial secondary visual area (V2LM). Local injection of GABA agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. These results suggest a key role of V2LM in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli.
Takashi Shinozaki 1 , Youichi Miyawaki 2 , Tsunehiro Takeda 1 1 Department of Complexity Science and Engineering, University of Tokyo, Chiba, Japan; 2 Mathematical Neuroscience, RIKEN BSI, Saitama, Japan Drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). We studied this double rivalry process based on psychophysical and magnetoencephalography (MEG) measurements. Pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. Reaction time (RT) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. We then compared MEG signals among those perceptual conditions and found a MEG response of interocular rivalry in the latency range expected from the result of RT measurement. These results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry.
Visual stimuli evoke several brain potentials with relatively precise time courses. The role of these brain potentials in visual object categorization is not clear. In this study we recorded Event Related Brain Potentials (ERP) while subjects participated in a face/non-face categorization task. Gray face and non-face natural object images were presented briefly (10 ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (SOA = 0-990 ms). Subjects reported presentation of face or non-face images by pushing one of the two assigned keys. We found that the face category discrimination performance significantly declined only in short SOA (10 and 20 ms) with a larger impact of masking on non-face discrimination. In ERP, the peak amplitude and latency of P1, N170 and area under curve of a late positive potential expanding from 300 to 500 ms were correlated with the subjects behavioral performance. The effect of backward masking on early ERP components may be due to altering sensory processing of visual stimuli while the effect on late ERP potential could be related to its impact on decision making processes.
Yasushi Naruse 1 , Ayumu Matani 1,2 , Tomoe Hayakawa 2,3 , Norio Fujimaki 2 1 University of Tokyo, Kashiwa, Japan; 2 NICT, Kobe, Japan; 3 Teikyo University, Tokyo, Japan
To study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. Alpha ringing is an evoked potential in alpha frequency band around 500 ms in latency. Eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of 1000 flash stimuli with their eyelids closed throughout the experiment. EEG was simultaneously recorded during the experiment. We classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. The result showed that the larger the amount of the alpha rhythm resetting is, the larger the P100 amplitude becomes. This suggested that a factor of the variability of the P100 amplitude is the amount of the prestimulus alpha rhythm resetting.
Research funds: a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (No. 16300083)
Hitoshi Sasaki 1 , Takuya Ishida 1 , Masayoshi Todorokihara 2 , Junichiro Miyachi 1 , Tahei Kitamura 3 , Ryozo Aoki 1 1 Dept. Physiol. & Biosignal., Osaka Univ. Grad. Sch. Med., Suita, Japan; 2 Dept. Phys. & Elec., Osaka Pref. Univ. Grad. Sch. Eng., Sakai, Japan; 3 Dept. Elec. Eng. & Elec., Col. Industri. Tech., Amagasaki, Japan
Recently it has been shown that noise can improve detection of sensory stimuli in several modalities. Here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. Contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. The contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. These findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception.
Narumi Katsuyama 1 , Nobuo Usui 1 , Izuru Nose 1,2 , Masato Taira 1,3 1 Department of Applied System Neuroscience, Nihon University School of Medical Science, Tokyo; 2 Faculty of Human Science, Bunkyo University, Saitama, Japan; 3 ARISH, Nihon University, Tokyo, Japan When an object is moving, perception of its 3D trajectory in depth can be strongly influenced by the trajectory of its cast shadow. For example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. Using fMRI, we found that several visual areas, including human MT and the posterior STS and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. Additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of STS and the posterior parietal cortex, including precuneus. These results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow.
PS2A-F083 Local area network in the gerbil's auditory cortex: Reversible focal inactivation with infrared laser irradiation Akira Yamamoto, Hiroshi Riquimaroux Gratuate School of Engineering, Doshisha University, SCNRL, Japan This study investigated local area networks in the primary auditory cortex (A1) and the anterior auditory field (AAF) by blocking neural activities with the near-infrared laser irradiation (wave length = 830 nm). In previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. By using this technique, the present study examined corticocotical relationships in the auditory cortex of the Mongolian gerbils (Meriones unguiculatus). CF (constant frequency) and FM (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. When irradiated AAF area and recorded neural responses from AI, the irradiation changed phasic responses into tonic responses, and vice versa. These results indicate that there are functional connections within AI or AAF, and between AI and AAF.
Takashi Doi, Hiroshi Riquimaroux Department of Knowledge and Engineering and Computer Sciences, Doshisha University, Kyoto, Japan
In a previous behavioral study, ablation of right auditory cortex (AC) made the discrimination between ascending and descending frequency modulated (FM) tones by Mongolian gerbil (Meriones unguiculatus) difficult (Wetzel et al., 1998) . This result indicates that some neurons in gerbil's right AC represent the directions of FM sweeps. Actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (AAF). In AAF, BFs are gradually shifted along the rostrocaudal direction, and the same BFs are arranged in dorsoventral direction (Thomas et al., 1993) . Moreover, AAF has dense synaptic connections within the area (Budinger et al., 2000) . We made network models based on this structure of AAF and could gain similar responses to the actual responses of directiondependent neurons. This result suggests that AAF in gerbil's AC has good structure to process FM tones.
Research funds: A grant to RCAST at Doshisha Univ. from MEXT, Innovative Cluster Creation Project by MEXT
It has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (SC). However, it is unclear as to how this auditory space map of the mammalian SC is formed in the auditory pathway. The present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (ICx) of anesthetized gerbils. The ICx is the major auditory nucleus that has projections to the SC. The stimuli were 50-ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. Single-unit responses were recorded from the ICx. The majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. For supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the ICx. However, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. The results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the ICx level.
Manabu Toyoshima 1 , Yasuo Takeda 2 , Yasushi Shimoda 1 , Kazutada Watanabe 1 1 Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Japan; 2 Department of Clinical Pharmacy and Pharmacology, Kagoshima University, Kagoshima, Japan NB-2 that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. We reported previously that NB-2 expression is prominent in the auditory system. NB-2 knockout mice exhibit impaired neural function in the auditory system. These findings indicate that NB-2 is indispensable for the function of auditory system. Here we report the detailed analysis of the NB-2 localization using anti-NB-2 monoclonal antibody that we produced recently. Immunohistochemical analysis of the rat brain showed that NB-2 was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (SN), caudate putamen (CPu) and fibers projecting from SN to CPu. The NB-2 immunopositive cells in SN are restricted to GABAergic neurons. Since GABAergic neurons play essential roles in the development and function of the auditory system, it is highly likely that NB-2 regulates the development and/or function of GABAergic neuron in the auditory pathway.
Reiko Nagashima, Kiyokazu Ogita Department of Pharmacology, Setsunan University Faculty of Pharmaceutical Sciences, Osaka, Japan Sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. There is compelling evidence that reactive oxygen species (ROS) are formed in the cochlea during acoustic stimulation. Glutathione (GSH) protects against the hearing loss through scavenging ROS generated by noise. In this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (GCS) gene, which is the rate-limiting enzyme in de novo GSH synthesis, in the cochlea following acoustic stimulation. Nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation (4 kHz octave band, 125 dB, 5 h), and then subjected to electrophoretic mobility shift assay to determine activator protein-1 (AP-1) DNA binding. AP-1 binding was increased 2-12 h after the exposure. RT-PCR and immunostaining revealed that noise exposure was effective in elevating the expression of GCS in the cochlea 2 h later. Taken together, AP-1 may participate in the expression of GCS gene in the cochlea after acoustic stimulation.
Masaharu Kudoh, Ryuichi Hishida, Katsuei Shibuki 1 Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan Multiple formants compose vowels. We have previously reported that bilateral lesions including in the auditory cortex (AC) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. In the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. Water-deprived rats were trained to discriminate between two sounds including four different formants. Licking a spout during presentation of one sound was rewarded with water while the other was not. Surprisingly, local lesions in the primary AC or the ventral association cortex had no clear effect on the discrimination learning. In contrast, the dorsal association areas impaired the discrimination learning. These findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants.
Hiroaki Tsukano, Yamato Kubota, Manavu Tohmi, Masaharu Kudoh, Katsuei Shibuki Department of Neurophysiol, Brain Research Institute, Niigata University, Niigata, Japan
We used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. C57BL/6 mice were anesthetized with urethane. The skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. Cortical images of green fluorescence in blue light were recorded by a cooled CCD camera. Responses in the auditory cortex elicited by sound stimuli (5-26 kHz for 500 ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (AI) and anterior auditory field. The activity patterns in AI elicited by 5 kHz were different from those elicited by 20 or 25 kHz. However, the areas activated by 5 kHz were also activated by the mixture of 20 plus 25 kHz but not by that of 19 plus 26 kHz, suggesting that cortical responses to missing fundamentals in AI were visualized using flavoprotein fluorescence imaging.
Hiroko Kosaki National Priting Bureau, Tokyo, Japan
We constructed a functional scheme of macaque auditory by distribution of Calcium Binding Protein, Parvalbumin (PV). Auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. Parvabumin showed a graduation, that is, inner core is most PV-rich, and outer rings showed the decrease of PV concentration. Comparing with PV staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by DeYoe and VanEssen by analysis of feed-forward and feedback connections.
Akihisa Kimura, Tomohiro Donishi, Keiichiro Okamoto, Yasuhiko Tamai Department of physiology, Wakayama Medical University, Wakayama, Japan Tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (TRN). In the present study, we determined how cortical and thalamic afferents intersect in the TRN with regard to tonotopic organization. In light of the fact that a subset of auditory cells in the TRN responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the TRN. Small injections of biocytin into the TRN, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. Retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. The results indicate anatomical nodes in the auditory TRN that would implement selective relay of auditory and/or polymodal sensory inputs.
PS2A-F092 Functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol Junsei Horikawa, Daisuke Uchiyama, Tatsunori Matsui, Shunji Sugimoto Department of Knowledge-Based Information Engineering, Toyohashi University of Technology, Toyohashi, Japan Guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye RH795 were recorded with a photodiode array. After determining the core (AI and DC) and belt fields, AI or DC was inhibited by putting a muscimol-containing agar piece on each field. The inhibition of AI resulted in reduction of responses in the belt fields by 55-90%, whereas the inhibition of DC resulted in reduction only by 20-50%. The reduction by AI inhibition was larger in the anterior and ventral belt fields and that by DC inhibition was larger in the posterior belt fields than in the other fields. Further inhibition of DC after the AI inhibition or vice versa resulted in suppression of the responses in all the fields. These results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from AI and DC.
Masataka Nishimura, Hiroyuki Kaizo, Wen-Jie Song Department of Electrical, Electronic, and Information Engineering, Graduate School of Engineering, Osaka University, Suita, Japan
The auditory cortex of many mammals has a core area and surrounding belt regions. In guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. However, the activity of the belt regions has not been fully examined. Using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. The auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye RH-795. A novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. The field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. We thus named the field as ventro-caudal field (VC). Smooth frequency gradient was observed in VC in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area.
Yoko Kato 1,2 , Kazuo Okanoya 1 1 Laboratory for Biolinguistics, RIKEN BSI, Wako, Saitama; 2 Graduate School of Humanities, University of Chiba, Chiba, Japan Bengalese finches sing complex courtship songs. To sing complex sequences, they require auditory feedback during singing. Song nucleus NIf has a projection from primary auditory area Field L and then it projects to sensory/motor nucleus HVC. Moreover, bilateral lesion of NIf cause song deterioration on complex sequences (Hosino and Okanoya, 2000) . We recorded auditory responses by multiunit activity from NIf and Field L. Auditory responses of NIf showed selectivity to Bird's own song (BOS) than its reversed song (REV). Comparing selectivity of NIf and Field L, NIf showed stronger selectivity than Field L. However, NIf did not show sequence dependent selectivity. These results suggest that NIf relays auditory information and enhances BOS selectivity. However, we did not observe a direct evidence that NIf related to generation of complex sequences. We started to record responses extracellularly from AC neurons of guinea pigs. In general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. During this period, animals do not appear to sleep but be sensitive to the environmental disturbance. Thereafter they usually fall asleep with their eyes closed. During this presleeping period, the best frequency tone was repeatedly presented 100-500 times at a fixed interval, through a speaker at 50-70 dB SPL. Responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. However, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. One phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. We suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus.
Masashi Sakai, Sohei Chimoto, Ling Qin, Yu Sato Department of Physiology, University of Yamanashi, Japan A periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (<10 Hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (>100 Hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". We physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. We found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). In addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. We will discuss functional consequences and spike evocation mechanisms of each population.
Atsuhito Toyomaki 1,2,3,4,5,6 1 Hokkaido University, Sapporo, Japan; 2 Hokkaido University, Sapporo, Japan; 3 Sakushin Gakuin University, Utsunomiya, Japan; 4 Kobe Shoin Women's University, Kobe, Japan; 5 RIKEN, Wako, Japan; 6 Sakushin Gakuin University, Utsunomiya, Japan Gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and Japanese special mora (sokuon). We recorded auditory evoked responses to short gaps and tones from children (8-12 years old, n = 8) and adults (19-23 years old, n = 12). There were six gap conditions with durations of 8, 16, 32, 64, 128 and 256 ms embedded in a continuous tone and six tone conditions with the same durations. The frequency of all the tone was 500 Hz. The responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. In contrast, an N1 and P2 complex followed the onset of all the tones in all the subjects. Thus development time course of neural process is conceivably different between gaps and tones.
PS2A-F098 An fMRI study on pitch control of voice using transformed auditory feedback method Akira Toyomrua 1 , Tamaki Miyamoto 2 , Atsushi Terao 2 , Sachiko Koyama 2 , Takashi Omori 2 , Harumitsu Murohashi 2 , Shinya Kuriki 2 1 JST, Saitama, Japan; 2 Hokkaido University, Sapporo, Japan Auditory feedback plays an important role in natural speech production. In the present study, we conducted an fMRI experiment while subjects performed a transformed auditory feedback (TAF) task to delineate the neural mechanism for control of pitch. The subjects were required to vocalize a and to hold the pitch of a feedback voice constant. In TAF condition, the pitch was altered suddenly two or three times, whereas in non-TAF condition the pitch was not modulated. Under the TAF condition, auditory feedback control is selectively expected to work more strongly than the non-TAF condition. Thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. As a result, right supramarginal gyrus, right frontal lobe (BA9), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation (12 subjects, p < 0.05 corrected). This result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere.
Sachiko Koyama-Takeichi 1 , Yuko Toyosawa 2 , Fumiya Takeuchi 3 , Michinao Matsui 4 , Shinya Kuriki 1 1 Research Institute for Elecronic Science, Hokkaido University, Sappro, Japan; 2 JST, Saitama, Japan; 3 Hokkaido University, School of Medicine, Japan; 4 Kobe Shoin Institute for Linguistic Science, Kobe, Japan Sounds with relatively long duration elicit a sustained component (Slow Field, SF). In the present study, we recorded cortical magnetic responses elicited by vowels and examined whether SF differs between native and non-native vowels (n = 11). Four synthesized vowels were used as stimuli (Stimulus duration 600 ms). Two of the vowels (a, o) are native for Japanese and the other vowels (ae, schwa) are not. Two inter-stimulus intervals were used (1200/4800 ms). For the native vowels, an early SF (250-300 ms) was larger for the long than for the short interval session in both hemispheres. For the non-native vowels, the early SF was larger for the long than the short interval session only in the right hemisphere. Neither an effect of interval nor hemisphere was significant for a late part of SF (400-600 ms) regardless of stimulus types.
Research funds: Japan Science and Technology Agent (Brain Sciences and Education), KAKENHI (16500166) PS2A-F100 Spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging Shunji Sugimoto 1,2 , Junsei Horikawa 1 1 Department of Knowledge-Based Information Engineering, Toyohashi University of Technology, Toyohashi, Japan; 2 RIKEN Brain Science Institute, Wako, Japan Optical imaging (voltage-sensitive dye, RH 795) showed spatiotemporal response patterns for frequency-modulated (FM) sounds in the multiple fields of the guinea pig auditory cortex. An FM sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. The location of the later response was corresponding roughly to the instantaneous frequency input of each FM sweep. On the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. The later response to an FM sound appeared clearly when the frequency of the FM sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. These results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds.
Yamato Kubota, Kuniyuki Takahashi, Ryuichi Hishida, Masaharu Kudoh, Katsuei Shibuki Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan Mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. If the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. We applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (AAF) to the primary auditory cortex (AI). Cortical responses in AAF and AI after sound stimuli (5-20 kHz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. After determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for 40 min, while the surface either AAF or AI was covered with a piece of carbon paper for preventing photo-inactivation. Although photoinactivation of AI had almost no effect on the responses in AAF, photo-inactivation of AAF significantly reduced the responses in AI. These results suggest the presence of auditory signal transmission from AAF to AI.
Kousuke Abe 1 , Go Ashida 1,2 , Kazuo Funabiki 2 1 Graduate School of Informatics, Kyoto University, Kyoto, Japan; 2 HMRO, Faculty of Medicine, Kyoto University, Kyoto, Japan Sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (NL) in birds. In vivo intracellular recording from owl's NL cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (Sound Analogue Potential; SAP). We simulated synaptic inputs to the owl's NL neurons by recruiting the convergence of phase-locked excitatory inputs. Several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of SAP, the amplitude of DC depolarization and the spectral features of synaptic noise in a complex manner. Biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed.
Takashi Nihashi 1 , Shigenori Takebayashi 2 , Masahiko Bundo 2 , Masazumi Fujii 3 , Toshihiko Wakabayashi 3 , Jun Yoshida 3 , Hiroyuki Fujisawa 1 , Kazunori Ando 1 , Kazumasa Hayasaka 1 1 Department of Radiology, National Hospital for Geriatric Medicine, Obu, Japan; 2 Department of Neurosurgery, National Hospital for Geriatric Medicine, Obu, Japan; 3 Department of Neurosurgery, Nagoya University School of Medicine, Nagoya, Japan
We identify SI, using fMRI in a routine scan for the patient who need a surgical approach. The activation of the brain with a tumor is complicated. We considered the pattern of the response. Twelve patients were participated in this study. Using 1.5 Tesla MR imager, tactile stimulation was applied to bilateral palm, respectively. The statistical threshold was set for individual. Contralateral activation on SI was found in 11 out of 12 patients in the affected hemisphere. When region is near central sulcus, the multiple sites were activated. On the other hand, when the tumor is from central sulcus, the activation is simple: contralateral SI. This method is useful to decide SI in affected hemisphere in a short time. However, there are great inter-individual differences due to the locations of the tumor.
Takayuki Iwano, Shinya Yamamoto National Institute of Advanced Industrial Science and Technology (AIST), Neuroscience Research Institute, Tsukuba, Japan
To examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. Subjects (n = 8) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. The subjects were required to identify the toe by verbal response. The subjects responded correctly when the great or fifth toe was touched (cf. Fein, 1987) . Surprisingly, subjects tended to misidentify the second toe as the third (47.2%), and the third toe as the fourth (37.2%), while the reverse misidentification rarely occurred (third as second, 4.5%; fourth as third, 7.1%). This unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields.
PS2A-G105 Effects of saccades on subjective temporal order of somatosensory signals Toshimitsu Takahashi 1,2 , Shunjiro Moizumi 1 , Ayami Okuzumi 1 , Humine Saito 1 , Shigeru Kitazawa 1,2 1 Department of Neurophysiology, Juntendo University Graduate School of Medicine, Tokyo, Japan; 2 CREST, JST, Saitama, Japan Morrone et al. (2005) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. In this study, we examined whether saccades affect temporal order judgments of tactile stimuli. Right-handed subjects were required to make a visually guided rightward saccade (24 • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. With a stimulation interval of 100 ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. However, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within 300 ms). The results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time.
PS2A-G106 Function-directed organization of the postcentral somatosensory cortex representing oral structures Takashi Toda 1,3 , Miki Taoka 2,3 1 Department Neuroscience Oral Physiology, Osaka University Graduate School of Dental Sciences, Suita, Japan; 2 Secondrary Cognitives Neurobiology, Tokyo Medical & Dental University, Tokyo, Japan; 3 Department Physiology, Toho University School of Medicine, Tokyo, Japan
The representation of oral structures in areas 3b and 1 of four conscious macaque monkeys was studied by recording single-neuron activities. A total of 115 electrode penetrations were made in areas 3b and 1. In 44 penetrations, pairs of adjacent neurons along the track had receptive fields (RFs) on continuous oral portions with or without overlapping, or otherwise on the same portion. In the remaining 71 penetrations, however, 14% of adjacent pairs (311/2153) had RFs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. We speculated that such an organization in areas 3b and 1 might be responsible for forming composite RFs of area 2 neurons. Those composite RFs often covered discrete but functionally-related oral portions as reported earlier.
Research funds: KAKENHI (12771111, 14771029)
Miki Taoka 1 , Michio Tanaka 1 , Hisayuki Ojima 1 , Atsushi Iriki 2 1 Secondrary Cognitives Neurobiology, Tokyo Medical and Dental University, Tokyo, Japan; 2 Laboratory of Symbolic Cognitive Development, RIKEN Brain Science Institute, Wako, Japan
We previously reported neuronal projections from the hand region of the second somatosensroy cortex (SII) to higher motor cortices (vetral premotor cortex etc.) suggesting that SII may be related to motor control of the hand movement. In the present study, we investigated the activities of SII hand neurons during voluntary movements. We recorded 168 neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. Among them (44% contra-, 55% bi-and 1% ipsilateral hand movements), we could determine receptive fields for only 43 neurons (25%). Most of activities (138 neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. We found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. Those results suggest that SII hand neurons code the start or end of a certain act of hand.
Takahiro Furuta 1,2 , Kouichi Nakamura 1 , Takeshi Kaneko 1 1 Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 2 CRULRG, Laval University, Canada
We investigated response properties of whisker-responsive neurons in the nucleus interpolaris (SpVi) combining juxtacellular recording and a Piezo-stimurator. The SpVi is one of the first relay stations in the vibrissal system. Rostral part of the SpVi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the SpVi projects to the ventral lateral part of the ventral posterior medial nucleus. In the rostral part of the SpVi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. In the caudal part of the SpVi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. Almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the SpVi, while neurons in the caudal part of the SpVi exhibited various angular tunings to all directions. These results indicate that the SpVi could be divided into two sectors by response properties.
Seiji Komagata 1,2 , Shanlin Chen 2 , Hiroki Kitaura 1 , Masaharu Kudoh 1 , Minoru Shibata 2 , Katsuei Shibuki 1 1 Department of Neurophysiology, Brain Research Institute, Niigata University; 2 Department Plastic Surgery, School of Medicine, Niigata University, Japan
We visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. Mice were anaesthetized with urethane (1.7 g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation (50 Hz for 1 s) applied to the left plantar forepaw. Changes in green fluorescence in blue light were observed in the right somatosensory cortex. Immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. However, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. The recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. The rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps.
Shin-ya Nakamura, Takaaki Narumi, Ken-Ichiro Tsutsui, Toshio Iijima Division System Neuroscience, Tohoku University Graduate School of Life Science, Sendai, Japan
In the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. The former includes the nucleus of trigeminal PrV and thalamic VPm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal SpVi and thalamic POm. In this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. Rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. The lesion or inactivation of PrV or VPm significantly impaired the task performance, whereas those of SpVi or POm did not. This result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination.
Kumiko Yokouchi 1 , Nanae Fukushima 1 , Tetsuhiro Fukuyama 2 , Akira Kakegawa 1 , Tetsuji Moriizumi 1 1 Department of Anatomy, Shinshu University School of Medicine, Matsumoto, Japan; 2 Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan
To know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (IO), mental (M) or lingual (L) nerves responsible for sensation of the upper and lower lips, and the tongue. For comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. The control, unilaterally IO or M nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. The bilaterally bulbectomized pups could not have access to the nipple. The bilaterally IO nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally M nerve-injured pups showed successful suckling. Suckling behavior of the bilaterally L nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. The results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling.
Takahiro Kawashima 1 , Takeshi Kawano 1 , Hidekuni Takao 1,2,4 , Kazuaki Sawada 1,2,4 , Hidekazu Kaneko 3,4 , Makoto Ishida 1,2,4 1 Department Electrical & Electronic Engineering, Toyohashi University of Technology, Aichi, Japan; 2 ISSRC, Toyohashi University of Technology, Aichi, Japan; 3 AIST, HSBE, Ibaraki, Japan; 4 JST, CREST, Japan A Si microprobe array (probe: Au-coated recording site at the tip, 2 m in diameter, 60 m in length; array: 40-m pitch) to record neuronal activities has been developed by using selective Si probe growth technique. To examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. Signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. However, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = 0.91). To record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance.
Research funds: KAKENHI (17206038), The 21st Century COE Program "Intelligent Human Sensing" PS2A-G113 A microelectrode positioning system for longterm extracellular recording of multiple neuronal activities Hidekazu Kaneko 1 , Hiroshi Tamura 2 , Shinya S. Suzuki 1 , Takahiro Kawashima 3 1 AIST, HSBE, Ibaraki, Japan; 2 Laboratory of Cognitive Neuroscience, Graduate School of Frontal Bioscience, Osaka University, Osaka, Japan; 3 Department Electrical & Electronic Engineering, Toyohashi University of Technology, Aichi, Japan A novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. The system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). The differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. The differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. Furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over 2 h.
Seiji Matsuda 1 , Takehiro Terashita 1 , Tetsuya Shimokawa 1 , Kyoujy Miyawaki 1 , Yuji Miguchi 1 , Takuya Doihara 1 , Jie Chen 1 , Shuang-yan Gao 1 , Chun-yu Li 1 , Min Wang 1 , Zhong Wang 1 , Bing Xue 1 , Naoto Kobayashi 1,2 , Kazuhiro Shigemoto 3 1 Department Anatomy, Ehime University of Medicine, Ehime, Japan; 2 Medical Education C, Ehime University of Medicine, Ehime, Japan; 3 Department of Hygiene, Ehime University of Medicine, Ehime, Japan This study shows the phylogenetic development of Cajal's initial glomeruli (IG) and Dogiel's pericellular nests (PCNs) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. The three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. The proportion of neurons having IG or PCNs increased with increasing phylogenetic complexity in the species examined here. In the chicks, the stem processes were longer and sometimes tortuous. Typical IG were observed not in frogs or chicks, but in rats and rabbits. Dogiel's PCNs also have been observed in the DRG of rats and rabbits. The nerve fibers in the PCNs were less than 1.2 m in diameter and had some varicosities. Some PCNs contain tyrosine hydroxylase-positive nerve fibers and varicosities.
Masayo Okumura, Eiji Kondo Matsumoto Dental University, Institute for Oral Science, Shiojiri, Japan
We established a rat nerve injury model using axotomy of the inferior alveolar nerve (IAN), and investigated its effect on gene expression in the trigeminal ganglion. Microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor STAT3, whereas other genes known to be regulated by STAT3 were not detected. STAT3 is expressed in many tissues and plays various roles. However, there have been few reports about the role of STAT3 in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. The aim of this study is to elucidate the role of STAT3 in gene expression in the trigeminal ganglion after IAN injury. At various time points, we analyzed and investigated changes of gene expression which are known to be influenced by STAT3 and STAT3phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and STAT3 are expressed. These results should help us understand injury-induced change mechanisms of the peripheral nerve.
Hirofumi Hashimoto 1 , Susumu Hyodo 2 , Makoto Kawasaki 1 , Minori Shibata 1 , Takeshi Saito 1 , Hiroaki Fujihara 1 , Takashi Higuchi 3 , Yoshio Takei 2 , Yoichi Ueta 1 1 Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan; 2 Laboratory of Physiology, Department of Marine Bioscience, Ocean Research Institute, University of Tokyo, Japan; 3 Department of Integrative Physiology, University of Fukui, Japan Adrenomedullin 2 (AM2) (identical to intermedin) belongs to the super family of AM. Centrally administered AM and AM2 activated oxytocin (OXT)-secreting neurons and increased plasma OXT level in rats. In the present study, we examined the effects of central administration of AM2 on OXT-secreting neurons and sympathetic outflow in comparison with that of AM in conscious rats. Effects of central administration of AM2 was stronger than those of AM and the effects of AM2 on OXT secreting neurons could not be blocked completely by pretreatment with CGRP or/and AM receptor antagonists. These data suggested that AM2 would have unknown receptor except CGRP and AM receptor.
Arata Oh-Nishi 1 , Makoto Saji 1 , Taku Uchida 1 , Sen-ichi Furudate 2 , Nobuyuki Suzuki 1 1 Division of Brain Science, Kitasato University, Graduate School of Medical Science, Kanagawa, Japan; 2 Division of Reproduction and Fetal Development, Kitasato University, Graduate School of Medical Science, Kanagawa, Japan
The mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. In this respect, NMDA receptors are thought to be crucially involved in cognitive and memory function. We have examined the effect of neonatal hypothyroidism and hyperthyroidism on the NMDA receptor function, using rats treated with methylmercaptoimidazole (MMI), which specifically blocks the biosynthesis of thyroid hormone and MMI-treated rats injected with thyroxine, respectively. Dose-response curves indicated that the sensitivity to NMDA of the NMDA receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. Concomitant with this observation, Western blot analysis showed that the NMDA receptor subunit NR1 expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats.
Our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (PNAS, 2004) . Here we report that the density and morphology of spines of pyramidal neurons in CA1 region are rapidly altered by treatments with nM estradiol and bisphenol A (xenoestrogen). Hippocampal slices are incubated with estradiol or bisphenol A for 2 h, and then neurons were injected iontophoretically with Lucifer Yellow. Three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by NeuroLucida Software. The results showed that in CA1, both estradiol and bisphenol A induce a significant increase in the total spine density, especially the density of thin spine. Synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. We investigated the effects of stress on enhanced green fluorescent protein (eGFP) expression in the arginine vasopressin (AVP)-eGFP transgenic (Tg) rats. After bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide eGFP fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. This Tg rat is a convenient tool to study dynamic changes of AVP expression in the hypothalamus under stressful condition.
Chitose Orikasa, Yasuhiko Kondo, Yasuo Sakuma Department of Physiology, Nippon Medical School, Tokyo, Japan
We report here a sex difference expression of somatostain mRNA within the sexually dimorphic nucleus of the preoptic area (SDN-POA), the volume of which depends on gonadal hormones during the ontogeny. In infant rats aged day 8-15, the volume of somatostain mRNA-positive region within the POA was significantly larger in males than in females and overlapped the SDN-POA in both sexes. The SDN-POA visualized by Nissl staining in adjacent sections agreed precisely with the extent of somatostain mRNA-positive cellular distribution. Orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day 15. The staining of somatostain mRNA in individual neurons was diminished when examined on day 35 or 70, albeit that the sex difference of the volume of somatostain mRNA-positive region persisted thoughtout the observed period. Somatostatin may play a role in the establishment of the SDN-POA, which lacks classic nuclear receptor for estrogen.
PS2A-G121 Short chain sugar acid, 2-buten-4-olide, activates oxytocin-secreting neurons in the hypothalamus of rats Makoto Kawasaki 1 , Tatsushi Onaka 2 , Hirofumi Hashimoto 1 , Hiroaki Fujihara 1 , Yoichi Ueta 1 1 Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan; 2 Department of Physiology, Jichi Medical School, Tochigi, Japan 2-Buten-4-olide (2-B4O), an endogenous sugar acid, which may be involved in the regulation of feeding. We examined the effects of 2-B4O on the hypothalamo-neurohypophyseal system in rats. The plasma oxytocin (OXT) levels were significantly increased at 15-60 min after intraperitoneal (i.p.) administration of 2-B4O (100 mg/kg), whereas plasma arginine vasopressin (AVP) levels did not change. Dual immunostaining revealed that Fos-like immunoreactivity (LI) was predominantly observed in OXT-secreting neurons in the paraventricular and the supraoptic nuclei 120 min after i.p. administration of 2-B4O. In addition, many Fos-LI neurons were also observed in the nucleus of the tractus solitarius (NTS) after i.p. administration of 2-B4O. These results suggest that peripherally administered high dose of 2-B4O activates OXT-secreting neurons in the hypothalamus through the activation of the NTS neurons.
PS2A-G122 Estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area Tomohiro Hamada, Yasuo Sakuma Department of Physiology, Nippon Medical School, Tokyo, Japan
The volume of the sexually dimorphic nucleus in the preoptic area (SDN-POA) is two to four times larger in male rat than in female, however function of this nucleus has not well known. In contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. Recently, transgenic rats expressing enhanced green fluorescent protein (EGFP) under the control of an estrogen receptor (ER) ␣ promoter were generated to tag ER␣-positive neurons in the brain. In the present study, we examined GFP expression could be used a marker for the SDN-POA. GFP labeled cells were distributed in the core of SDN-POA of male and female transgenic rats and in the majority of these cells included ER␣, immunohistochemically. Both area and number of GFP expressed cells in the SDN-POA were larger in male than in female, however, female GFP cells in the SDN-POA showed concentrated distribution than male. These results suggest that GFP labeled cells in SDN-POA could be useful marker to make clear the function of the SDN-POA. Recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. Although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. In the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. The neurons were transfected with Venus-actin. Time-lapse images were taken by laser scanning confocal microscope during steroid treatment. Testosterone but not estradiol increased the number of spines/filopodia of the dendrites within 4 h. These results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term.
Tetsuya Kimoto 1,2 , Shinpei Higo 1,2 , Yasushi Hojo 1,2 , Kouhei Nakajima 1,2 , Hironori Nakanishi 1,2 , Hirotaka Ishii 1,2 , Suguru Kawato 1,2 1 Department of Biophysics & Life Science, University of Tokyo, Tokyo, Japan; 2 CREST, JST, Japan Hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). Neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. However, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. In the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with HPLC system.
PS2A-G125 GABA depolarizes GnRH neurons isolated from adult GnRH-EGFP transgenic rats Chengzhu Yin, Nobuyuki Tanaka, Masakatsu Kato, Yasuo Sakuma Nippon Medical School, Department of physiology, Tokyo, Japan GnRH neurons are essential in the reproductive neuroendocrine system. In regulation of GnRH neurons, GABA may be one of the major players, especially in relation to GnRH/LH surge. We, therefore, performed a cell-physiological analysis of GABA action on rat GnRH neurons. Cells were dispersed from adult GnRH-EGFP transgenic rats and cultured overnight. GnRH neurons, were applied to the perforated patch-clamp configuration with gramicidin D. GABA evoked Cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. The reversal potential of the response was ranged from −40 to -10 mV in identified GnRH neurons in both sexes. There was no difference in the reversal potential among the stages of estrous cycle. In unidentified neurons, however, the reversal potential was more negative than -50 mV and most of them were ∼−80 mV. In conclusion, GnRH neurons isolated from adult rats express GABAA receptor and its reversal potential is more positive than the resting potential. Although the neural activation in the subfornical organ (SFO) by angiotensin II (ANGII) is widely regarded for the increments of ANGII-induced water intake and vasopressin release, galanin (GAL) have been reported to inhibit them. Therefore, GAL may inhibit neural activity of ANGII-sensitive SFO neurons. RT-PCR analysis demonstrated existences of all mRNAs of GAL receptor subtypes, GalR1, GalR2 and GalR3, in the SFO. In extracellular recording on SFO slice preparation, GAL dose-dependently led to inhibition of neural activity. All GAL sensitive neurons showed excitatory response by ANGII. GalR1 selective agonist M617 induced inhibitory responses, as well as GAL. In patch-clamp recordings, GAL induced outward current in some neurons. These results suggest that GAL inhibits neural activity in SFO neurons through, at least partially, outward current following activation of GalR1.
Hiroaki Fujihara 1 , Tomoki Fujio 1 , David Murphy 2 , Yoichi Ueta 1 1 Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan; 2 Molecular Neuroendocrinology Research Group, The Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, UK We have generated transgenic (Tg) rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. In this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking 2% saline for 5 days in 3, 6, 12 and 24 months old Tg rats. In 3 and 6 months, there were no difference between Tg rats and Tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. In aged Tg rats (12 and 24 months old), there were no obvious changes in water balance. These results suggest that the expression of AVP-eGFP transgene does not disturb body fluid homeostasis in Tg rats.
PS2A-H128 Prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus Takashi Mera 1 , Hiroaki Fujihara 2 , Hirofumi Hashimoto 2 , Makoto Kawasaki 2 , Tatsushi Onaka 3 , Takakazu Oka 1 , Sadatoshi Tsuji 1 , Yoichi Ueta 2 1 Department of neurology (Division of psychosomatic medicine), School of medicine, University of Occupational and Environmental Health, Japan; 2 Department of physiology, School of medicine, University of Occupational and Environmental Health, Japan; 3 Department of physiology, Jichi Medical School
We examined the effects of restraint stress (RTS), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (PrRP) gene expression in the hypothalamus and brainstem. Moreover, we examined the effects of pretreatment with an anti-PrRP antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (PVN). RTS, nociceptive stimulus and acute inflammatory stress upregulated the PrRP gene expression in the brainstem. Pretreatment with anti-PrRP antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the PVN. These results suggested that PrRP is a potent and important mediator of stress response in the brain through the hypothalamic PVN.
Taieb Bousejin 1 , Afsaneh Eliassi 1 , Nasser Naghdi 2 , Ali ghanbari 1 1 Ghsemi; 2 Pastor Institute, Tehran, Iran
The purpose of this study was to consider the role of the ventromedial hypothalamus (VMH) D1 Receptors on histamine-induced gastric acid secretion (GAS). The animals were anasthetized and guide cannulas were implanted unilaterally above (0.5 mm) VMH. Animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. IV infusion of histamine in sham grup induced marked increase in GAS with a peak response that started from 30 min up to the end of experiments (90 min). At the peak acid response, the VMH microinjection SKF38393 (0.1,1) significantly reduced the amount of GAS (p < 0.001). There was no any effect by microinjected SCH23390 (0.1) into the VMH. Injecting SKF38393 into the VMH, 5 min after SCH23390, had no effect on GAS in compare with control. But, the acid suppressant effect of SKF38393 was completely removed by peripheral injection of SCH23390 (p < 0.01). Our results show that the VMH D1 dopamine receptors have regulatory mechanisms of GAS by interaction with H2 receptors through an inhibitory neural pathways.
Zhilin Song, Celia D. Sladek Department of Physiology, UCHSC, Aurora, CO, USA Although prior studies demonstrated expression of P 2X purinoceptors in supraoptic neurons (SON) and indicated their importance in ATP stimulated vasopressin release, in studies monitoring the effect of ATP on intracellular Ca ++ ([Ca ++ ] i ), we have obtained evidence that P 2Y purinoceptors (P 2Y R) are important in the response to ATP. ATP stimulated [Ca ++ ] i increase was maintained in Ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [Ca ++ ] i stores. P 2Y R agonists increased [Ca ++ ] i in SON, with P 2Y1 R agonist being the most effective. The possibility that P 2Y1 R mediates ATP induced [Ca ++ ] i increase in SON was further evaluated using a P 2Y1 R antagonist, MRS2179. ATP stimulated increase in [Ca ++ ] i was greatly attenuated by MRS2179 (100 M) in 2 mM Ca ++ medium. In Ca ++ -free medium, there was no significant response to ATP in the presence of MRS2179. Furthermore, combined treatment with MRS2179 and PPADS (10 M, a P 2X R antagonist) also abolished the [Ca ++ ] i response to ATP. These results demonstrated that P 2Y1 R mediates a large portion of the [Ca ++ ] i response to ATP challenge in SON.
PS2A-H131 Analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus Hirotaka Ishii 1,2 , Yasuhiro Sonoki 1,2 , Aizo Furukawa 2,3 , Yasushi Hojo 2 , Tetsuya Kimoto 1,2 , Suguru Kawato 1,2 1 Department of Biophysics and Life Science, University of Tokyo, Tokyo, Japan; 2 CREST, JST, Japan; 3 Kurihama National Hospital, Japan Brain neurosteroids are steroids synthesized endogenously in the brain. Our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. To define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. This study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day 1(PD1) to the adult stage using RT-PCR/Southern blotting. Enzymes required for the synthesis of estradiol from cholesterol were expressed form PD1 to PD14 with a higher level than in the adulthood. These results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function.
Hideo Mukai 1,2 , Gen Murakami 1 , Shirou Kominami 3 , John H Morrison 4 , William G.M Janssen 4 , Tetsuya Kimoto 1,2 , Suguru Kawato 1,2 1 Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro, Tokyo 153-8902, Japan; 2 CREST Project, JST, Japan; 3 Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739, Japan; 4 Kastor Neurobiology of Aging Laboratories, Fishberg Research Center for Neurobiology, USA Estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. However, the existence of estrogen receptor alpha (ER␣) still remains elusive. With highly purified antibody RC-19, mass spectrometric analysis identified ER␣ in the hippocampus and immunohistochemistry showed ER␣ localization in principal neurons of CA1, CA3, and granule cells in dentate gyrus. Further, Western blot revealed that ER␣ is contained in PSD fraction, confirming the observation with immunoelectron microscopy. These results imply that the synaptic ER␣ mediates the effects of estrogen in hippocampal neurons.
Ken Takumi GnRH neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. GnRH neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of GnRH is controlled by a complex of excitatory and inhibitory inputs. However, anatomical studies which quantified the direct input on GnRH neuron are few. In this study, we quantitatively analysed glutamatergic and GABAergic input onto GnRH neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between GnRH neuron and axon terminals immunoreactive for either VGluTs or VGAT were counted and the densities of the appositions on the dendrites and soma were calculated.
Sabine GOURAUD 1 , Song T. Yao 1 , Jing Qiu 1 , Julian FR Paton 2 , David Murphy 1 1 University of Bristol, HW-LINE, UK; 2 Department of Physiology, Bristol Heart Institute, University of Bristol, United Kingdom
The neuropeptide hormone vasopressin (VP) is produced in the magnocellular neurons of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei and stored in the posterior pituitary (PP). Dehydration evokes an increased expression of the VP gene in magnocellular neurons and a massive release of VP from the PP in the circulation to promote the water conservation at the kidney level. In parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (HNS) is observed but poorly understood. We investigated this activity dependent plasticity of the HNS using proteomic (2D Fluorescence Difference Gel Electrophoresis (DIGE)) combined with MALDI mass spectrometry approaches to identify proteins that change in abundance in the SON and the PP from 3 days dehydrated rats. A truncated form of ProSAAS, a Granin-Like Neuroendocrine Peptide Precursor known as a potent inhibitor of the prohormone convertase 1, has been found decreased in the PP and increased in the SON.
Ichiro Nishimura, Masakatsu Kato, Yasuo Sakuma Department of Physiology, Nippon Medical School, Tokyo, Japan Function of Gonadotropin-releasing hormone (GnRH) neurons is regulated by gonadal steroid estrogen. However, the precise mechanism of estrogen action upon these cells has not been clarified. We investigated a direct action of estrogen on the regulation of potassium current in GnRH neuronal cell line GT1-7. Delayed rectifier potassium current (I K ) and large-conductance calcium-activated potassium (BK) current were recorded by patch clamp configuration in GT1-7 cells cultured in DMEM supplemented with 10% FBS for 3 days. BK current was increased by addition of 17-estradiol (E2) in culture medium in a physiological concentration range. This action of E2 was blocked by ICI-182,780, a potent estrogen receptor (ER) antagonist. We further examined whether E2 acted through ER ␣ or ER  by using selective agonists PPT and DPN, respectively. The DPN augmented the BK currents similar to the effect of E2 but PPT had no effect. E2 had no effect on the I K . These results indicate that E2 increases the BK current by activating ER without affecting the I K .
Research funds: KAKENHI 16590180, 16086210 PS2A-H136 Myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary Katsutoshi Taguchi 1 , Haruko Kumanogoh 2 , Shun Nakamura 2 , Seiji Miyata 3 , Shohei Maekawa 1 1 Department of Biosystems Science, Kobe-University, Kobe, Japan; 2 Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, NCNP, Tokyo, Japan; 3 Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan A membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. Here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. One of them, 25 kDa protein, was identified as myelin protein zero (P0) from mass analysis and this result was confirmed by western blotting that a specific antibody to 25 kDa band reacted to an authentic P0 prepared from rat sciatic nerve myelin. P0 is a type I transmembrane glycoprotein and a member of the immunoglobulin superfamily. The expression of P0 has been believed to be restricted to the peripheral myelin in mammals. Our result, however, indicates that P0 expresses more widely and participates in cell communications.
Mari Ogiue-Ikeda, Norio Takata, Suguru Kawato Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan 17-estradiol (E2) has a rapid effect on synaptic transmission. Recently, we found that hippocampal neurons synthesize E2 (Hojo et al., 2004) , and express estrogen receptor ␣ (ER␣) at synapses. Endocrine disrupters are representative estrogenic industrial compounds. While their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. In this study, we investigated the effects of E2 and endocrine disrupters (DES, BPA, NP, OP and TBT) on NMDA-induced LTD in the rat hippocampal CA1, CA3 and DG with a custom made multi-electrode measuring system (MED64). LTD was enhanced by E2 dose-dependently in CA1, CA3 and DG. DES, BPA, NP, OP and TBT had similar or different effects on LTD dose-dependently. Our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of LTD of the hippocampal neurons. We examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (GFP), arginine vasopressin (AVP) and oxytocin (OXT) genes and body fluid balance in AVP-enhanced (e) GFP transgenic rats. Chronic salt loading caused marked increase of the eGFP fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. There were no differences of the AVP and OXT gene expressions in the hypothalamus, plasma AVP and OXT levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. Humoral responses to chronic salt loading were maintained in AVP-eGFP transgenic rats.
Takeshi Saito 1 , Takushi X. Watanabe 2 , Tomoko Urabe 2 , Hirofumi Hashimoto 1 , Hiroaki Fujihara 1 , Yukio Hirata 3 , Yoichi Ueta 1 1 Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan; 2 Peptide Institute, Inc., Osaka, Japan; 3 Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University, Tokyo, Japan Salusin- was newly discovered as a bioactive endogenous peptide. Low concentration of salusin- stimulates the secretion of arginine vasopressin (AVP) from perifused rat hypophysis. Salusin- coexists with AVP, but not oxytocin, in the rat magnocellular supraoptic (SON) and paraventricular nuclei (PVN). To further investigate the physiological role of salusin- in body fluid homeostasis, we examined the effects of salt loding for 5 days on salusin--like immunoreactivity (LI) in the SON and PVN of rats by immunohistochemistry. The marked increase of salusin--LI in the SON and PVN were observed in the salt loaded rats. The result suggests that salusin- may play a role of body fluid balance by regulating AVP release.
Naoyuki Yamamoto 1 , Hao-Gang Xue 1 , Yuji Ishikawa 2 , Yoshitaka Oka 3 , Hitoshi Ozawa 1 1 Department of Anatomy and Neurobiology, Nippon Medical School, Tokyo, Japan; 2 National Institute of Radiological Sciences, Chiba, Japan; 3 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
The terminal nerve GnRH (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. The major afferent source to the GnRH neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. Here, we examined the presence of such an afferent pathway in medaka Oryzias latipes. Injections of a DiI crystal into the cluster of GnRH neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the GnRH neurons. These results suggest that the afferent pathway to the GnRH neurons is a character shared by "advanced" teleosts like medaka and percomorphs.
Takeshi Yamazaki 1 , Eiji Munetsuna 1 , Asuka Kamogawa 1 , Suguru Kawato 2 , Shiro Kominami 1 1 Graduate School of Integrated Arts Science, Hiroshima University of Higashihiroshima, Japan; 2 Graduate School of Arts and Science, Tokyou University of Tokyo, Japan Tributyltin, TBT, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. To analyze molecular mechanism of stimulation of estrogen synthesis, we determined mRNA contents of estrogen biosynthetic enzymes and activity of P450arom in the hippocampus.
Method: The cultured hippocampus slices from 10 days male rat were treated with 100-1000 nM of TBT for 48 h. After the treatment, total RNA was extracted and the levels of mRNA of estrogen synthetic enzymes were quantified by real-time RT-PCR. Activity of P450arom was determined by quantification of [3H]estradiol from [3H]testosterone.
Result: Forty-eight hours treatment of hippocampal slices with 100 nM TBT induced increases in mRNA contents of P450arom, and with 1000 nM TBT induced that of 3-HSD. Estradiol content was increased by the treatment with 100 nM TBT, but not affected by 1000 nM TBT. TBT may modulate estradiol synthesis by alteration of expression of P450arom. The medial preoptic area (mPOA) is an important neural site for regulation and maintenance of sleep. Studies have indicated that GABAergic neurons and terminals at the mPOA are active during sleep. Present study was carried out to elucidate the contribution of GABA-A receptor at the mPOA in sleep-wakefulness (SW) in male Wistar rats. The SW was assessed by chronically implanted electrodes for EEG, EOG, and EMG. A bilateral guide cannula was also implanted for drug injection into the mPOA. After recovery, three baseline sleep recordings were taken for 4 h on different days. Bicuculline methoiodide (GABA-A receptor antagonist) at a dose of 30, 60, 90 and 120 ng in 200 nl was injected bilaterally into the mPOA in different groups of rats and their SW was studied for subsequent 4 h. The 30 ng dose of bicuculline methoiodide had minimum effect whereas 60 and 90 ng produced arousal. Maximal wakefulness was observed at dose of 90 ng with no further increase in wakefulness at higher dose of 120 ng. The results suggest the involvement of GABA-A receptors at the mPOA in SW.
Yoshiaki Isobe 1 , Hiroyuki Tsuda 2 1 Department of Neuro-physiology and Brain Science, Nagoya City University, Graduate School of Medical Sciences, Nagoya, Japan; 2 Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences, Japan Locomotor activity in rodents shows free-running circadian rhythms even under the constant light. Constant light exerts a promoting effect on hepatic carcinogenesis. After the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via Wee1). To know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (DEN), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. DEN was injected (i.p., 200 mg/kg) on rats during the free-running condition under the constant dim light (DD) and constant light (LL). The effects of DEN were gentle under the DD. However, under the LL, phase delay accompanying the elongation of circadian period () was observed. Decrement of an amount of activity in 24 h after the DEN administration was obvious under the LL compared with that under the DD. This study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. Although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, (1 antagonist) were effective, naltrindole (␦ antagonist) and nor-BNI ( antagonist) did not interrupt the hibernation. The increment of c-Fos expression was observed in arcuate nucleus (ARC) at 1 h after from hibernation onset compared with before hibernation. In addition, a localized -endorphin-like immunoreactivity (-End IR) was observed in neuronal perikarya in ARC at 1 h after from hibernation onset. Although -End IR in ARC got weak, the -End IR of nerve fibers in preoptic nucleus (PON) got strong with progression of hibernation. These results suggest that the -endorphin was transported to PON from ARC by axonal flow and then played an important role in maintenance of hypothermia via 1-opioid receptors in hibernation.
Wei-Min Qu, Zhi-Li Huang, Naomi Eguchi, Yoshihiro Urade, Osamu Hayaishi Department of Molecular and Behavioural Biology, Osaka Bioscience Institute, Osaka, Japan Prostaglandin (PG) D 2 is a potent somnogenic substance, and isomerized from PGH 2 through the action of PGD synthase (PGDS). PGDS has two distinct types, the lipocalin-type PGDS (L-PGDS) and hematopoietic PGDS (H-PGDS). Selenium compounds have been reported to decrease sleep by inhibiting PGDS in rats. To clarify what type of PGDS inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected SeCl 4 into L-PGDS and H-PGDS knockout (KO), and their wild-type (WT) mice. In WT mice, SeCl 4 decreased rapid eye movement (REM) and non-REM sleep for 5 h after injection and, concomitantly, increased wakefulness. Similar results were observed in H-PGDS KO mice. In contrast, L-PGDS KO mice did not exhibit any significant changes in sleep-wake profiles after SeCl 4 administrations. These findings indicate that PGD 2 plays an essential role in the maintenance of the sleep state under physiological conditions, and L-PGDS is a key enzyme for the production of PGD 2 involved in sleep-wake regulation. Under baseline conditions, H 1 R KO mice showed essentially identical sleep-wakefulness cycles to those of wild-type (WT) mice but with fewer incidents of brief awakening (<16 s epoch), prolonged duration of non-rapid eye movement (NREM) sleep episodes, a decrease in the number of state transitions between NREM sleep and wakefulness, and a shorter latency for initiating NREM sleep after an intraperitoneal injection of saline. The H 1 R antagonist pyrilamine mimicked these effects in WT mice. These results indicate that H 1 R is involved in the regulation of behavioral state transitions from NREM sleep to wakefulness (Huang et al., 2006) .
PS2A-H147 Dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice Koichi Fujimura, Ai Fukushima, Takahiro Nakamura, Toshihiro Jogamoto, Kazuyuki Shinohara Division of Neurobiology & Behaviour, Nagasaki University, Graduate School of Biomedical Science, Nagasaki, Japan
The involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (SCN) neuron was investigated by extracellular single unit recordings from the optic chiasma and the SCN in mice. Recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power LED (λ = 500 nm). The SCN neurons responded to the light in intensities above 10 11 photons/cm 2 /s and were activated maximally at around 10 15 photons/cm 2 /s, they were about 1.6 log units less sensitive than optic fibers with high sensitivity. A sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the SCN neuron did not suppress the subsequent light response in the SCN neurons, except in a few neurons. These results suggest that the most of the light responsive SCN neurons are driven by any inputs independent of the high sensitive optic fibers.
Masayuki Ikeda, Tomoyoshi Kojiya Department of Biology, Faculty of Science, Toyama University, Japan
The hypothalamic suprachiasmatic nucleus (SCN) has a pivotal role in the mammalian circadian clock. SCN neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. We have found robust circadian rhythms in the cytoplasmic concentration of Ca 2+ in SCN neurons. Since cytosolic Ca 2+ regulates diverse cellular systems, we have hypothesized that the cytosolic Ca 2+ rhythms may mediate the cellular output from the clock gene oscillations. Here, to address the clock gene functions on the Ca 2+ rhythms, mouse Bmal1 and its dominant negative sequence (mBMF1R5) are transfected into the organotypic culture of SCN with a yellow cameleon Ca 2+ sensor by the gene gun. The results demonstrated that over-expression of Bmal1 or mBMF1R5 significantly inhibited the circadian Ca 2+ rhythms and thus we concluded that the native Bmal1 rhythm is essential for cellular output processes of the murine clock system.
PS2A-H149 The activation of ␣2 adrenergic receptor increases the frequency of carbachol-induced  oscillation in rat hippocampal slices Masafumi Nakano, Jun Arai, Kiyohisa Natsume Kyushu Institute of Technology, Kyushu, Japan
Recently it is found that locus ceruleus (LC) activation suppresses  rhythm in hippocampus in vivo. Noradrenergic fibers derived from LC project to hippocampus. Carbachol, a cholinergic agent, can induce  oscillation in rat hippocampal slices like  rhythm in vivo. In the present study, the effect of epinephrine on the generation of carbachol-induced  oscillation in CA3 region of rat hippocampal slices. Carbachol (30 M) induced  oscillation with the frequency and the amplitude of 14.8 ± 0.1 Hz, and 0.7 ± 0.1 mV, respectively (mean ± S.E.M.; n = 3). Epinephrine (50 M) significantly increased the frequency of 16.2 ± 0.1 Hz (**p < 0.01), not change the amplitude. Clonidine (50 M), an ␣2 receptor agonist, alone significantly increased the frequency at the concentration of 50 M (*p < 0.05). Yohimbine, an ␣2 receptor antagonist, suppressed the oscillation. These results suggest that the application of adrenaline will increase the frequency of hippocampal  rhythm via ␣2 receptor. Attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. We recorded transient activities of two neuron groups by two tetrodes apart 0.4 mm from each other in the hippocampal CA3 region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. It was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. Peri-stimulus time histogram (PSTH) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency 100-300 ms), oscillatory (2-4 Hz) and sustained responses. Following the pairing stimuli, the PSTH showed change in transient response properties (8/9 slices). These results suggest the pairing stimuli would change attractor dynamics of the neural network in the CA3 region.
Ryozo Aoki 1 , Hiroshi Wake 2 , Hitoshi Sasaki 1 , Kiyokazu Agata 3 1 Dept. Physiol. & Biosignal. Osaka Univ. Grad. Sch. Med., Suita, Japan; 2 Dept. Elec. Eng. & Elec. Col. Industri. Tech., Amagasaki, Japan; 3 Dept. Biophys., Kyoto Univ. Grad. Sch. Sci., Kyoto, Japan By insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. The frequency spectrum showed an almost monotonously decreasing distribution likely as 1/f, ranging from 10 − 1 to 10 + 2 Hz. During the EEG recordings the planarian was kept still by cooling in several degrees. When it was cooled down to lower temperatures the amplitude of EEG was suppressed, and by warming again restored with spikes provably due to motions. This EEG active state continued beyond 40 min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to 60 min. By observing the sample it turns out the sticked head was degraded. Strong photo stimulation suppressed this EEG signals and recovered after over 30 min. However little response to light pulse stimuli was observed on the EEG spectrum.
Mariko Uchida 1 , Hiroki Sato 1,2 , Naoki Tanaka 2 , Atsushi Maki 1,2 1 Japan Science and Technology Agency, CREST, Saitama, Japan; 2 Advanced Research Laboratory, Hitachi, Ltd., Saitama, Japan Previous studies about electroencephalography (EEG) described that alpha-wave power (the frequency band from 8 to 12 Hz) decreases and the sleep spindle power (from 12 to 16 Hz) increases in falling asleep. The purpose of this study is to analyze the crosscorrelation between the EEG power changes (EEGPC) of each band and the cortical hemoglobin concentration changes (HbCC) during sleep. We measured optical topography (OT) and EEG simultaneously. The HbCC was measured at eighty-eight positions covering whole head of subject by OT probes. Five females and eight males participated in this measurement. The results showed the high correlation between EEGPC and HbCC at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. The time lag from EEGPC to HbCC was from 1 to 8 s in (i), and from 8 to 15 s in (ii). We examine these differences between (i) and (ii) in detail. Carnitine deficiency disturbs fatty acid oxidation under the fasting condition (FC). We show herein that nocturnal locomotor activity (LA) was reduced under FC and recovered to normal by carnitine injection in Jvs −/− mice, a model of systemic carnitine deficiency. As judged from EEG/EMG profiles, Jvs +/+ mice showed prolonged wakefulness under FC, but Jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-REM sleep. As the orexinergic arousal system plays an important role in LA, we determined orexin neuronal activity in the fasted mice. Fasted Jvs −/− mice had fewer c-Fos + orexin neurons in their lateral hypothalamus and a reduced orexin-A content in their CSF, suggesting that the fasted Jvs −/− mice exhibited reduced LA and fragmented of wakefulness due to suppressed orexin neuronal activity.
Juhyon Kim 1 , Kazuki Nakajima 1 , Yutaka Oomura 2 , Kazuo Sasaki 1 1 Div. of Bio-Information Eng., Univ. of Toyama, Toyama, Japan; 2 Dept. of Integrat. Physiol., Kyushu Univ., Fukuoka, Japan Novel peptide, orexin, identified in the lateral hypothalamus (LH) participates in the regulation of sleep-wakefulness. Orexin-containing neurons in the LH project to the pedunculopontine tegmental nucleus (PPT). The PPT is one of brain sites which control sleepwakefulness. Thus, we examined effects of orexin on PPT neurons electrophysiologically using brain slice preparations in rats. Applications of orexin depolarized the membrane potential of PPT neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. When extracellular K + concentration was increased, the magnitude of the depolarization significantly decreased. When extracellular Na + was replaced by N-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. These results suggest that the ionic mechanism for orexininduced depolarization includes K + channel, non-selective cation channel and/or Na + /Ca 2+ exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on PPT neurons.
Ben-Shiang Deng 1 , Wei Zhang 1 , Akira Nakamura 2 , Masashi Yanagisawa 3 , Yasuichiro Fukuda 2 , Tomoyuki Kuwaki 1,2 1 Dept. Molec. Integ. Physiol., Chiba Univ., Japan; 2 Dept. Autonom. Physiol., Chiba Univ., Japan; 3 Dept. Molec. Genet., Univ. Texas, USA We examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (KO) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. We also studied whether pharmacological blockade of orexin in the wildtype mice (WT) resulted in a similar abnormality. Ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-A, -B, SB-334867 (an orexin receptor antagonist), or vehicle. Data were examined for only awake periods because sleeping distorts the chemoreflex. Hypercapnic ventilatory responses but not hypoxic responses were attenuated in KO. Similar abnormality was reproduced in WT treated with SB-334867. Icv injection of orexin partially restored the hypercapnic chemoreflex in KO. Our findings suggest that orexin plays a crucial role for CO2-sensitivity at least during waking periods.
Research funds: KAKENHI 16590162,17590183
Junko Hara 1 , Taizo Matsuki 2 , Katsutoshi Goto 1 , Masashi Yanagisawa 3 , Takeshi Sakurai 1,2 1 Department of Pharmacology, Basic Medical Science (COE), University of Tsukuba, Ibaraki, Japan; 2 Yanagisawa Orphan Receptor Project, ERATO, JST, Tokyo, Japan; 3 Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas, Dallas, Texas, USA When the production of inflammatory cytokines is stimulated by acute inflammatory, the nonREM-sleep amount of animals increases. This is possibly due to changes in the biological activity of the TNFalpha system. Besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. Orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. Recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. Here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. These effects might also possibly be involved in the pathophysiology of narcolepsy.
PS2A-I157 Prenatal exposure to bisphenol A enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons Tetsuya Fujimoto 1 , Kazuhiko Kubo 2 , Shuji Aou 1 1 Dept. Brain Sci. Eng., Kyushu Inst. Technol., Kitakyushu, Japan; 2 Dept. Otorhinolaryngol., Chidoribashi Hospital, Fukuoka, Japan Prenatal exposure to Bisphenol A (BPA) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (Fujimoto et al. 2006) . In this study, the effects of BPA on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. The smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by BPA. In the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. This type of neurons did not respond to plant odors. In contrast female amygdala neurons did not show such selectivity. The sex difference in this neuronal response pattern was attenuated by BPA exposure. These findings suggest that BPA impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators.
Research funds: Grants-in-aid for Scientific Research (no. 16209006, S.A.)
Shuji Aou 1 , Tetsuya Fujimoto 1 , Yumi Ichihara 1 , Kimiya Narikiyo 1 , Toru Ishidao 2 , Hajime Hori 2 , Yukiko Fueta 2 1 Dept. Brain Sci. Eng., Kyushu Inst. Technol., Kitakyushu, Japan; 2 Dept. Environm. Manage., Sch. Health Sci., Univ. Occup. Environm. Health, Kitakyushu, Japan 1-Bromopropane (1-BP), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. In this study, we investigated the effects of prenatal exposure to 1-BP on sexual differentiation of reproductive and non-reproductive behaviors. Pregnant rats were exposed to 700 ppm of 1-BP during prenatal period. The open-field test, Lashley III maze test and sexual behavior were evaluated at adult age. 1-BP significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. In sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. These results suggest that 1-BP is the potential candidate of endocrine disruptors which affect brain development. (16651027) PS2A-I159 Changes in hippocampal excitability of rats prenatally exposed to 1-bromopropane Yukiko Fueta 1 , Toru Ishidao 1 , Susumu Ueno 2 , Yasuhiro Yoshida 3 , Hajime Hori 1 1 Department of Environmental Management, School of Health Sciences; 2 Department of Pharmacology; 3 Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan Inhalation exposure to 1-bromopropane (1-BP), a substitute for ozone depleting compounds, alters the function of GABAergic system in the hippocampus of adult male rats. But the neurotoxcitiy induced by prenatal exposure has not been well investigated. In this study pregnant rats were exposed to 1-BP (700 ppm) during gestational day 1-20 (6 h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. Basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. Offspring, however, exhibited an enhancement of averaged S/R curve of PS in the CA1 at the PND 12-15. Conversely, S/R curves of fEPSP as well as PS in the CA1 were inhibited at the age of 6-8 weeks. Our results suggest that 1-BP causes hyperexcitability in pregnant rats, and disrupts basic excitability in the CA1 of the offspring during development.
Research funds: Grant-in-Aid for Exploratory Research (16651027) PS2A-I160 Effects of endocrine disrupting chemical bisphenol A on the development of mouse cerebral cortex Keiko Nakamura 1,2 , Kyoko Itoh 1 , Takeshi Yaoi 1 , Tohru Sugimoto 2 , Shinji Fushiki 1 1 Dept. Pathol. Appl. Neurobiol., Kyoto Pref. Univ. Med, Kyoto, Japan; 2 Dept. Pediatr., Kyoto Pref. Univ. Med, Kyoto, Japan Bisphenol A (BPA), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. We have thus studied whether prenatal exposure to low-doses of BPA affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. Pregnant mice were injected subcutaneously 20 g/kg of BPA daily from embryonic day 0 (E0). Control animals were injected vehicle alone. For evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (BrdU) was given to pregnant mice and processed for immunohistochemistry. The total RNA was extracted from embryonic telencephalons at different embryonic period. BrdU-labeled cells were decreased in the ventricular zone at E14.5 and E16.5, whereas those cells increased in the cortical plate at E14.5, as compared with control mice. Some of the genes associated with neurogenesis and thyroid hormone function were upregulated in BPA-treated group.
Research funds: JSPS grant 15390334
Keiko Ikemoto 1 , Teruko Uwano 2 , Hisao Nishijo 2 , Taketoshi Ono 2 , Masayuki Ito 3 , Ikuko Nagatsu 4 , Katsuji Nishi 5 , Shin-ichi Niwa 1 1 Dept. Neuropsychiat., Fukushima Med. Univ. Sch. Med.; 2 Toyama Med. Pharm. Univ.; 3 Faculty Med., Mie Univ., Mie, Japan; 4 Fujita Health Univ. Sch. Med., Toyoake, Japan; 5 Dept. Leg Med., Shiga Univ. Med. Sci., Japan
We examined the effect of maternal repeated cold stress (RCS) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (TH) immunohistochemistry. RCS was loaded to pregnant rats between day 9 and 19 after fertilization. Pups were perfused at postnatal day 8. In the frontal cortex, the number of largesized (more than 7 m in diameter) TH-immunoreactive (-ir) varicosities was significantly smaller in prenatally RCS rats than controls. In the locus coeruleus of prenatally RCS rats, TH immunoreactivity was less than that of controls. In the medullary C1/A1 catecholaminergic field, the size of TH-ir neurons was smaller and the quantity of THir fibers were less in prenatally RCS rats, although there were not significant differences. It was suggested that prenatal RCS impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates.
PS2A-I162 Developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response Maiko Kawaguchi 1,2,3 , Kaori Morohoshi 3,4 , Rie Yanagisawa 3 , Erina Saita 5 , Gen Watanabe 6,7 , Masatoshi Morita 3 , Kazuyoshi Taya 6,7 , Hirohisa Takano 3,4 , Toshiyuki Himi 1,2 , Hideki Imai 3,8 1 Dept. Toxicol and Pharmacol., Facul. Pharmacy, Musashino Univ., Tokyo, Japan; 2 Res. Inst. Pharmaceut. Sci., Musashino Univ., Tokyo, Japan; 3 Nation. Inst. for Environ. Stud., Ibaraki, Japan; 4 Grad. Sch. Environ. Sci., Univ. Tsukuba, Ibaraki, Japan; 5 Wildlife Rescue Veterinarian Associ., Tokyo, Japan; 6 Facul. Agriculture, Tokyo Univ. Agriculture & Technol., Tokyo, Japan; 7 The United Grad. Sch. Veterinary Sci., Gifu Univ., Gifu, Japan; 8 Div. Environ. Health Sci., Dep. Social Med., Facul. Med., Miyazaki Univ., Miyazaki, Japan Antiseptic pentachlorophenol (PCP) treatment to rats affects thyroid hormone (TH) system, which is essential for normal development of central nervous system. In this study, we show the exposure to PCP during gestation and lactation suppressed plasma TH level, and induces gene expression of neurogranin and TH receptor , which play a role in neural formation. The present data suggest that PCP may affect central nervous system development, though stress response was not affected by PCP exposure.
PS2A-I163 The effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the Fos expression in the brain, and the level of plasma corticosterone in offspring rat Hiroshi Abe, Noriko Hidaka, Kei Odagiri, Yuko Watanabe, Yasushi Ishida Dept. of Psychiatry, Miyazaki Med. Coll., Univ. of Miyazaki, Japan One group (PSY) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. The other group (C) was born from the dams not exposed to such stress. PSY, comparing to C, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. On the other hand, there were no significant differences between the number of Fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. However, plasma corticosterone was elevated in PSY compared with C. These results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate.
Akane Nakasato, Yasushi Nakatani, Yoshinari Seki, Hideho Arita Department of Physiology, Toho University school of Medicine, Tokyo, Japan
To evaluate roles of DA and serotonergic (5-HT) systems in stressinduced anxiety, we measured brain DA and 5-HT levels before, during and after a forced swimming test (FST) in autistic model of the rat. The model rat was made by exposing a pregnant rat to valproic acid (VPA). Our previous study demonstrated that the autistic model exhibited abnormality of 5-HT system and behavioral impairments related to autism. In the present experiment, we gave a prolonged FST for 60 min in the model rat, which frequently experienced to be drowned after the immobility time during FST. Brain DA and 5-HT levels were measured from samples collected from the prefrontal cortex (PFC). We found a gradual and steady increase in PFC DA level during FST, although 5-HT level showed only transient augmentation. Behavioral alteration after FST was characterized by an increased appetite during light phase (sleep) of circadian cycle. We suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical DA system.
Shigeo Masaki, Eiko Aoki, Satoshi Yonezawa, Atsuo Nakayama Dept. Embryology, Inst. Developmental Res., Kasugai, Aichi, Japan Neuroligin (NL1-4) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. Recently, several studies have implicated NL3 and NL4 in autistic disorders. NLs have a relative identical structure (∼70%); NL1 and NL2 localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while NL3 seems express in the olfactory glia, but NL4 distribution is unknown. Here we have generated antibody against human NL4, and explored its distribution in the post mortem human tissues. In the central and peripheral nervous system, NL4 was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. NL4 was observed in paraneurons and some endocrine cells outside the CNS. These results suggested that NL4 is important for neuroendocrine function. NL4 cDNA was transfected to in neuroblastoma. Formed spine-like structures on the cells expressing NL4 were rough and thicker than those of NL1 or NL3 transformants. It suggested the unique activity of NL4 for synapse formation. Motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. Recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. However, the underlying mechanisms are yet to be elucidated. We report here that the knockout (KO) mice deficient in the expression of motopsin exhibit morphological abnormality. Golgi-Cox staining revealed that spine density on both apical and basal dendrites of hippocampal CA1 pyramidal neurons in the KO mice significantly decreased than in the wild type mice. Similarly, spine density tended to decrease at cingulate cortex of the KO mice than wild type. Our results suggest that motopsin affects dendritic spine formation and/or stabilization. Mental retardation is a frequent disorder affecting 1-3% of the population. Recently the truncation of motopsin/neurotrypsin gene has been identified in Algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an IQ below 50. Here we report that knockout (KO) mice lacking motopsin gene mildly impaired water maze performance and social behavior. The KO mice significantly delayed the latency to the platform area on a probe test of hidden version of Morris water maze although they showed the similar performance to wild-type mice during training session. In a social memory test, the KO mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. Our results suggest that the KO mice provide insights into the molecular mechanisms important for development of cognitive functions.
Natsue Yoshimura 1 , Daisuke Horiuchi 1 , Tomoyuki Miyashita 2 , Minoru Saitoe 2 , Hitoshi Okazawa 1 1 Department of Neuropathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; 2 Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan Polyglutamine tract binding protein-1 (PQBP-1) was originally isolated as one of the candidates for polyglutamine disease related protein. Recently, several groups has reported about PQBP-1 disease that PQBP-1 mutant causes X-linked mental retardation (XLMR). To investigate the function of PQBP-1 in XLMR pathology, we produced two kinds of flies, human PQBP-1 overexpression flies (hPQBP1 flies) and drosophila PQBP-1 knock-down flies (dPQi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (STM) to long-term memory (LTM). The hPQBP1 flies showed memory impairment in LTM. In current study, we analyze memory abilities of the dPQi flies to observe detailed function of PQBP-1 in memory formation.
Seiji Hayashizaki, Masahiko Takada Tokyo Metropolitan Institute for Neuroscience, USA When two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. This has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (BLA) and the nucleus accumbens (NA). It has been shown that the BLA and NA are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. Thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. To this end, we implanted cannulae bilaterally and electrodes into the BLA and NA to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer.
Xue-Zhi Sun 1 , Sentaro Takahashi 1 , Yoshihisa Kubota 1 , Rui Zhang 2 , Chun Cui 2 , Yoshihiro Fukui 2 1 Natl. Inst. Radiol. Sci., Chiba, Japan; 2 Sch. Med. Tokushima Univ., Tokushima, Japan Heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like X-or gamma-rays. Using this special feature, heavy ion irradiation has been applied for cancer treatment. The safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. For instance, brain tumors treated by heavy-ion beams became smaller or disappearance. However, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. In order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. The characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation.
Yukio Imamura Department of psychiatry, University of Ottawa, ON, Canada NMDARs contain two NR1 subunits paired with two NR2 subunits. NR1 and NR2 (A-D) subunits harbor the glycine and glutamate binding sites, respectively. NMDARs are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. After the peak of synaptogenesis, the NR1/NR2A complex, characterized by rapid offset kinetics, dominates at the synapse, while the NR1/NR2B complex, characterized by slow kinetics, predominates in the extra-synaptic area. The activation of extrasynaptic NMDARs by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. Using whole-cell voltage-clamp recordings from CA1 pyramidal neurons from mice (at 12 weeks of age), we found that following induction of ischemia, ifenprodil, a selective NMDAR-NR2B antagonist, reduced the inward current of the isolated NMDAR at extra-synaptic site while it had less effect at the synaptic NMDAR. The molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. In the present study, we observed expression and changes of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the gerbil hippocampal CA1 region after ischemia. In blood, corticosterone levels increased biphasically at 30 min and 12 h after ischemia, and thereafter its levels decreased. In the sham group, MR and GR immunoreactivities were weakly detected in the CA1 region. By 3 days after ischemia, MR and GR were not significantly altered in the CA1 region. From 4 days after ischemia, MR and GR immunoreactivities were detected in astrocytes and microglia in the CA1 region, and at 7 days after ischemia. The specific distribution of corticosteroid receptors in glia may be associated with the differences of MR and GR functions against ischemic damage. The present study was investigated the effects of early treadmill training after cerebral infarction in rats. We determined whether treadmill exercise changes cellular expression of caspase-3 and midkine in the MCA area. Stroke was induced by a 90-min MCA occlusion using an intraluminal filament. Rats were exercised for 20 min each every day on a treadmill. Brain damage in ischemic rats was evaluated by infarct volume. Exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and MKimmunoreactive calls. No significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. Cellular expressions of MK were significantly increased in striatum (glia) of the exercised rats. Treadmill exercise was shown to suppress the decrease in caspase-3 expression in the penumbra. The present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery.
PS2A-J174 Reversed astrocytic glutamate transporter GLT-1 crucial to the Ca 2+ paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures Tatsuro Kosugi, Koichi Kawahara, Takeshi Yamada, Motoki Tanaka Lab. of Cellular Cybernetics, Graduate School of Information Science and Technology, Hokkaido Univ., Sapporo, Japan "Ca 2+ paradox" is the phenomenon whereby the intracellular concentration of Ca 2+ paradoxically increases during reperfusion with normal Ca 2+ -containing media after brief exposure to a low Ca 2+ solution. The present study aims to characterize the Ca 2+ paradoxinduced cell injury in neuron/astrocyte co-cultures. Prior exposure of the cultures to a low Ca 2+ solution for 60 min significantly injured only neurons after reperfusion with a normal Ca 2+ medium for 24 h, but astrocytes remained intact. After the onset of reperfusion, the intracellular concentration of Na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic GLT-1. The present findings suggested that Ca 2+ paradox-induced accumulation of Na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic GLT-1 during the reperfusion episode. Common genetic mutation in Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) has been associated with missense mutations of Notch3 concerning cysteine residues within the extracellular amino-terminal region. We report new mutations of two Japanese CADASIL families, which did not directly involve a cysteine residue. Exons of the Notch3 were amplified by PCR and subsequently analysed for DHPLC and direct sequence. The first patient carried the missense mutation C577T, which results in pro167ser. The second patient carried the missense mutation C302G, which results in arg75pro. New mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the Notch3 receptor which may involve an alteration in the ligand binding or putative dimerisation properties.
PS2A-J177 MCI -186, a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3kinase Madinyet Niyaz 1 , Tadahiro Numakawa 2 , Yoshinori Matsuki 1 , Emi Kumamaru 2 , Yuki Yagazaki 2 , Harumi Kitazawa 1 , Hiroshi Kunugi 2 , Motoshige Kudo 1 1 Pathology department of Tokyo Medical University, Tokyo, Japan; 2 Department of Mental Disorder Research, National Institute of Neuroscience, NCNP, Tokyo, Japan
The role of MCI -186, a radical scavenger, in the central nervous system (CNS) has not been fully elucidated. In the present study, we found that treatment with MCI -186 prevented the cultured cortical neurons from cell death induced by serum deprivation. Furthermore, we found that MCI -186 exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the MCI -186-dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). These results suggested that MCI -186 exerts a protective effect on CNS neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger.
Osamu Tokumaru 1 , Noriko Yoshimura 1 , Tetsuro Sakamoto 1 , Takaaki Kitano 2 , Naoko Nisimaru 1 , Isao Yokoi 1 1 Dept. Physiol., Sch. Med., Oita Univ., Japan; 2 Med. Edu. Ctr., Sch. Med., Oita. Univ., Japan Protective effects of ethyl pyruvate (EP) on energy metabolism of rat brain exposed to ischemia were investigated by 31 P-nuclear magnetic resonance (25 • C). Brain slices were incubated in standard artificial cerebrospinal fluid (ACSF) with 2 mM EP (EP-1), ACSF replaced by ACSF with 2 mM EP after ischemia (EP-2), or ACSF only (Control). The brain slices were exposed to ischemia by stopping the perfusion for 1 h. High-energy phosphate, creatine phosphate (PCr) and ␥-ATP, levels were measured. Decrease in PCr level was not different among the three groups when exposed to ischemia. But increase in PCr level after the reperfusion was significantly larger in EP-1 than in Control (p < 0.01). These results indicate that EP is effective in the reperfusion period and is more protective when administered before ischemic exposure. The importance of timing of administration of EP in clinical use was suggested.
Research funds: Grant-in-Aid for Scientific Research (C) #17591639 from MEXT to T.K.
Hideaki Tamai 1 , Kuniko Shimazaki 2 , Norimasa Seo 1 1 Department of Anesthesiology and Critical Care Medicine, Jichi Medical University, Graduate School, Tochigi, Japan; 2 Department of Physiology, Jichi Medical University, Tochigi, Japan
We investigated the effects of acupuncture on cell proliferation in the dentate gyrus (DG) and the lateral ventricle (LV) of adult rats. In this study, acupuncture was performed at the acupoints Neiguan (PC6), Yintang (Ex-HN3) and Sanyinjiao (Sp6), which have been used for the enhancement of conscious and functional recovery in stroke patients. Eight weeks old male Wistar rats were used in the experiment. Through 5-bromo-2,-deoxyuridine (BrdU) immunohistochemistry, a significant increase in cell proliferation in the DG of the acupunctured group was observed. However, the cell proliferation in the LV was not affected with the acupoints PC6, Ex-HN3 and Sp6. The present findings indicate that the sensitivity on cell proliferation in the DG by acupuncture stimulation is higher than in the LV.
Yukio Ago 1 , Keiko Takahashi 1 , Shigeo Nakamura 1 , Akemichi Baba 2 , Toshio Matsuda 1 1 Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan; 2 Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan This study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. The staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. In group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. The anxiogenic drug -CCA increased the number of rearing, but did not affect the number of steps climbed. On the other hand, methamphetamine increased the number of steps climbed to the second step. In these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. These findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice.
Tomonori Fujiwara 1 , Tatsuya Mishima 1 , Takefumi Kofuji 2 , Kimio Akagawa 1 1 Department of Cell Physiology, Kyorin University School of Medicine, Mitaka, Tokyo, Japan; 2 Radio Isotope Laboratory, Kyorin University School of Medincine, Mitaka, Tokyo, Japan HPC-1/syntaxin 1A is believed to regulate the exocytosis of synaptic vesicles. In order to examine the neurophysiological function in vivo, we have produced HPC-1/syntaxin1A knock-out mice. Surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. However, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. However, once memory consolidation was acquired, the extinction process was disturbed in homozygote.
We further examined latent inhibition of cued fear memory (LI) to access behavioral property. Interestingly, LI was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. Implication of these behavioral abnormalities in HPC-1/syntaxin1A knock-out mouse will be discussed.
Research funds: KAKENHI (15700292) PS2A-K182 Effects of local administration of the GABA agonists into the hippocampus CA1 area on active avoidance learning and serotonergic systems in the administration area in rats Satoko Hatakenaka 1 , Hiroko Miyakubo 1 , Junichi Tanaka 1 , Yasushi Hayashi 2 , Yukio Hattori 2 , Masahiko Nomura 3 1 Department of Curriculum, Teaching and Memory, Naruto University of Education, Tokushima, Japan; 2 Department of Human Nutrition, Notre Dame Seishin University, Okayama, Japan; 3 Department of Physiology, Saitama Medical School, Saitama, Japan
In Fischer 344 male rats, bilateral injections of the ␥-aminobutyric acid (GABA) A agonist muscimol into the CA1 area slightly decreased the avoidance rate in an active avoidance task. Similar injections of the GABA B agonist baclofen enhanced the avoidance rate. There are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that GABA A and GABA B receptors have the opposite action on the performance of avoidance learning. Perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite 5-hydroxytryptamine (5-HIAA) concentration in the CA1 area, whereas baclofen perfusion had no effect, suggesting that the GABAergic system may exert to inhibit the serotonin release in the CA1 area through GABA A receptors.
Sawako Arai, Taku Nagai, Kenji Takahashi, Hiroyuki Kamei, Kazuhiro Takuma, Kiyofumi Yamada Lab. Neuropsychopharmacol, Kanazawa Univ., Kanazawa, Japan
We performed immunohistochemical c-Fos mapping after a prepulse inhibition (PPI) test of the startle reflex in mice. Startle stimulus increased the number of c-Fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (PnC), while prepulse trials without startle stimulus increased c-Fos expression in the lateral globus pallidus (LGP). In mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-Fos expression was diminished but c-Fos expression remained in the LGP. Prepulse-induced c-Fos expression in the LGP was colocalized with GAD-67. Fluoro-gold infusion into the PnC and the pedunculopontine tegmental nucleus (PPTg) retrogradely labeled neurons in the PPTg and LGP, respectively. Microinjections of phaclofen, but not picrotoxin, into the PPTg impaired PPI of the startle reflex. These results suggest that GABAergic neurons in the LGP which project to the PPTg play a crucial role through the activation of GABA B receptors in the PPI of the startle reflex.
Shiho Kitaoka 1 , Sho Koyasu 1 , Akinori Nishi 2 , Tomoyuki Furuyashiki 1 , Toshiyuki Matsuoka 1 , Shuh Narumiya 1 1 Department of pharmacology, University of Kyoto, Kyoto, Japan; 2 Department of Physiology, University of Kurume, Kurume, Japan Prostaglandins E2 (PGE 2 ) exert their actions in various organs through specific receptor, EP1 to 4. The previous study suggests that EP1 modulates DA system. To investigate the roles of EP1 in DA system, we examined EP1KO mice with behavioral sensitization induced by cocaine. The administration of cocaine elevated DA concentration in the nucleus accumbens up to ∼300% in both wild-type and EP1KO mice. However, increase of locomotor activity in EP1KO mice was significantly lower than that in wild-type mice. Because locomotor activity is closely related to dopamine D1 receptor (D1R) signaling, we tested the density of D1R and D1R signaling with phosphorylation of DARPP-32. There were no differences in D1R binding. D1R signaling was significantly attenuated in the striatal slices from EP1KO mice. The effect of D1R agonist on locomotor activity was also attenuated in EP1KO mice. These results indicate that PGE 2 has enhancing effects on locomotor activity via EP1 by potentiating the D1R signaling. Central serotonin (5-HT) function has been implicated in impulsivity. The present study examined rats with 5-HT depletion by parachloroamphetamine (PCA) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of 5-HT and its metabolites (5-HIAA) in the brain. For both tasks, significant negative correlations between learning performance and 5-HT and 5-HIAA concentrations were observed in the medial prefrontal cortex and nucleus accumbens. In contrast, for reversal task only, significant correlations between learning performance and 5-HT and 5-HIAA concentrations were observed in the orbitofrontal cortex and amygdala. These data suggest the regional difference of 5-HT roles on selective indices of impulsivity.
Yuki Sato 1,2,3 , Tatsushi Onaka 2 , Norimasa Seo 1 , Eiji Kobayashi 3 1 Dept. Anesthesiol., Jichi Med. Univ., Tochigi, Japan; 2 Dept. Physiol., Jichi Med. Univ., Tochigi, Japan; 3 Div. Organ Replacement Research, Center for Mol. Med., Jichi Med. Univ., Tochigi, Japan Cyclosporine is widely used for preventing allograft rejection. However, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. Cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. It is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. Here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. Calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. On the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. All these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. To investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in Sprague-Dawley rats (P1-21; adult). Conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. Neuronal activation within brainstem nuclei was defined by the expression of c-fos. Labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered Fos-expressing neurons. With rotational stimulation that comprised cycles of constant angular acceleration and deceleration, Fos-labeled neurons were observed by P4 in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei DMCC, IOA, IOC, IOK). A later maturation time was evidenced for the utricular system. Fos-labeled neurons were only identifiable in the vestibular nucleus by P7; in the prepositus hypoglossal nucleus and inferior olive (subnuclei DMCC and IO) by P11. Within the vestibular nucleus of P7-9 rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. In P21 and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. However, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. Taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. To determine whether perineuronal nets (PN) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of PN within the vestibular nuclei during development (rats, P1 to adult) and after unilateral labyrinthectomy (UL) in the adult. Histochemistry with the lectin Wisteria floribunda agglutinin was used to map PN about NeuN-immunopositive neurons within the vestibular nuclei. In normal postnatal rats, PN was detectable by P3 in the vestibular nucleus as fuzziness about neuronal cell bodies. From P9 onwards, the fuzzy PN progressively consolidated into a network organization. The fuzziness was no longer observable after P12. During postnatal development, the number of neurons showing PN increased with age, reaching the adult level by P21. With UL, the PN network on the lesioned side remained compact until 5 days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. By 11 days after UL, the PN of some neurons resumed the network pattern as was observed in normal adult rats. This phenomenon was found in the PN of the remaining neurons by 14 days after UL. The PN on the labyrinth-intact side showed the compact network of uninjured age-matched rats. Taken together, our findings indicate PN changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear.
Minori Ueda, Takayuki Suzuki, Hiroyoshi Miyakawa Laboratory of Cellular Neurobiology, Tokyo University of Pharmacy and Life Science, Tokyo, Japan Dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. To better understand these processes, we analyzed AMPAR-and NMDARmediated EPSCs and synaptically induced transporter currents (STCs) elicited with high-frequency stimuli. Recordings were made from pyramidal cells and astrocytes in the CA1 region of rat hippocampal slices, 100 Hz/20 pulse tetanic stimulations were delivered to Schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. The decay time course of the last isolated STC during the tetanic stimulation was not significantly different from that of the first. While the amplitude of the AMPAR-mediated EPSCs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the NMDAR-mediated EPSCs. These findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at 100 Hz.
Ikuko Yao 1 , Hiroshi Takagi 1 , Hiroshi Ageta 1 , Tomoaki Kahyo 1 , Ken Hatanaka 1 , Kaoru Inokuchi 1 , Mitsutoshi Setou 1,2 1 Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan; 2 University of Tokyo, Tokyo, Japan; 3 Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Okazaki, Japan
We identified and characterized a novel ubiquitin ligase named Scrapper. Scrapper is an F-box protein which has leucine rich repeat and C-terminal membrane localization sequence, highly expressed in neurons throughout the brain. To investigate the physiological role of Scrapper in the neuron, we recorded mEPSCs from the neuron over-expressed the EGFP-tagged full-length Scrapper construct or truncated form of Scrapper constructs. They exhibited a strong suppression or enhancement in the frequency of mEPSCs while showing a non-significant change in mEPSC amplitude, rise, and decay time compared with neurons expressing EGFP. The passive membrane properties of neurons such as Membrane resistance (Rm), series resistance (Rs), and membrane capacitance (Cm) were not statistically different from those of control. These data suggests a presynaptic effect of Scrapper protein.
PS2P-A003 Presynaptic membrane potential-dependent regulatory mechanism of transmitter release Tetsuya Hori, Tomoyuki Takahashi Department of Neurophysiology, University of Tokyo Graduate School of Medicine, Tokyo, Japan
In simultaneous pre-and postsynaptic recordings at the calyx of Held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. A weak sustained depolarization (e.g., 10 mV, 1 s) of calyceal nerve terminal potentiated EPSCs despite that it diminished presynaptic action potential (a.p.) amplitude. As we further depolarized the terminal EPSCs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. When presynaptic Ca 2+ currents (I pCa ), induced by an a.p.-waveform command pulse, were used to evoke EPSCs, a weak sustained depolarization enhanced I pCa and EPSCs in parallel. This EPSC facilitation was robust at the calyx of Held both in rats and mice, but was almost absent in P/Q-type Ca 2+ channel knockout mice. We conclude that the P/Q-type specific Ca 2+ channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization.
Hiroshi Takagi 1 , Koji Ikegami 1 , Ken Hatanaka 1,2,3 , Yoko Fujiwara-Tsukamoto 1 , Mineo Matsumoto 1 , Ikuko Yao 1 , Mitsutoshi Setou 1,2,4 1 Mitsubishi Kagaku Institute of Life Sciences, Japan; 2 PRESTO, Japan; 3 School of Pharmaceutical Sciences, The University of Tokyo, Japan; 4 Okazaki Institute for Integrative Bioscience, Japan A variety of post-translational modifications to the exposed Cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. However, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. Of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. In the ROSA22(−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient A-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. We provide herein, using ROSA22 mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated A-type K current.
Satoshi Kawasaki 1 , Shingo Kimura 1 , Reiko Fujita 2 , Shuji Watanabe 1 , Kazuhiko Sasaki 1 1 Dept. of Physiol., Sch. of Med., Iwate Medical Univ., Morioka, Japan; 2 Dept. of Chem., Sch. of Lib. Arts & Sci., Iwate Medical Univ., Morioka, Japan Application of dopamine (DA) induces a slow Na + -current response in the identified neurons of Aplysia ganglia under voltage clamp. This type of response is produced by the activation of trimeric G-protein sensitive to cholera toxin (CTX) as previously reported. The Na +current response to DA was gradually and irreversibly depressed after intracellular injection of Clostridium difficile toxin B, which is known to inactivate all types of Rho family G-proteins. Intracellular application of Clostridium botulinum exoenzyme C3, a specific toxin to RhoA-C, also depressed the DA-induced response irreversibly. Furthermore, the DA-induced current response was significantly depressed by GAP domain of p50RhoGAP applied intracellulary. In contrast, GEF domain of RhoGEF Dbs had a tendency to increase the response. These results suggest that the DA-induced Na + -current response may be regulated by the activation of Rho family G-protein. The ␦2 glutamate receptor (␦2R) plays a crucial role in cerebellar functions. Although ␦2R has a putative channel pore domain, and ␦2R displayed Ca 2+ -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦2R functions as a channel. Here we introduced a ␦2R transgene, which had a mutation (Gln618Arg) in the putative channel pore conserved in Ca 2+ -permeable glutamate receptors, into ␦2 −/− mice. Surprisingly, a mutant ␦2R transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦2 −/− mice, such as ataxia and loss of long-term depression. These results indicate that Ca 2+ influx through ␦2R is not required for its function in the cerebellum in vivo, and that wild-type ␦2R may not function as a Ca 2+ -permeable ion channel.
Research funds: KAKENHI (17700316) and Takeda Science Foundation PS2P-A007 Distribution of TARP -8 on hippocampal neurons and its key role in synaptic and extrasynaptic expression for AMPA receptors Masahiro Fukaya 1 , Mika Tsujita 2 , Maya Yamazaki 2 , Etsuko Kushiya 2 , Manabu Abe 2 , Kaori Akashi 2 , Masanobu Kano 3 , Haruyuki Kamiya 4 , Kenji Sakimura 2 , Masahiko Watanabe 1 1 Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan; 2 Department of Cellular Neurobiology, Brain Research Institute, Niigata, Japan; 3 Department of Cellular Neuroscience, Graduate School of Medical Science, Osaka University, Suita, Japan; 4 Department of Molecular Anatomy, Hokkaido University School of medicine, Sapporo, Japan
The -8 is one of four transmembrane AMPAR regulatory proteins (TARPs). Pre-and post-embeding immunogold visualized -8 on excitatory synaptic and extrasynaptic membrane. In -8-KO mice, AMPARs were reduced in hippocampal homogenates (46% of control) and PSD fraction (35%). Immunogold labeling also exhibited reduction of extrasynpatic (47%) and synaptic (35%) AMPARs in CA1 pyramidal cells. The reduction of extrasynaptic receptors was particularly severe on dendrites (36%) and spines (38%). AMPAR-mediated responses were reduced at CA1 synapses (52%). Therefore, -8 is the major auxiliary subunit of hippocampal AMPARs.
Etsuko Tarusawa 1 , Yugo Fukazawa 1 , Elek Molnar 2 , Masahiko Watanabe 3 , Ryuichi Shigemoto 1,4 1 Div. Cerebral structure, NIPS, Okazaki, Japan; 2 MRC, Univ. of Bristol, Bristol, UK; 3 Hokkaido Univ., Sapporo, Japan; 4 SORST, JST, Kawaguchi, Japan Relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (RG) and corticogeniculate (CG) synapses. It has been shown that the synaptic transmission at both RG and CG synapses is mediated via AMPA and NMDA receptors. However, how AMPA and NMDA receptors are expressed in these two types of synapses have not been elucidated. We examined the expression pattern of AMPA and NMDA receptors in RG and CG synapses using SDS-digested freeze-fracture replica labeling (SDS-FRL). The SDS-FRL revealed that synaptic size of individual RG synapses was significantly smaller than that of CG synapses. RG synapses expressed 1.7 to 3 times higher density of AMPA receptors than CG synapses. On the other hand, CG synapses expressed 1.6 to 3 times more NMDA receptors than RG synapses. These results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through AMPA and NMDA receptors.
Katsuyuki Kaneda 1,2,3 , Hitoshi Kita 1 1 Dept. of Anat. & Neurobiol., Univ. of Tennessee, Memphis, TN, USA; 2 Japan Society for Promotion of Science, Tokyo, Japan; 3 Dept. of Developmental Physiology, NIPS, Okazaki, Japan
To investigate the properties of synaptically induced slow responses in globus pallidus (GP) neurons, whole-cell recordings were performed using rat brain slice preparations. Repetitive stimulation of the GP and internal capsule induced mixed fast EPSPs/IPSPs followed by a slow IPSP (sIPSP), and a long-lasting slow depolarization (sDEPO). Bath application of NBQX, CPP, and gabazine blocked the mixed EPSPs/IPSPs. The GABA B receptor antagonist CGP55845 abolished the sIPSP. An mGluR1 antagonist, but not an mGluR5 antagonist, partially blocked the sDEPO. In addition, CGP55845 enlarged the amplitude of fast IPSCs, but not of EPSCs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic GABA B receptors in GABA release. These results indicate that synaptically released GABA and glutamate can evoke GABA B receptor-and mGluR1-mediated responses in the GP. Contribution of these responses to the control of GP activity will be discussed.
Research funds: NIH and the JSPS PS2P-A010 Essential contribution of glutamate to GABA depolarization involved in hippocampal seizure-like activity Yoko Tsukamoto 1 , Yoshikazu Isomura 1,2 , Michiko Imanishi 1 , Tomoki Fukai 2 , Masahiko Takada 1 1 System Neurosci., Tokyo Met. Inst. Neurosci., Tokyo, Japan; 2 Neural Circuit Theory, RIKEN BSI, Saitama, Japan
We have previously shown that neuronal synchronization is achieved by excitatory GABAergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. However, it still remains unclear how the GABA response is converted from inhibitory to excitatory in the process of afterdischarge induction. Here we traced the time-course of amplitude and reversal potential of GABAergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of GABA response. The GABA reversal potential in pyramidal cells rose to spike-threshold levels for >20 s after the induction. GABA Amediated Cl-influx lasted for 1.5 s, and then glutamate enhanced the conversion effectively in a GABA A -independent manner, which was dependent on an extracellular K increase. Coapplication of GABA and glutamate caused a similar oscillatory activity. The results show GABA and glutamate may cooperatively induce as well as maintain seizure-like activity.
Michiko Nakamura 1 , Yuko Sekino 1,2 , Toshiya Manabe 1,2 1 Division of Neuronal Network, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 2 CREST, JST, Japan Profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. In the present study, we found that this synaptic strengthening was partially mediated by presynaptic GABA A receptor activation during the developmental period (P < 30), using electrophysiological methods and optical imaging. In immature animals (P10), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. This fiber volley facilitation was significantly decreased by either inhibition of GABA A receptors or suppression of GABA release from interneurons. These results suggest that GABA released from inhibitory interneurons and GABA A receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development.
Takuya Nishimaki, Il-Sung Jang, Jyunichi Nabekura Dep. Dev. Physiol., NIPS, Sokendai, Okazaki, CREST, JST, Japan Lateral superior olive (LSO) is the first auditory center. During the early postnatal period, the inhibitory synaptic inputs to LSO neurons from medial nucleus of the trapezoid body (MNTB) change from predominantly GABAergic to glycinergic. We focused on metabotropic GABA B receptor (GABA B R) as the key molecule of difference between GABA and glycine. In immature LSO neurons postsynaptic GABA B R could activate K + channels, but this effect ceased by the third postnatal week. Baclofen, a GABA B R agonist, reduced IPSC amplitude at MNTB-LSO synapses in neonate (<P5) with a significant change in the paired-pulse ratio. The presynaptic effect of baclofen was gradually decreased with development (P2-18). In situ hybridization and immunohistochemistry experiment revealed GABA B R expression in MNTB neurons was also gradually decreased. The property of MNTB-LSO synapses in GABA B R knock out mouse remained immature at P14-16 compared with control mouse. Thus, GABA B receptor seems to play an important role in development of synaptic property. Pyramidal cells in the hippocampal CA1 area receive GABAergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (AIS). Here, we used the SDSdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣1, ␣2, and 2/3 subunits quantitatively on distinct subcellular compartments of pyramidal cells. Labelings for these subunits were accumulated over clusters of intramembrane particles (IMP) on the protoplasmic face (P-face) of the plasma membrane, indicating that many IMP clusters of a certain size represent GABAergic synapses. The synaptic labelling densities for GABA-A receptor subunits were not significantly different on the tested subcellular compartments. Double labelling experiments showed that the majority of synapses contains ␣1, ␣2, and 2/3 subunits.
In the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. Therefore, we investigated the effects of the ␣ 1 -or ␣ 2 -adrenoceptor agonist on firing rates of presynaptic interneurons (INs). Presynaptic cell-attached recordings showed that spontaneous activity of GABAergic INs was enhanced by the ␣ 1 -adrenoceptor agonist phenylephrine, while reduced by the ␣ 2 -adrenoceptor agonist clonidine. Immunohistochemical studies showed that ␣ 1 -adrenoceptors were expressed in the molecular layer and the Purkinje cell (PC) layer, while ␣ 2 -adrenoceptors were observed in cell bodies of PCs and INs. These results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ 1 -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of INs via ␣ 2 -adrenoceptors, which were located in the presynaptic IN soma. Thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of GABAergic inputs from INs to PCs.
Research funds: KAKENHI (16700344) PS2P-B015 Involvement of basal PKC and ERK 1/2 activities in constitutiveinternalization of AMPA receptors in cerebellar Purkinje cells Tetsuya Tatsukawa 1 , Takahiko Chimura 2 , Azumi Matsumoto 2 , Kazuhiko Yamaguchi 2 1 Lab. for Motor Learning Control, RIKEN, BSI, Japan; 2 Lab. for Memory & Learning, Japan AMPA receptor (AMPAR) internalization provides a mechanism for cerebellar LTD, which is the underlying basis of motor learning and motor coordination. However, the relationship between cerebellar LTD and constitutive AMPAR internalization in parallel fiber (PF)-Purkinje cell (PC) synapses remains unclarified. In the present study, we demonstrated that tetanus toxin (TeTx, a blocker of exocytosis mediated by VAMP) infusion into PCs caused the rundown of PF-EPSC amplitude through the constitutive AMPAR internalization, and then addressed question whether intracellular signals involved in cerebellar LTD induction modify the constitutive AMPAR internalization at PF-PC synapses using electrophysiological and immunocytochemical techniques. Our results suggest that the regulation of actin polymerization is involved in the surface expression of AMPARs and the surface expressing AMPARs are constitutively internalized depending on both basal PKC and MEK-ERK1/2 activities at PF-PC synapses.
Taisuke Miyazaki 1 , Kohichi Tanaka 2 , Masahiko Watanabe 1 1 Department of Anatomy, School of Medicine, Hokkaido University, Sapporo, Japan; 2 Department of Molecular Neuroscience, Tokyo Medical and Dental University, Tokyo, Japan Glutamate released to the synaptic cleft has to be removed by glutamate transporter. In the cerebellum, glutamate transporter GLAST is richly expressed in Bergmann glial cells (BG) that enwrap synapses, dendrites and somata of Purkinje cells (PCs). In this study, anatomical analysis was applied to GLAST-deficient mice. In the mutant mice, lamellate processes from BG fibers became atrophic, resulting in incomplete sealing of synaptic cleft. Furthermore, the distribution of climbing fiber (CF) terminals was decreased proximally. By anterogradely labeling, multiple CF innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. These results suggest that GLAST is essential for complete ensheathment of PC synapses and for the establishment of CF mono-innervation by suppressing local ectopic innervation between nearby PC dendrites.
Miwako Yamasaki 1,2 , Kouichi Hashimoto 2 , Taisuke Miyazaki 1 , Masahiko Watanabe 1 , Masanobu Kano 2 1 Dept. of Anatomy, Grad. Sch. of Med., Hokkaido Univ., Sapporo, Japan; 2 Dept. of Cellular Neuroscience, Grad. Sch. of Med., Osaka Univ., Suita, Japan
The ␥ isoform of protein kinase C (PKC␥) is highly expressed in cerebellar Purkinje cells (PCs), and its deficiency impairs the late phase of climbing fiber (CF) synapse elimination. In the present study, we analyzed multiple CF innervation in PKC␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of CFs. In general, CF innervation regressed proximally in the PKC␥-deficient mouse. In some mutant PCs, a considerable part of the proximal dendrites lost association with CF terminals. In the majority of multiply innervated PCs, single main CF innervated broad region of proximal dendrites, whereas surplus CFs innervated the somata and basal portion of the proximal dendrites. Moreover, immunoelectron microscopy revealed synaptic contact of CF terminals to somatic spines. These results suggest that PKC␥ is crucial for strengthening innervation by a main CF and elimination of surplus CFs from proximal somatodendritc compartments of PCs.
Keiji Imoto 1,2 , Takashi Kodama 1,2 , Ryuichi Shigemoto 2,3,4 , Yugo Fukazawa 2,3 , Yasuo Mori 5 1 Division of Neural Signaling, NIPS, Okazaki, Japan; 2 School of Life Science, Graduate University for Advanced Studies (Sokendai), Okazaki, Japan; 3 Division of Cerebral Structure, NIPS, Okazaki, Japan; 4 SORST, Japan Science and Technology Agency, Kawaguchi, Japan; 5 Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Japan A murine ataxic mutant, rocker, has a point mutation in the Ca V 2.1 (P/Q-type) ␣ subunit gene and shows the mildest phenotype among the series of Ca V 2.1 mutants. Although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-Purkinje cell synapses was considerably impaired. We demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of AMPA receptors in PSD, whereas the presynaptic function remained normal. We also found the dendrites of Purkinje cells showed poor arborization in rocker mice. These lines of evidence indicate that Ca V 2.1 exerts its strong influence on postsynaptic maturation and dendritic structure.
Bai-Chuang Shyu, Chia-Ming Lee, Wei-Chih Chang Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan ROC
The aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the Anterior cingulate cortex (ACC) using a novel mouse brain slice preparation protocol. The mono-and poly synaptic responses in the ACC were excited by direct thalamic activation. The synaptic transmission involves both AMPA and NMDA glutamate receptors. Several thalamus-evoked responses were identified: the early negative component (N1) emerged in layer VI near cingulum. Subsequently a positive component (P) appeared in layer VI. The second negative component (N2) became apparent in layers II/III and V. Then the activities spread downward to layer VI and developed as N3 component in a medial-to-lateral direction. We confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. The thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the ACC and this process was modulated extensively by glutamatergic synapses.
PS2P-B020 Target-dependent extracellular Ca-dependence of the short-term plasticity of the solitary complex synapses in the rat
Lab. Neurophysiol., Dept. Neurosci., Jikei Univ. Sch. Med., Tokyo, Japan
The discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (SC) forms the primary filter of the visceral information. Such frequency filtering of the synaptic transmissions from the solitary tract (TS) to neurons in the SC depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (PPR). Here we analyzed the mechanism underlying distinct PPRs among distinct types of synapses. In synapses from TS to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, PPR significantly increased from 0.4 ± 0.2 to 1.1 ± 0.2 (n = 6; mean ± S.D.) by reducing [Ca 2+ ] o from 2 to 0.5 mM, whereas that in low-pass synapses from TS to the neurons in dorsal motor nucleus of the vagus was affected only slightly. These results suggest that the synaptic frequency filtering in the SC is under distinct control depending on the function of the postsynaptic target systems.
PS2P-B021 Response of spinal monosynaptic reflex to high frequency stimulation in newborn rat
The susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. In an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (MSRs) evoked by dorsal root stimulation, were mediated by both NMDA and non-NMDA glutamate receptors. MSRs were constant in amplitude at 1/15 s, and were depressed completely by CNQX. When stimulus rate was increased to 1/s, MSR amplitudes were greatly reduced initially, and recovered later. This recovery of MSR was eliminated by APV. Non-NMDA component of MSRs was eliminated completely at 1/s, but appeared in constant amplitudes in the presence of cyclothiazide. From these results, Non-NMDA glutamate receptor mediated almost most of MSRs at 1/15 s, but not at 1/s due to desensitization. In contrast, NMDA glutamate receptor does not mediate MSRs, but activated at 1/sec, in place of non-NMDA receptor. In conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission.
Kenichi Ono 1 , Keisuke Shiba 2 , Ken Nakazawa 3 , Ichiro Shimoyama 4 1 Department of Otolaryngology, Chiba University, Chiba, Japan; 2 Department of Otolaryngology, Kimitsu Central Hospital, Chiba, Japan; 3 Department of Integrative Neurophysiology, Chiba University, Chiba, Japan; 4 Section for Human Neurophysiology, Research Center for Frontier Medical Engineering, Chiba University, Chiba, Japan
We determined synaptic source of the respiratory-related activity of laryngeal motoneurons (LMs) using spike-triggered averaging of LM membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. In inspiratory (I) LMs, monosynaptic EPSPs were evoked by spikes of I neurons with augmenting firing patterns, and monosynaptic IPSPs were evoked by spikes of expiratory (E) neurons with decrementing firing patterns and of I neurons with decrementing firing patterns. In ELMs, monosynaptic IPSPs were evoked by spikes of I neurons with decrementing firing patterns and of E neurons with augmenting firing patterns. We conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of LM membrane potentials.
PS2P-B023 In-vivo gene transfer of exogenous protein to the primary afferent neurons Chiaki Yamada 1,2 , Eiji Shigetomi 1,3 , Fusao Kato 1 1 Lab. Neurophysiol., Jikei Univ. Sch. Med., Tokyo, Japan; 2 Dept. Basic Biol. Sci., Kyoritsu Univ. of Pharm., Tokyo, Japan; 3 JSPS, Japan
In order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. Here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. In the anesthetized young Wistar rats, we injected pCAGGS-EGFP plasmid vector (through courtesy of Drs. J. Miyazaki and K. Nakajima) into the nodose ganglion (NG) at the neck and transferred it by electroporation. Two days after transfection, the NG was extirpated and fixed to observe EGFP signals. A large portion of somata in the NG as well as the centrally and peripherally projecting afferent fibers expressed high levels of EGFP with few damaged cells. This technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain.
Research funds: KAKENHI (17650116, 176865) PS2P-B024 Laminar sources of synaptic input to layer 1 neurons in rat visual cortex Takuma Mori, Edward M. Callaway Salk Institute, USA Layer 1 of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. Very little is known about connectivity of these cells with cells in other cortical layers. We examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer 1 neurons of rat visual cortex, using scanning laser photostimulation. We find that axon descending cells get excitatory synaptic inputs primarily from layer 2/3. Neurogliaform cells receive more excitatory inputs from layers 4 and 5. The dominant inhibitory synaptic inputs onto both types of cells originate from layer 2/3. These neurons also get inhibitory inputs from layers 1 and 4-6. The presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer 1 are highly integrative.
Takako Nishi 1 , Tsukasa Gotow 2 , Shiro Nakagawa 2 1 Ins. Nat. Sci., Senshu Univ., Kawasaki, Japan; 2 Dept. Neurol., Kagoshima Univ. Grad. Sch. Med.-Den. Sci., Kagoshima, Japan Four extra-ocular photoreceptors, the photoresponsive neurons, A-P-1, Es-1, Ip-1 and Ip-2 which respond directly to light, exist in the abdominal ganglion of the mollusc Onchidium. A-P-1/Es-1 of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other Ip-1/Ip-2. The present study was conducted to examine the functional significance of the above Ip-1/Ip-2, having a peak sensitivity at 510 nm light. The body wall of the animal transmitted enough 510 nm to hyperpolarize Ip-1/Ip-2 in the ganglion covered by the body wall. Ip-1/Ip-2 were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. Ip-1/Ip-2 were not coupled to A-P-1/Es-1 one another. Finally, Ip-1/Ip-2, the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera.
PS2P-C026 Cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken Tatsuo Sato, Iwao Fukui, Harunori Ohmori Department of Physiology, Faculty of Medicine, Kyoto University Interaural level difference (ILD) and timing difference (ITD) are the major two cues to localize the sound source, and are processed separately. Although the details of ITD processing are well investigated, not much is known about ILD. In the barn owl, it is proposed that ILD is calculated in LLDp (posterior part of the dorsal lateral lemniscal nucleus). We confirmed already that in vivo LLDp calculates ILD in the chicken as well. In this work using the patch clamp recording with the brainstem slice preparation, we found that LLDp neurons fire tonically under the regulation of two K + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. We are intending to clarify the cellular mechanism of ILD calculation as the interaction of both excitatory and inhibitory synaptic inputs. To understand the functional organization of the CNS, it is essential to know how sensory information is processed within the CNS. We have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. In this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. Stimulation applied to the olfactory nerve elicited EPSP-related optical signals in the olfactory bulb from the 7-day old embryonic stage. The EPSP was mediated by glutamate, and NMDA-and non-NMDA-receptor components were identified. In more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. The results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing.
Yoko Momose-Sato, Katsushige Sato Dept. Physiol., Tokyo Med. Dent. Univ. Sch. Med., Tokyo, Japan
In an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around E7. It is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. We found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. At early stages of development (E7-E8), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. As development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. The oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. We examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics.
Akiyoshi Shimada 1 , Nyberg Tobias 1 , Nahoko Kasai 1 , Yuriko Furukawa 2 , Keiichi Torimitsu 1,2 , Kunihiko Obata 3 , Yuchio Yanagawa 2,4 , Tadaharu Tsumoto 2,3 1 NTT Basic Research Laboratories, NTT Corporation, Kanagawa, Japan; 2 SORST, JST, Saitama, Japan; 3 Neuronal Circuit Mechanisms Research Group, Brain Science Institute, RIKEN, Saitama, Japan; 4 Department of Genetic and Behavioral Neuroscience, Graduate School of Medicine, Gunma University, Gunma, Japan
In the immature stage of cerebral cortex, GABA A receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. Cortical neurons of glutamic acid decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mice at postnatal day 0-1 were cultured on a microelectrode array. Spontaneous firing of cortical neurons appeared within the first 5 days in vitro (DIV). When a GABA A receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least 14 DIV: increase, decrease, disappearance and no change. These results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of GABA A receptors.
PS2P-C030 Pathway specific signal modulation by purinergic receptors in the parallel processing system Makoto Kaneda 1 , Toshiyuki Ishii 2,4 , Yasuhide Shigematsu 3 , Toshihiko Hosoya 2 , Yukio Shimoda 3 1 Dept. Physiol., Keio Univ. Sch. Med., Tokyo, Japan; 2 BSI, RIKEN, Wako, Japan; 3 MRI, Tokyo Womens' Medical Univ., Tokyo, Japan; 4 Dept. Biomolecular Science, Univ. Toho, Funabashi, Japan
The retina divides visual information into ON-and OFF signals, and processes them with independent subsets of neurons. So far no difference between these pathways has been found for neurotransmitter functions. We have previously found that P2X2-purinoceptors are selectively present on the OFF-type, but not on the ON-type cholinergic amacrine cells of the mouse retina. We therefore asked if ATP has different functions in the two pathways. As expected, only the OFF-type, but not the ON-type, cholinergic amacrine cells responded to ATP. This response was mediated by the P2X2-purinoceptors. Furthermore, a P2X-purinergic receptor antagonist activated the tonic phase firing of the OFF ganglion cells, but not of the ON cells. Thus ATP has OFF-pathway specific modulatory functions mediated by P2X-purinergic receptors.
Bhupesh Mehta, Gulnaz Begum, Preeti G. Joshi Department of Biophysics, National Institute of Mental Health and Neuro Science, Bangalore, India Cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic Ca 2+ . These oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. The synchronized Ca 2+ oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. These spontaneous oscillations are inhibited by activation of purinergic receptors. We observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists ATP and UTP, but an enhanced oscillatory pattern was observed when the action of 2MeSATP was observed. The inhibition of 2MeSATP response by P2Y1 receptor specific antagonist MRS 2179 indicates that different subtypes of Purinergic receptors play different role in the regulation of synchronized Ca 2+ oscillations. Data will be presented on the P2 receptor mediated regulation of synchronized Ca 2+ oscillations in cultured hippocampal neurons.
Mahomi Kuroiwa, Akinori Nishi Department of Pharmacology, Kurume University School of Medicine, Kurume, Japan Presence of additional D1-like dopamine receptors that selectively couple to Gq/phospholipase C (PLC) has been proposed. In recent studies, SKF83959 was shown to selectively stimulate PLC-linked D1-like dopamine receptors. DARPP-32 is a phosphoprotein that regulates the efficacy of D1 receptor signaling. Activation of D1 receptors that couple to Golf/PKA signaling leads to DARPP-32 phosphorylation at Thr34. We investigated the effect of SKF83959 on DARPP-32 phosphorylation at Thr34 by using mouse neostriatal slices. Treatment of neostriatal slices with SKF83959 increased Thr34 phosphorylation. The effect of SKF83959 was enhanced by a PLC inhibitor, U73122. In contrast, the effect of SKF83959 was partially blocked by a D1 receptor antagonist, SCH23390, and the SCH23390-insensitive increase in Thr34 phosphorylation was completely abolished by an adenosine A 2A receptor antagonist, ZM241385. Thus, analysis of DARPP-32 phosphorylation revealed that SKF83959 activates at least three signaling cascades in the neostriatum: (1) PLC, (2) D1 receptor/Golf/PKA, (3) adenosine A 2A receptors.
PS2P-C033 Thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus Naoshi Fujiwara Division of Medical Technology, Niigata University School of Health Sciences, Japan Effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. Cortical and hippocampal slices of the C57BL6 mouse were dyed with a voltage-sensitive dye RH414. In cortical slices, electrical single-pulse stimulation to the layer V evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers II-III and widely expanded in these layers. The excitation propagation in the layers II-III was significantly inhibited by thiamylal (200 mol/l). In hippocampal slices, excitation propagation elicited by the same stimulation to the CA1 stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. On the other hand, an AMPA/kainate receptor antagonist CNQX (20 mol/l) inhibited excitation propagation in both cortical and hippocampal slices. The results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus.
Research funds: Grant-in-Aid from JSPS No.15390471 PS2P-C034 Brain-derived neurotrophic factor regulating AMPA receptor translocation to postsynaptic density through IP3R and TRPC calcium signaling Hiroko Nakata, Shun Nakamura National Institute of Neuroscience, Tokyo, Japan
The change in the number of postsynaptic AMPA-type glutamatergic receptors (AMPARs) by neuronal activity is implicated in the excitatory synaptic plasticity. Here, we showed that BDNF induced AMPAR translocation to postsynaptic site of the dissociated cortical pyramidal neurons. Using calcium imaging, we located the spines that were activated by BDNF. With thus identified spines, we analyzed the AMPAR subunit GluR1 localization to postsynaptic density (PSD) by immunostaining. We found that BDNF increased the ratio of surface GluR1 at PSD within 20 min. BDNF selectively translocated AMPAR, but not NMDAR. The essential signaling was Ca 2+ transients evoked by the activation of TrkB/PLC␥ pathway. Both the intracellular Ca 2+ rise, that is, Ca 2+ release from IP3R and Ca 2+ influx through storeoperated cation channel TRPC contributed to the increase of the surface expression of GluR1 at PSD. These results suggest an important role of BDNF in the regulation of AMPAR trafficking by spatial and temporal Ca 2+ signaling regulation.
Research funds: Health sciences research grant of nano-1
Hiroyuki Konishi 1 , Kazuhiko Namikawa 1,2 , Keiji Shikata 3 , Yuji Kobatake 1 , Taro Tachibana 3 , Hiroshi Kiyama 1 1 Dept. Anat. & Neurobiol., Osaka City Univ., Grad. Sch. Med., Osaka, Japan; 2 Dept. Anat., Asahikawa Med. Col., Hokkaido, Japan; 3 Dept. Bioeng., Osaka City Univ., Grad. Sch. Eng., Osaka, Japan Activation of Akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. In this study, we attempted to identify novel substrates for Akt by using an antibody, which recognized phosphorylated consensus motif by Akt. Using PC12 cells, which overexpressed constitutively active Akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. Using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. We then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. The truncated TrkB receptor, T1, is involved in the control of cell morphology via the regulation of Rho proteins via Rho GDI1. However, it is unclear whether T1 regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, ROCK, PAK and ERK. Moreover, BDNF resulted in the disappearance of stress fibers in the cells treated with LPA, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with CFP fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho signaling pathways and the actin cytoskeleton.
Anjana Kalita Eukaryotic Gene Expression Laboratory, National Institute of Immunology, New Delhi, India JAK and STAT are activated in response to many cytokines and growth factors. This pathway has been primarily studied in non-neuronal origin cells. It has been shown that STAT activated cellular transformation, occurs through transcription regulation of specific genes like c-Myc, Cyclin D1, Bcl2. The present study was undertaken to identify target genes of JAK/STAT pathway in response to TNF-alpha & IGF-1 in neuronal origin cells. We also looked at the mechanism of activation of these target genes. We saw that JAK/STAT pathway was involved in upregulation of CyclinD1 in response to IGF-1 both at protein & RNA levels but no change was observed in c-Myc, Bcl2 levels. Interestingly, MAPK-erk, which is also known to activate CD1 in other systems, had no effect on the CD1 level in neuronal cells. On the other hand, PI3Kinase not only activated JAK/STAT pathway but also affected on CyclinD1 level. Thus it appears that JAK/STAT is activated by PI3K, which in turn upregulates CyclinD1 level. PI3K also activated erk without changing CD1 level, thus we show that JAK/STAT pathway is important for survival of neurons, activated by PI3K. CD1 is a one of the major downstream targets of JAK/STAT.
Hiroko Inoue 1 , Haruhisa Kawasaki 2 , Ritsuko Fujii 2 , Tohru Yoshioka 2 , Kazunori Sango 3 , Toshihiko Kadoya 4 , Hidenori Horie 2 1 Sch. of Sci. and Eng., Waseda Univ., Tokyo, Japan; 2 Advanced Res. Center for Biol. Sci., Waseda Univ., Tokyo, Japan; 3 Tokyo Metropolitan Inst. for Neurosci., Tokyo, Japan; 4 Pharmaceut. Res. Lab. Kirin Brewery Co., Ltd., Gumma, Japan Oxidized galectin-1 (GAL-1/Ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. But, the mechanism how GAL-1/Ox stimulates macrophages remains to be clarified. In the present study with RT-PCR and Western blotting, we searched for the molecules contributing to the mechanism. RT-PCR analysis with cultured rat peritoneal macrophages revealed that the application of GAL-1/Ox enhanced mRNA expression of nerve regeneration-related factors such as IL-6, IL-1 . However, those of GDNF, NGF, BDNF, NT-3, NT-4, IGF-I, and IGF-II scarcely detected in control condition were not changed by the application of GAL-1/Ox. Since IL -6, IL-1, and LIF can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with GAL-1/OX may enhance nerve regeneration after nerve injury.
Kennichi Katou, Taku Iwamoto, Satoshi Kida Dep. of Bioscience, Tokyo Univ. of Agriculture, Japan ␣CaMKII has been known to play essential roles in synaptic plasticity. In response to increase in Ca 2+ concentration, ␣CaMKII is activated by the interaction with Ca 2+ /CaM. Prolonged increase in Ca 2+ level leads to further activation of ␣CaMKII through autophosphorylation at T286. To understand the molecular dynamics of ␣CaMKII activation in vivo, we have tried to visualize and monitor the ␣CaMKII activity in various types of cells using FRET. In Hela cells, increase in Ca 2+ level induces continuous interaction between ␣CaMKII and CaM and conformational change of ␣CaMKII, indicating that the conformational change of ␣CaMKII mainly reflects the interaction with CaM. In SH-SY5Y cells, the increase in Ca 2+ level induces only transient interaction between ␣CaMKII and CaM but the conformational change of ␣CaMKII is maintained following the termination of the interaction with CaM, suggesting that conformational change of ␣CaMKII is induced by the interaction with CaM and then maintained by autophosphorylation. Thus, mechanisms for regulation of ␣CaMKII activation is cell-type dependent.
Akifumi Kamata, Yusuke Takeuchi, Kohji Fukunaga Dept. Pharmacol., Grad. Sch. Pharmaceu. Sci., Tohoku Univ., Sendai, Japan Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is highly expressed in brain including dopaminergic neurons, however, the role of CaMKII in the dopaminergic neurons remains to be unclear. CaMKII consist of four isoforms, ␣, , ␥ and ␦, and differential isoforms are implicated in the different neural functions. We have examined the isoforms of CaMKII expressed in rat substantia nigra (SN). RT-PCR and immunoblot analyses revealed that the ␥ and ␦ isoform mRNAs including ␦3 isoform, a nuclear isoform of CaMKII, were predominantly expressed in SN. The immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the SN dopaminergic neurons, and immunoreactivity against anti-CaMKII␦1-4 antibody was detected in both nucleus and cytoplasm. Furthermore, we defined expression of BDNF mRNAs having the exon II and IV in SN. Taken together with our previous observation, the CaMKII␦3 isoform was involved in the expression of BDNF in SN dopaminergic neurons.
PS2P-D041 The role of Ca 2+ /calmodulin-dependent protein kinase II in neuronal functions revealed by inactivated CaMKII␣ knock-in mouse Yoko Yamagata 1,2 , Hiroyuki Sakagami 3 , Keiji Imoto 1,2 , Kunihiko Obata 4 , Yuchio Yanagawa 5 1 Okazaki, Japan; 2 SOKENDAI, Okazaki, Japan; 3 Tohoku Univ., Sendai, Japan; 4 RIKEN, Wako, Japan; 5 Gunma Univ., Maebashi, Japan Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. We engineered knock-in mice with the inactivated ␣ subunit of CaMKII by replacing K42 with R42 to study functional significance of CaMKII activity in intact animals. As expected, CaMKII activity was selectively reduced, while CaMKII subunit protein levels were comparable to those of wild type controls in homozygous mutants. In situ hybridization revealed normal expression patterns of CaMKII subunit mRNAs, including dendritic localization of CaMKII␣ mRNA. Further analyses of these mice will be presented in the meeting.
Research funds: A Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (KAKENHI , #17500218) PS2P-D042 Characterization of Ca 2+ -dependent membrane binding proteins of rat brain Shohei Maekawa 1 , Katsutoshi Taguchi 1 , Haruko Kumanogoh 2 , Shun Nakamura 2 1 Division of Biosystems Science, Grad. Sch. of Sci/Tech, Kobe-U Kobe 657-8501, Japan; 2 Division of Biochemistry and Cellular Biology, National Inst. Neurosci. Kodaira, Tokyo 187-0031, Japan Ca 2+ -dependent membrane binding proteins were purified through a Ca 2+ -dependent membrane binding and EGTA-induced membrane dissociation protocol from a crude Triton solubilized membrane fraction. Molecular characterization of these Ca 2+ -dependent membrane binding proteins through LC-MS/MS identified annexin VII, annexin XI, and copin isoforms, in addition to annexin VI and neurocalcin␣. Since the membrane fraction contains phosphatidylethanolamine (PE), cholesterol, and phosphatidylcholine (PC), the membrane binding activity of annexin VI, a major protein in this fraction, was studied using artificial liposomes. Annexin VI showed a PE-dependent, but not cholesterol-dependent liposome binding activity. Immunostaining of annexin VI in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. These results suggest the participation of annexin VI in the membrane cycling at the presynaptic region.
PS2P-D043 Generation of dominant negative mutants of transcription factor CREST Takuzou Simo, Kenichi Kato, Hiroshi Hosoda, Satoshi Kida Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan Transcription factor calcium-responsive transactivator (CREST) has been shown to be required for dendritic growth of neuron. Previous studies have shown that CREST contains two functional domains; multifunctional domain (MFD; aa251∼322) required for transactivation, nuclear localization and homodimerization and C-terminal transactivation domain (CTD; aa238∼402). To further understand the function of CREST on brain function, we tried to generate the dominant negative mutant of CREST to use mouse genetics study. Indeed, we generated fusion proteins of GAL4-DBD with mutants of CREST; CREST C ( 238∼402) lacking C-terminal transactivation domain, MFD and MFD-NLS that is a fusion protein of MFD with nuclear localization signal (NLS). From the experiments using GAL4 one hybrid system, we found that MFD-NLS shows the strong dominant negative effects on transcriptional activation by CREST.
PS2P-D044 Characterization of IQ-ArfGEF, a novel exchange factor for Arf6, in mouse brain Hiroyuki Sakagami, Hisatake Kondo Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai, Japan ADP-ribosylation factor 6 (Arf6) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. To understand its neuronal functions, we have focused on guanine nucleotide exchange factors (GEF) for Arf6. In this study, we determined the entire sequence of a partially identified cDNA (mKIAA0522) encoding a novel Sec 7 domain. The deduced protein contained coiled coil, IQ, Sec7, plekstrin homology domains. Interestingly, it contained a type I PDZ domain-binding motif at its Cterminal tail. ARF pull-down assay demonstrated its ability to active ARF6 more selectively than ARF1. Thus, we named this protein as IQ-ArfGEF. In situ hybridization analysis demonstrated the preferential expression of IQ-ArfGEF in the forebrain and cerebellar granule cells. Furthermore, IQ-ArfGEF mRNA was localized not only in the hippocampal neuronal layers but also in their dendritic fields. Our present findings suggested that IQ-ArfGEF may play important roles in dendrites through activation of ARF6 and interaction with various PDZ proteins.
Eriko Miura 1 , Masahiro Fukaya 1 , Tokiharu Sato 2 , Kazushi Sugihara 3 , Masahide Asano 3 , Katsuji Yoshioka 2 , Masahiko Watanabe 1 1 Dept. Anat., Hokkaido Univ. Sch. Med., Sapporo, Japan; 2 Dept. Mol. Cell. Biol., Cancer Res. Inst., Kanazawa Univ., Kanazawa, Japan; 3 Adv. Sci. Res. Center, Kanazawa Univ., Kanazawa, Japan
The c-Jun N-terminal kinase (JNK) is one of the major mitogenactivated protein kinases (MAPKs), and JSAP1 (or JIP3) is a JNKassociated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression and distribution in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1∼3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with sER and cell membrane. This wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to JNK MAPK pathway and other intracellular machineries.
Yasukazu Hozumi, Kaoru Goto Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Japan Diacylglycerol kinase (DGK) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. To address the functional implications of DGK isozymes in the brain, we have raised specific antibodies against the isozymes. By immunoblot analysis, we found that the immunoreactive bands for DGK, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. Immunohistochemical examination on rat brain revealed that DGK was detected as dot-like structure in the cell membrane, and DGK␥ as round-shaped structure in the juxtanuclear region of neurons. DGK was detected in the perinuclear region and in the dendrites of Purkinje cells. These data show that DGK isozymes localize differentially to the internal membrane system in neurons, suggesting that DGK isozymes play unique roles in distinct internal membrane system.
Kotaro Hattori 1 , Shigeo Uchino 2 , Tomoko Isosaka 1,3 , Shinichi Kohsaka 2 , Takeshi Yagi 3 , Shigeki Yuasa 1 1 Dept. Ultrastractural Res., Nat. Inst. Neurosci., NCNP, Kodaira, Japan; 2 Dep. Neurochem., Nat. Inst. Neurosci., NCNP, Kodaira, Japan; 3 KOKORO Biology Group, FBS, Osaka Univ., Suita, Japan Recently, we have found that Fyn-mediated tyrosine-phosphorylation of NMDA-R subunits is required for haloperidol-induced catalepsy. Haloperidol induced catalepsy in the control mice, but such response was significantly reduced in Fyn-deficient mice. Fyn activation and enhanced tyrosine-phosphorylation of the NMDA-R NR2B subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in Fyn-deficient mice. Both NR2B phosphorylation and the NMDA-induced calcium responses increased after dopamine D 2 -R blockade in cultured striatal neurons of the control, but not in Fyn-deficient neurons. Accordingly, D 2 -R antagonist activates striatal Fyn and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing catalepsy. We also report further proteomics analysis on this molecular pathway.
Hiroaki Okuda, Shingo Miyata, Tsuya Taneda, Atsushi Yamaguchi, Shinsuke Matsuzaki, Natsuko Kumamoto, Masaya Tohyama Department of Anatomy & Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan
The schizophrenia is a well-known debilitating psychiatric disorder. Recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including DYSBINDIN (DTNBP1: dystrobrevin binding protein 1) at 6p22.3. However it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. Since, there is an important another factor, an environmental factor, for the schizophrenia. Although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. Therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the N-terminal region of DYS-BINDIN as a bait. As a result, we detected several transcriptional factors. Thus, we further examine that these interactions regulate the expression level of particular genes in the neurons.
Ryota Adachi 1 , Naoko Oda 2 , Ryuzo Shingai 1,2 1 21st COE Program, Iwate University, Morioka, Japan; 2 Department of Welfare Engineering, Faculty of Engineering, Iwate University, Morioka, Japan
The response to environmental stimuli is important to live for organisms. The soil nematode Caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. C. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. The sensory neurons detected these stimuli are in the head region. The neural network and genes required in each neuron are well known. However, it is under investigation about the integration of chemical and thermal stimuli. To understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. Wild type worms cultivated at 10 • C showed decreased chemotaxis response to water-soluble chemicals and alcohol. Alteration of assay temperature did not affect response to Cl − but that to Na + . To discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay.
Takashi Takekawa 1 , Masaki Nomura 2,3 , Toshio Aoyagi 2 , Tomoki Fukai 1 1 RIKEN Brain Science Institute, Saitama, Japan; 2 Graduate School of Informatics, Kyoto University, Kyoto, Japan; 3 CREST, Japan Science and Technology Agency, Kawaguchi, Japan
The neuron model proposed by Izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. However, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. In fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. Here, we study the phase responses of the Izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. Furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. Therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks.
Honghai Cui, Hirotaka Sakamoto, Mitsuhiro Kawata Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto, Japan
The amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. In the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (SPS) paradigm as PTSD (post-traumatic stress disorder) model. Rats were perfused 7 days after SPS, and intracellular injections of Lucifer Yellow were carried out in neurons of central (CeA) and basolateral amygdala (BLA). At the cellular level, we observed a significant increase of dendritic arborization in BLA pyramidal neurons but no effect on the CeA neurons. These results suggest that SPS-induced structural plasticity of the BLA provides a cellular substrate for affective disorders that are triggered in PTSD.
Takahiko Kawasaki 1,2 , Tatsumi Hirata 1 1 Division of Brain Function, National Institute of Genetics, SOK-ENDAI, Mishima, Japan; 2 PREST, Kawaguchi, Japan
In macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively. The secondary projections from the MOB and AOB first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. Although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. Therefore, We investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of MOB and AOB neurons.
PS2P-D053 An essential role for CLICK-III/CaMKI␥ in regulation of neurite extension Natsumi Ishihara, Sayaka Takemoto-Kimura, Hiroyuki Okuno, Haruhiko Bito Dept. of Neurochem., Univ. of Tokyo, Tokyo, Japan CLICK-III/CaMKI␥, a membrane-anchored CaMK, is a novel member of the CaMKI family that acts downstream of CaMKK. In order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged CLICK-III was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. Phenotypic studies in PC-12 cells expressing various mutants of CaMKK and CLICK-III revealed that depolarization-induced activation of CaMKK-CLICK-III signaling is sufficient to robustly induce neuritogenesis. Interestingly, CLICK-III localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. Furthermore, lipidification mutants of CLICK-III were impaired in their ability to facilitate activity-dependent neuritogenesis. Our results thus indicate a novel role of CLICK-III in activity-dependent neurite formation and extension.
Takuro Maruyama 1 , Masahiro Matsuura 2 , Nobuhiko Yamamoto 1 1 Grad. Sch. of Frontier Biosci., Osaka Univ., Suita Japan; 2 Riso Kagaku Corp., Tsukuba Japan During development, thalamocortical (TC) axons invade into the cortex and stop growing in layer 4. To study TC axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. The dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered Fc-tagged extracellular domains of several candidate molecules. After four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-A5, Sema7a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. A tendency for TC axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. These results suggest that ephrin-A5, Sema7a and kit ligand may contribute to regulating TC axonal growth in the developing cortex.
Research funds: KAKENHI 17023030, KAKENHI 15300107 PS2P-E055 Expression pattern of the guidance molecules and their receptors during GnRH neuron migration from the nose to the ventral forebrain Shizuko Murakami 1 , Katsuhiko Ono 2 1 Dept. Anat., Juntendo Univ. Sch. of Med., Tokyo, Japan; 2 Div. of Neurobiol. and Bioinfo., Nat. Ins. for Physiol. Sci., Okazaki, Japan Using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of GnRH neurons of chick embryos. Netrin-1 expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. A few GnRH neurons expressed netrin-1 receptor neogenin. Slit-1 was expressed in the forebrain, but its receptor robo-1 was not expressed in GnRH neurons. Semaphorin (sema) 3A expression was detected in the olfactory-forebrain region. Within the forebrain, sema3A was absent in the restricted region of the medial forebrain where GnRH neurons migrated in association with a subset of olfactory fibers. At the end of this pathway, sema3A expression was observed in the caudal forebrain, and then GnRH neurons changed their course ventrally. Many GnRH neurons expressed sema3A receptor neuropilin-1, suggesting that sema3A may act as a repellent during the migration of GnRH neurons.
Kazuto Fujishima 1,2 , Qing-Hua Jin 1 , Junko Kurisu 1 , Tomoo Hirano 2 , Mineko Kengaku 1 1 RIKEN, BSI, Lab. for Neural Cell Polarity, Saitama, Japan; 2 Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan During development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. Dendrite morphology is also regulated by cell-cell contacts with neighboring cells. Recent studies have implicated Notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. We previously showed that Delta/Notch-like EGF-related receptor (DNER), neuron-specific transmembrane protein, acts as a Notch ligand and mediates neuron-glia interaction during cerebellar development (Eiraku et al., 2005) . DNER is expressed in the dendrite of developing cortical pyramidal neurons. Here, we analyzed the role of DNER in the development of pyramidal neuron dendrites. We quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and DNER knock out mice.
Reelin signal controls neuronal migration in the developing brain. Because the binding of Reelin to ApoER2/VLDLR induces Dab1 phosphorylation, Dab1 is a critical component of Reelin signal. Recent studies suggested that Reelin signal is involved not only in neuronal migration but in other aspects of neural development. We investigated a possible function of the Reelin signal in the filopodia and neurite formation. Administration of Reelin protein increased number of filopodia in a lesser extent than BDNF treatment. PP2 suppressed effects of Reelin and BDNF on filopodia formation, suggesting that both signal pathways involve Src kinase function. In spite of these similarities of Reelin and BDNF functions in the filopodia formation, BDNF did not phosphorylate Dab1. Reelin treatment of the neurons derived from Dab1 deficient mice did not induce significant increase in the number of filopodia. These results suggest that the filopodia and neurite formation involves the Reelin-Dab1 signal pathways.
Hidenobu Mizuno 1,2 , Tomoo Hirano 1,2 , Yoshiaki Tagawa 1,2 1 Dept. Biophys., Kyoto Univ. Grad. Sch. Sci., Kyoto, Japan; 2 CREST, JST, Kawaguchi, Japan
The left and right hemispheres are interconnected via the corpus callosum. In mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area 17/18 border of the other hemisphere, where they terminate in layers 2/3 and 5. We used in utero electroporation-mediated gene transfer of GFP to assess distribution of callosal projections during development. GFP-labeled callosal axons arrived at the 17/18 border and started to invade the cortical plate around P5. They generated exuberant axonal arborizations by P13 then underwent remodeling to form the adult pattern by P15. Neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. Exogenous expression of a potassium channel Kir2.1 in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers 2/3. Area-specific targeting to the 17/18 border was not affected. Results suggest that certain stages of callosal axon development require neuronal activity in cortex.
PS2P-E060 Identification of a novel tetraspan membrane protein, NPLP, that is up-regulated after the facial nerve axotomy Chihiro Akazawa, Yoh Sasaki, Masato Hoshi, Yasuko Nakamura, Shinichi Kohsaka Department of Neurochemistry, National Institute of Neuroscience, NCNP, Japan Facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. To identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cDNAs expressed in the injured facial nucleus. We identified a novel putative tetraspan protein designated as neuronal PMP22like protein (NPLP) that has a distant homology to PMP22/claudin family proteins. The mRNA expression of NPLP was detected on the cell bodies of neurons meanwhile mRNA of PMP22 resided on the cell bodies of Schwann cells. The NPLP mRNA level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. NPLP has a large extracellular domain that may interact with other membrane molecules. Overexpression of NPLP showed the microspike formation in PC12 cells. Our results suggest that NPLP plays an important role in the regeneration of injured motor neurons.
Shinsuke SHIBATA 1 , Shin-ichi SAKAKIBARA 2 , Hirotaka J OKANO 1 , Hideyuki OKANO 1,3 1 Dept. Physiol., Keio Univ., Sch. Med., Japan; 2 Dept. Histol. Neurobiol., Dokkyo Med. Univ., Japan ; 3 CREST-JST, Japan Musashi (Msi) is an evolutionarily conserved RNA-binding protein.
Drosophila-Msi regulates asymmetric cell division in neural development. In mammalian CNS we identified two members, Msi1 and Msi2, which are co-expressed in neural precursor cells including neural stem cells, suggesting Msi proteins regulate the immaturity of CNS stem cells. To reveal the roles of Msi family in vivo, we generated Msi1 and Msi2 deficient mice. Msi1−/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. Msi2−/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of PNS. Msi1−/− Msi2−/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. In vitro and in vivo analysis show Msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian CNS and PNS development.
Youhei Koshimizu 1 , Satoshi Yamagoe 2 , Kazuo Suzuki 2 , Michiko Ohtomi 1 1 Department of Biomolecular Science, Graduate School of Science, Toho University, Chiba, Japan; 2 Department of Bioactive Molecules, National Institute of Infections Diseases, Tokyo, Japan
We have previously suggested that LECT2 (leukocyte cell-derived chemotaxin 2) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. However, the mechanisms of these regulations have not yet been clarified. In this study, we carried out multi-immunostaining against LECT2 and Tau or MAP2 in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of LECT2 in neurons. At the early stages in cultured neurons, LECT2 was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. In tissues of adult mouse brain, LECT2 was localized to soma, axons and dendrites in neurons. These results suggest the possibility that LECT2 influences the extension of dendrites and axons from the early stage of neuritic development. Furthermore, it is thought that LECT2 may regulate the dendritic and axonal morphology in mature neurons.
Mitsuru Noda 1,2 , Takahiro Moriya 1 , Hideyuki Terazono 1 , Suguru Kudo 5 , Masahiro Yamaguchi 3 , Kenji Yasuda 4 , Izumi Nagata 2 , Kazuyuki Shinohara 1 1 Div. Neurobiol. & Behav., Nagasaki Univ. Grad. Sch. Biomed. Sci., Nagasaki, Japan; 2 Dpt. Neurosurg. Radiol. Nagasaki Univ. Nagasaki, Japan; 3 Dpt. Life Sci., Grad. Sch. Arts & Sci., Univ. Tokyo, Tokyo, Japan; 4 Dpt. Physiol., Grad. Sch. Med., Univ. Tokyo, Tokyo, Japan; 5 Special Division for Human Life Technology, National Institute of Advanced Industrial Science and Technology (AIST), Japan Neural stem cells (NSCs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. Using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of BDNF. The individual NSCs from E12.5 nestin-promoter GFP transgenic mice were placed into 32 pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of BDNF. We observed that a part of NSCs exhibited neuron-like morphology and extended some neurites. The application of BDNF increased the rate of neurite outgrowth. Thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of BDNF.
Ayumi Kyuka, Ryoichi Yoshimura, Yasuhisa Endo Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan
In order to investigate the relationship between neurite outgrowth and target cells, NG108 cells were co-cultured with vascular smooth muscle cells (SM-3). SM-3 cells promoted the neurite outgrowth, but repelled the contact of their growth cones. The DNA microarray analysis indicated that neuropilin-1 mRNA was specifically up regulated in NG108 cells. But the Western Blotting analysis did not show the significant increase of neuropilin-1. Sema3A, a candidate of ligands for neuropilin-1, was found in NG108 cells rather than in SM-3 cells. The immunocytochemical analysis revealed that neuropilin-1 was increased in the surface of NG108 cells co-cultured with SM-3 cells or cultured with the conditioned medium of SM-3 cells. These results suggest that neuropilin-1 may be transferred from cytoplasmic pool to surface of NG108 cells by some soluble factors released by SM-3 cells. We will discuss about kinetics of neuropilin and chemorepellents.
Yoshikuni Edagawa 1 , J. Nakanishi 2 , H. Hamada 1 , Y. Yokomachi 1 , K. Yamaguchi 3 , N. Takeda 1 1 Inst. Biomed. Eng., Waseda Univ., Tokyo, Japan; 2 RIKEN, Japan; 3 Kanagawa Univ., Japan
We show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of UV light. This molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. For the neurite outgrowth, PC12 cell was recruited as a model of neuron. By irradiating UV light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ECM. To draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. NGF stimulated each PC12 cell to extend a neurite along the narrow path: the elongation required both photo-activated path and NGF. The successive irradiation in front of developing neurite achieved bending or branching of the neurite. These results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system.
Tetsuhiro Kakimoto, Hironori Katoh, Manabu Negishi Lab. of Molecular Neurobiology, Grad. Sch. of Biostudies, Kyoto Univ., Kyoto, Japan N-WASP is important for actin polymerization and is strongly expressed in brain. Toca-1 was shown to be required for Cdc42 to activate N-WASP in vitro, although its physiological role is unknown. Here we studied neural function of Toca-1. Toca-1 is strongly expressed in developing brain. Knockdown of Toca-1 in PC12 cells significantly enhances neurite elongation. Consistently, overexpression of Toca-1 suppresses neurite elongation through its amino terminus including the F-BAR/EFC domain, which induces plasma membrane invagination. In addition, knockdown of N-WASP also enhances neurite elongation in PC12 cells, in clear contrast to the previous report using dominant negative mutants. On the other hand, knockdown of Toca-1 in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of N-WASP enhances both axon elongation and branching. These results suggest that a vesicle trafficking regulating protein Toca-1 regulates different aspects of neuronal morphology from N-WASP.
Research funds: KAKENHI (17079003) PS2P-E067 Pragmin, a novel effector of Rnd2 GTPase, stimulates RhoA activity and regulates neurite outgrowth Hironori Katoh, Hiroko Tanaka, Manabu Negishi Kyoto University, Japan
The Rho family small GTPase Rnd2 is specifically expressed in brain. Although Rnd1 and Rnd3 display an antagonistic action for RhoA, signaling pathways of Rnd2 are not well known. Here we have performed a yeast two-hybrid screen using Rnd2 as bait and identified a novel Rnd2-effector protein, Pragmin (pragma of Rnd2). Pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. In in vitro and in vivo binding assays, Pragmin specifically bound to Rnd2 among the Rho family GTPases in a GTP-dependent manner. Rnd2-bound Pragmin significantly stimulated RhoA activity and induced cell contraction through RhoA-and Rho-kinase-dependent pathway in HeLa cells. In PC12 cells, expression of Pragmin inhibited the NGF-induced neurite outgrowth in response to Rnd2, and knockdown of Pragmin by a Pragmin-specific small interfering RNA enhanced the neurite elongation. Therefore, these results suggest that Rnd2 controls neurite outgrowth by regulating RhoA activity through Pragmin.
PS2P-E068 Regulation of growth cone motility by collapsin response mediator protein-2 (CRMP-2) Masumi Iketani 1 , Yanai Shigeki 1 , Fumio Nakamura 1 , Yoshio Goshima 1,2 , Kohtaro Takei 1,2 1 Dept. of Mol. Pharmacol. & Neurobiol., Yokohama City Univ. Grad. Sch. of Med., Yokohama, Japan; 2 CREST, Japan Sci. & Tech. Agency, Kawaguchi, Japan Nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. Collapsin response mediator protein-2 (CRMP-2) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. The local functions of CRMP-2 within growth cones, however, remains unclear. Chromophoreassisted laser inactivation (CALI) that can inactivate selectively a target protein with high spatial and temporal resolutions. Acute localized loss of function of CRMP-2 in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. Asymmetric inactivation of CRMP-2 in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. These findings suggest that a spatial distribution of CRMP-2 activity within the growth cone can regulate growth cone motility and direct neurite outgrowth.
Research funds: CREST, JST PS2P-E069 Eph receptors are negatively controlled by protein tyrosine phosphatase receptor type O Takafumi Shintani 1,2 , Masaru Ihara 1,2 , Hiraki Sakuta 1,2 , Hiroo Takahashi 1,2 , Ikuko Watakabe 1 , Masaharu Noda 1,2 1 Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Japan; 2 CREST, JST, Kawaguchi, Japan Eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. Eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (PTPs) responsible for the negative regulation of Eph receptors have not been elucidated. Here, we identified protein tyrosine phosphatase receptor type O (Ptpro) as a specific PTP that efficiently dephosphorylates both EphA and EphB receptors as substrates. Using the retinotectal projection system, we show that Ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections.
PS2P-E070 Neogenin acts as a displacement factor that releases Rho from Rho-GDI Katsuhiko Hata, Toshihide Yamashita Department of Neurobiology, Graduate School of Medicine, Chiba University, Chiba, Japan Repulsive gudiance molecule (RGMa) is a potent inhibitor of axon regeneration in the adult central nervous system. RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on activation of the Rho/Rho-kinase pathway. Here we show that direct interaction of the Rho GDP dissociation inhibitor (Rho-GDI) with Neogenin which is a receptor of RGMa initiates the activation of RhoA, and this interaction between neogenin and Rho-GDI is strengthened by RGMa. We also found that Neogenin facilitates the release of prenylated RhoA from Rho-GDI. Thus, understanding of the molecular mechanisms of RGMa may be useful for developing methods to overcome the inhibitory signals.
Nobuyuki Fukushima, Masumi Nakashima, Ryou Tokunaga, Ryutaro Moriyama Department of Life Science, Kinki University, Higashiosaka, Japan Lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through G protein-coupled LPA receptors, including LPA 1 and LPA 2 . Here we examine how LPA 3 , third LPA receptor subtype, is involved in neuronal development by using pheochromocytoma 12 (PC12) cells. PC12 cells expressed low levels of endogenous LPA 3 mRNA. When PC12 cells were infected with retrovirus expressing LPA 3 and treated with nerve growth factor under serum-free conditions for 3 days, neurite formation was observed in 55% of total infected cells, which was similar to that in control virus-infected PC12 cells. However, the mean length of neurites was increased in PC12 cells expressing LPA3, compared with that in control PC12 cells. Interestingly, multiple neurite sprouts were frequently observed in LPA-treated LPA 3 -expressing cells. Indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. These results indicated that LPA 3 was involved in neuritogenesis and branch formation.
Masao Sakurai 1 , Tomoko Aoki 1 , Kyoko Ishikawa 1 , Shingo Yoshikawa 2 , John B. Thomas 2 , Chihiro Hama 1 1 CDB, Riken, Hyogo, Japan; 2 Salk Institute, USA In Drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (AL). Each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (ORNs) and projection neurons (PNs). In an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified Wnt5 as a regulatory factor for glomerular patterning. In the Wnt5 mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by Wnt5 expression in ORNs. Previous studies in embryos have shown that Wnt5 repels the axons expressing Derailed (Drl). In the ALs of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the Wnt5 mutant. This and other genetic data suggest that Drl is an antagonist for Wnt5 signaling. We propose that Wnt5 signal transmitted from ORNs to PNs provide one of mechanisms for glomerular patterning in Drosophila.
PS2P-E073 A homeodomain transcription factor, Homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the Drosophila brain Mai Ando, Yoko Totani, Katsuo Furukubo-Tokunaga Grad. Sch. Life Envir. Sci., Univ. Tsukuba, Japan
The precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. Temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. We are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. Here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. MARCM mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. Currently, we are looking for genes that are either downstream or work cooperatively with hth. Based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons.
PS2P-E074 Differential microarray analysis of mushroom body transcripts using chemical ablation Ayako Ino 1 , Masatomo Kobayasi 1 , Taiki Nakajima 1 , Lydia Michaut 2 , Indrayani Ghangrekar 1 , Kuniaki Takahashi 3 , Ryu Ueda 3 , Kaoru Saigo 4 , Walter J. Gehring 2 , Katsuo Furukubo-Tokunaga 1 1 Grad. Sch. Life Envir. Sci., Univ. Tsukuba, Japan; 2 Biozentrum, Univ. of Basel, Switzerland; 3 Genet. Strains Res. Ctr., Natl. Inst. Genet.; 4 Dept. Biophys. Biochem. Grad. Sch. Sci., Univ. Tokyo, Japan Mushroom bodies (MBs) are the centers for olfactory associative learning in the Drosophila brain. We have surveyed MB transcripts using a Drosophila whole-genome microarray and identified 1,465 genes that exhibited reduced expression levels with pharmacological MB ablation. Among the identified genes, we have further selected 102 genes that exhibited a most consistent reduction of expression levels in the ablated brains. In situ hybridization analyses confirmed preferential MB expression patterns for the majority of the identified genes. Moreover, we also demonstrate that RNAi of many of the identified genes causes mild to strong MB defects. These results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the MB neurons in brain development and plasticity.
Mikiko Inaki 1 , Yoshie Suzuki 1 , Hiroyuki Aburatani 2 , Akinao Nose 1 1 Dept. Phys., Univ. Tokyo, Tokyo, Japan; 2 RCAST, Univ. Tokyo, Tokyo, Japan
In Drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. To understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. We focused on two neighboring muscles, 12 and 13, which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. By comparing gene expression data from the two muscles, we identified ∼170 genes with more than two-fold difference in expression level. For 26 candidate genes out of 33 (78%), the differential expression was confirmed by in situ hybridization and/or quantitative PCR. We focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. Ectopic expression and/or knockdown of some of them altered the target specificity of muscles 12 or 13, implicating them in neuronal target recognition.
Makoto Aoki, Hiroshi Segawa, Mayumi Naito, Hitoshi Okamoto Laboratory for Developmental Gene Regulation, Brain Science Institute, RIKEN, Saitama, Japan
The sensory neurons have two axons, the central and peripheral axon. Previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the LIM domains of Islet-2. To find the molecules which regulate the formation of peripheral axons of sensory neurons in the Islet-2 signaling cascade, we have created cDNA library from trigeminal sensory neurons of zebrafish larvae. Most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. Most of them show CNS or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the LIM domains of Islet-2. Loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry.
Hiroshi Yamamoto, Kiyokazu Agata Development of Biophysics, Graduate school of Science, Kyoto University, Kyoto, Japan
Highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. They act through receptors of the Deleted in Colorecal Cancer (DCC) and UNC5 receptor families. Freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. The planarian central nerve system (CNS) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. We have identified eight netrin related genes from planarians, Dugesiajaponica, including four different netrin homologues and three UNC5 homologuesand one DCC homologue. We analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted RNAi experiments to investigating their functions in the process of regeneration of visual neurons. Here we will summarize these results and speculate their molecular actions during eye regeneration in planarians.
PS2P-E078 Analysis of the significance and mechanism of neurite elimination using C. elegans as a model Yu Hayashi 1 , Takaaki Hirotsu 2 , Eriko Kage 1 , Hideaki Takeuchi 1 , Hirofumi Kunitomo 3 , Yuichi Iino 3 , Takeo Kubo 1 1 Dept. Biol. Sci., Grad. Sch. Sci., Univ. Tokyo, Tokyo, Japan; 2 Dept. Biol., Fac. Sci., Kyushu Univ. Grad. Sch., Japan; 3 Mol. Genet. Res. Lab., Univ. Tokyo, Japan During brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. We previously showed that elimination of neurites occurs in the nematode C. elegans and that a novel transcription factor MBR-1 is involved in this process. In the present study, we attempted to clarify the significance of this phenomenon. The mbr-1 mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. By executing cell-specific rescue experiments, we suggest that mbr-1 functions in a set of interneurons involved in olfactory processing. We are now examining whether neurite elimination occurs in these neurons using GFP reporters. We expect that our research provides a useful model for genetic dissection of neurite elimination.
Taro Asakura 1 , Ken-ichi Ogura 1 , Yoshio Goshima 1,2 1 Dept. of Mol. Pharmacol. and Neurobiol., Yokohama City Univ., Grad. Sch. of Med., Yokohama, Japan; 2 CREST, Japan Sci. and Tech. Agency, Kawaguchi, Japan Netrin is an evolutionary conserved axon guidance molecule. In C. elegans, Netrin/UNC-6 is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. One of the neurons guided by Netrin/UNC-6 is the HSN neuron. The HSN neuron of C. elegans has interesting axon guidance. Netrin/UNC-6 is required for the first ventral growth of the HSN neurons. However, what molecules participate in the axon guidance is largely unknown. In this study, we found that the vulval precursor cells strongly expressed Netrin/UNC-6 during the axonal guidance of the HSN neurons. Silencing of Netrin/UNC-6 only at the vulval precursor cells resulted in abnormal axon guidance of HSN. In addition, expression of Netrin/UNC-6 only at the vulval precursor cells in unc-6 null mutants partially restored the HSN guidance defects. These results suggest that Netrin/UNC-6 expressed in vulval precursor cells plays essential roles on axon guidance of the HSN neuron.
Tomomi Sato, Takanori Hamaoka, Hidenori Aizawa, Hitoshi Okamoto Brain Science Institute, RIKEN, Saitama, Japan
The optic tectum is a visual center in vertebrates. It receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. Although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. Here, we established a stable transgenic line Tg(brn3a-hsp70:GFP) in zebrafish to visualize the retinotectal and the tectobulbar projections. To explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxP and Gal4/UAS systems. Using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. In addition, we showed that ephrinB2a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere 2. These results suggest a neuroanatomical and a molecular basis for the motor map in the tectum.
Research funds: Grant-in-Aid for Young Scientists (B) 16700296
Masahiko Taniguchi 1 , Yoshinori Mikami 2 , Tomoyuki Masuda 3 , Tomoyuki Yoshida 2 , Naoto Matsuda 2 , Masayoshi Mishina 2 , Takao Shimizu 4 1 Department of Biochemistry, Cancer Research Institute, Sapporo Medical University, Sapporo, Japan; 2 Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, Japan; 3 Department of Anatomy, Fukushima Medical University, Japan; 4 Dapartment of Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, Japan Semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. Here we report the identification of zebrafish semaphorin 6D (Sema6D). The ORF of Sema6D is 3054 bp (1018 aa). Zebrafish Sema6D protein showed a 70.8% identity with mouse Sema6D protein. To clarify the expression pattern of Sema6D, in situ hybridization was performed. In the embryos, Sema6D is expressed in the lens and hindbrain. We also found that Sema6D has the repulsive activity. These results indicate that Sema6D repels specific types of the axons and functions in the nervous system development.
Research funds: KAKENHI on Priority Areas from the MEXT (17023013)
Mayumi Yamada 1 , Shohei Maekawa 2 , Seiji Miyata 1 1 Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan; 2 Division of Bioscience, Graduate School of Science and Technology, Kobe University, Kobe, Japan OBCAM belongs to the immunoglobulin superfamily cell adhesion molecule (CAM) and was synapse-specific CAM in cortical and hippocampal neurons in vivo. However, it is uncertain how OBCAM is involved in synaptic functions. In this study, we showed that OBCAM was highly concentrated on spines of cultured mature neurons. The inhibition of OBCAM function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. The suppression of OBCAM mRNA expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. In contrast, overexpression of OBCAM augmented the formation of synapses. Moreover, OBCAM was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. These results suggest that OBCAM, a spine-specific CAM, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity.
PS2P-F083 Semaphorin 4D inhibits 1 integrin activity through the R-RasGAP activity of Plexin-B1 Izumi Oinuma, Hironori Katoh, Manabu Negishi Lab. Mol. Neurobiol., Grad. Sch. Biost., Kyoto Univ., Kyoto, Japan Plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. We have recently reported that the semaphorin 4D (Sema4D) receptor, Plexin-B1 functions as a GAP for R-Ras, a member of Ras family GTPases implicated in regulation of integrin activity and cell migration. Here we characterized the role of R-Ras downstream of Sema4D/Plexin-B1 in cell migration. Activation of Plexin-B1 by Sema4D suppressed the ECM-dependent R-Ras activation, R-Ras-mediated PI3-K activation, and 1 integrin activation through its R-Ras GAP domain, leading to inhibition of cell migration. In addition, inactivation of R-Ras by overexpression of the R-Ras-specific GAP or knockdown of R-Ras by RNA interference was sufficient for suppressing 1 integrin activation and cell migration in response to the ECM stimulation. Thus, we conclude that R-Ras activity is critical for ECM-mediated 1 integrin activation and cell migration, and that inactivation of R-Ras by Sema4D/Plexin-B1-mediated R-Ras GAP activity control cell migration by modulating the activity of 1 integrins.
Shun Hamada 1 , Emi Fukuda 2,3 , Shota Katori 2,3 , Takeshi Yagi 2,3 1 Dept. Nutrition Health Sci., Fukuoka Women's Univ., Fukuoka, Japan; 2 Grad. Sch. Frontier Biosci., Osaka Univ., Osaka Japan, 3 CREST, JST, Japan CNR/Protocadherin (Pcdh)␣ is a subfamily of the clustered Pcdhs that comprise >50 cadherin-related molecules and are encoded by three gene clusters (␣,  and ␥). The molecular features and synaptic localization of the clustered Pcdhs have raised the possibility that they are synaptic recognition molecules. We have demonstrated that overexpressed Pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. Furthermore, we found that a stretch of about fifty amino acids located at the C-terminus of Pcdh␣s interfered the trafficking to the cell surface. In the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking 34 or more C-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the C-terminus of Pcdh␣s in the intracellular retention. MDGA1 is a novel cell surface glycoprotein similar to Ig-containing cell adhesion molecules (IgCAMs) with functions in migration and process outgrowth. MDGA1 is expressed by layer 2/3 neurons throughout the neocortex at P7 mice, but is absent in adults. Between E15.5 and late P0, stages that span the generation and radial migration of layer 2/3 neurons, MDGA is expressed in patterns consistent with its expression by migrating layer 2/3 neurons, suggesting a role for MDGA1 in controlling their migration and settling in the superficial cortical plate. We performed loss-of-function studies using RNA interference (RNAi) with in utero electroporation into the lateral ventricle at E15.5 to transfect progenitors of superficial layer neurons. We found that an RNAi suppressing MDGA1 protein blocks proper migration of superficial layer neurons to the superficial cortical layer. We conclude that MDGA1 acts cell autonomously to control the migration of superficial layer cortical neurons. In various pathological conditions, activated microglia mediate immune responses to injured CNS neurons. However, it is not clear whether and how activated microglia affect neurons via direct contacts. This study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following CNS injury and whether telencephalin (TLCN), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. Hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (KA). Compared to control animals, KA-injected mice showed higher density of contacts between activated microglia and dendrites of CA1 pyramidal neurons. Contacts with longer interface appeared in KA-injected mice. These results suggest the importance of direct contacts for the immune response of microglia to injured neurons. Similar contact formation was also observed in TLCN-deficient mice, indicating that the direct contacts are mediated by other molecules than TLCN. Kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three IgG-like domains. Western analysis revealed that the expression levels of Kilon is low at early neuronal culture and increased with progress of culture days. Immunocytochemical observation showed that Kilon was localized at elongating axon and growth cones but not at dendrites on 7 days in vitro (DIV), while Kilon was observed at synapses, mainly at presynaptic terminals on 14 DIV. Similar tendency was observed in Kilon immunohistochemistry of brain sections in vivo. Kilon was observed at axonal fibers of the cerebral cortex on postnatal day 2, but it was seen at synapses in adult brains. These results suggest that Kilon is axonal cell adhesion molecule to control axonal guidance and/or extension.
PS2P-F088 Analysis of mice that show abnormal expression of neuroglycan C, a central nervous system-specific transmembrane proteoglycan Sachiko Aono 1 , Yoshiyuki Kuroda 1 , Fumiko Matsui 1 , Yoshihito Tokita 1 , Keiko Nakanishi 1 , Michiru Ida 1 , Masahito Ikawa 2 , Masaru Okabe 2 , Katsuhiko Ono 3 , Atsuhiko Oohira 1 1 Institute for Developmental Research, Aichi Human Service Ctr., Kasugai, Japan; 2 Research Institute for Microbial Diseases, Osaka University, Suita, Japan; 3 National Institute for Physiological Sciences, Okazaki, Japan Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. To study the role of NGC in the brain, we produced two strains of NGC-mutant mouse by gene-targeting; a mouse strain with no NGCexpression and a strain with low expression (knockdown mice). Both mice were viable and fertile. They did not show obvious abnormalities in gross brain anatomy. To examine their behavioral phenotype precisely, the NGC-knockdown mice were subjected to several kinds of behavioral tests sequentially. They displayed obvious abnormalities in Morris water maze and passive avoidance tests, suggesting that NGC is involved in learning and memory. We are now carrying out the same experiments using the NGC-knockout mice.
Research funds: KAKENHI (17500246) PS2P-F089 Phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation Koichi Iwata 1 , Tatsuhisa Watanabe 2 , Ikuko Suzuki 1 , Junichi Kitagawa 1 , Akiko Ogawa 3 , Kenro Kanda 4 , Kazunao Kuramoto 5 1 Dept. of Physiol., Sch. of Dent., Nihon Univ., Tokyo, Japan; 2 Dept. of Oral and Maxillofacial Surgery, Sch. of Dent., Nihon Univ., Tokyo, Japan; 3 Dept. of Oral Diagnosis, Sch. of Dent, Nihon Univ., Tokyo, Japan; 4 Shinjuku Vocational School of Acupuncture, Moxibustion and Judo Therapy, Tokyo, Japan; 5 Division of Research animal Center, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
The capsaicin-induced pERK expression was studied in the aged rats (30-34 months) following noxious face stimulation. A large number of pERK-LI cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. The larger number of pERK-LI cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. The present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age.
Hirokazu Katsura 1 , Koichi Obata 1 , Masafumi Sakagami 2 , Koichi Noguchi 1 1 Department of Anatomy and Neuroscience, Hyogo College of Medicine, Hyogo, Japan; 2 Department of Otorhinolaryngology, Hyogo College of Medicine, Hyogo, Japan
Recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) in dorsal root ganglion (DRG) neurons contributes to the development of inflammatory and neuropathic pain. In the present study, we examined whether the newest member of the MAPK family of proteins, ERK5 (also known as big MAPK 1 or BMK1) is activated in the DRG and participate in pain-related behaviors in the complete Freund's adjuvant (CFA) model. Peripheral inflammation induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-containing small-to-medium-diameter DRG neurons at days 1 and 3 after CFA injection. Furthermore, time course of phosphorylated-ERK5 level in the DRG matched the emergence of CFA-induced pain hypersensitivity. Our data suggest that activation of ERK5 in DRG neurons may contribute to the development of inflammatory pain.
PS2P-F091 Activation of ERK5 in DRG neurons contributes to acute pain Toshiyuki Mizushima 1,2 , Koichi Obata 1 , Takashi Mashimo 2 , Koichi Noguchi 1 1 Department of Anatomy and Neuroscience, Hyogo College of Medicine, Hyogo, Japan; 2 Department of Anesthesiology, Osaka Univ.
Recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of ERK and p38 in dorsal root ganglion (DRG) neurons. However, there has been no study examining ERK5 (also known as big MAPK 1 or BMK1) activation in DRG neurons after noxious stimulation of normal tissue. Here, we report intensity-dependent ERK5 phosphorylation in DRG neurons by painful stimulation. Noxious stimulation induced phosphorylated-ERK5 in small-to-medium diameter sensory neurons with a peak at 2 min after stimulation. Furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. Our data suggest that activation of ERK5 in DRG neurons may contribute to acute pain induced by noxious stimulation.
Koichi Obata, Koichi Noguchi Department of Anatomy and Neuroscience, Hyogo College of Medicine, Japan There is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogenactivated protein kinase (MAPK) in the dorsal root ganglion (DRG) and spinal cord contributes to the development of inflammatory and neuropathic pain. In the present study, we examined whether the newest member of the MAPK family of proteins, ERK5 (also known as big MAPK 1 or BMK1) is activated in the DRG and spinal cord and participate in pain-related behaviors in the L5 spinal nerve ligation (SNL) model. L5 SNL induced an increase in the phosphorylation of ERK5 not only in the injured L5 DRG, but also in the spared L4 DRG at day 7 after surgery. Furthermore, L5 SNL induced a striking increase in ERK5 phosphorylation in glial cells in the ipsilateral dorsal horn. Our data suggest that activation of ERK5 in the DRG and spinal cord may contribute to the development of neuropathic pain.
Atsushi Sakai 1 , Minoru Asada 1 , Naoki Seno 2 , Hidenori Suzuki 1 1 Department of Pharmacology, Nippon Medical School, Tokyo, Japan; 2 Pharmaceutical Research Center, Kyowa Hakko Kogyo Co., Shizuoka, Japan Glial cell line-derived neurotrophic factor (GDNF) has been known to alleviate the neuropathic pain. However, the mechanisms of GDNFinduced analgesia remain almost unclear. GDNF binds to GFR␣-1, which forms receptor complex and signals intracellularly through RET. Recently, neural cell adhesion molecule (NCAM) has been found to be an alternative signal-transducing receptor for GDNF. Here, we report that NCAM is involved in GDNF-induced analgesia in a rat model of the neuropathic pain. NCAM mRNA expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but GDNF treatment returned its expression to the normal level. Treatment with NCAM antisense oligodeoxynucleotide blocked the analgesic effect of GDNF without affecting RET phosphorylation. These results suggest that activation of NCAM signaling may provide a new strategy to relieve intractable chronic pain.
PS2P-F094 Interleukin-1 enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation Mamoru Takeda 1 , Jun Kadoi 1 , Msanori Nasu 2 , Masayuki Takahashi 1 , Shigeji Matsumoto 1 1 Dep. Physiol and 2 Res. Cent. for Odont. Nippon Dent. Univ., Japan
The present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (TRG) neuronal activity via the IL-1 paracrine mechanism following inflammation. Two days after CFA into the whisker pad area, the mean number of TRG neurons that were encircled by glial fibrillary acidic protein and IL-1-immunoreative satellite cells were significantly increased compared with those in the control. FG labeling was used to identify the TRG neurons innervating the site of inflammation. In the FG-labeled small TRG neurons, the occurrence of IL-1 induced depolarization in inflamed rats was larger than that in control rats. IL-1 application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. These results suggest that activation of TRG satellite glial cells modulates the excitability of TRG neuronal activity via the IL-1 paracrine mechanism following peripheral inflammation.
Junichi Kitagawa 1 , Mamoru Takeda 2 , Jun Kadoi 2 , Yoshiyuki Tsuboi 1 , Shigeji Matsumoto 2 , Koichi Iwata 1 1 Dept. of Physiol., Sch. of Dent., Nihon Univ., Tokyo, Japan; 2 Dept. of Physiol, Sch. of Dent. at Tokyo, Japan, Nippon Dental Univ., Tokyo, Japan
The present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ION-CCI). The mechanical escape threshold was significantly lower in ION-CCI rats at day 3 after ION treatment and the threshold decrement was lasting more than day 14. Single unit activities of ION were recorded from the ION-CCI rats. The firing frequency was significantly higher in A␦ fibers in ION-CCI rats as compared with naive at day 3-14 after ION-CCI. Whole cell patch clamp recording was performed from the middle TRG neurons. IK and IA currents were significantly smaller and Ih current was larger in ION-CCI rats than that of naive rats. The present results suggest that Ik, IA and Ih currents are involved in abnormal firing of TRG neurons in the rats with ION-CCI, resulting in neuropathic pain in trigeminal region following peripheral nerve injury.
Hisako Urai, Munehiro Uda, Katsuya Kami Graduate Schools of Sport and Exercise Science, Osaka University of Health and Sport Sciences, Osaka, Japan Mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (DHR). The present study examined the number of c-Fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following DHR in rats. Furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (SAN). Rats performed an intermittent bout of DHR for 90 min. At 6, 12, 24, 48 and 120 h post-DHR, the rats were applied a weight on the right triceps surae muscle. Immunohistochemical staining for c-Fos and GFAP on spinal cords was performed by Freefloating ABC method. The number of c-Fos-positive neurons detected in superficial dorsal horn were increased at 6 h, peaked at 12 h and then decreased. Intense GFAP immunoreactivities were also detected at 6 and 12 h post-DHR. These results suggest that DHR generates mechanical hyperalgesia by increasing responsiveness of SAN, and moreover astrocytes may regulate excitability of SAN.
Katsuya Kami, Hisako Urai, Munehiro Uda Department of health science, Osaka University of Health and Sport Sciences, Osaka, Japan A production of inflammatory cytokines is increased in injured skeletal muscles. The present study examined relationship between production of inflammatory cytokines in skeletal muscles and Fospositive neurons in spinal dorsal horn following downhill running in rats. The rats performed the downhill treadmill running for 90 min at 25 m/min. After the running, rats were applied the weight on the gastrocnemius muscles for 30 min, and then spinal cord and soleus muscles were removed from the rats. Productions of IL-1beta, IL-6 and TNF-alpha in soleus muscles and expression of Fos protein in dorsal horn were examined using immunohistochemical approach. At 6 h post-running, number of Fos-positive neurons was increased, peaked at 12 h and then decreased to control level at 24 h post-running. Vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at 2 days post-running. These results indicated that increased numbers of Fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles.
Shinichi Sugiyo 1 , Yusuke Sakai 2 , Aya Masawaki 3 , Takashi Shimoda 2 , Masayuki Moritani 1 , Motohide Takemura 1 1 Dept. Oral Anatomy and Neurobiology, Osaka University Grad. Sch. of Dentistry, Osaka, Japan; 2 Dept. of Fixed Prosthodontics, Osaka University Grad. Sch. of Dentistry, Osaka, Japan; 3 Dept. of Dental Anesthesiology, Osaka University Grad. Sch. of Dentistry, Osaka, Japan Diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. We examined if there exist the diabetic rat (DM)-specific difference in nociceptive behavioral and c-Fos immunoreactivity (IR) by formalin test. Injection of formalin into the upper lip 2 weeks before streptozotocin injection induced biphasic specific pain related behavior (PRB) for 90 min. First phase was greater in DM than in the control rat (Ctrl). In DM, second phase was much greater than Ctrl. c-Fos IR in the trigeminal caudal nucleus was also greater in DM than in Ctrl. These results indicate that DM induced greater PRB and c-Fos expression following formalin injection into the rat upper lip.
Yasuko Kozaki, Satoshi Hurune, Fukushi Kambe, Hisao Seo, Kazue Mizumura Res. Inst. Environ. Med., Nagoya University, Nagoya, Japan
We have reported that prostaglandin EP3 receptor (EP3R) activation attenuates the desensitization of bradykinin (BK)-induced increase of intracellular calcium ([Ca 2+ ] i ) in a PTX-sensitive manner in CHO cells expressing canine EP3R and mouse BK B2 receptor (B2R). In this study, we examined the involvement of protein kinase A (PKA) in the desensitization of the BK response. When BK (1 nM) was applied twice with a 6-min interval to the cells expressing B2R, the second [Ca 2+ ] i increase by BK was markedly attenuated. However, the pretreatment with a specific inhibitor of PKA, H-89 (1 M) restored the second response. To further confirm cAMP increase by BK, the expression of a cAMP responsive reporter gene was examined. BK (10 pM) treatment for 1 h significantly increased the reporter gene expression. It is likely that BK increases the level of intracellular cAMP, and thus activates PKA, resulting in the desensitization of the BK response. These results suggest that the desensitization of BKinduced increase in [Ca 2+ ] i was at least in part mediated by PKA.
PS2P-F100 Contribution of peripheral 5HT2A or 5HT3 receptors to Fos expression in the trigeminal spinal nucleus (VSP) produced by the masseter muscle injury of rats Keiichiro Okamoto, Akihisa Kimura, Tomohiro Donishi, Yasuhiko Tamai Dept. Physiology, Wakayama Med Univ., Japan
We have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (MM) injury is attenuated by blocking peripheral 5HT2A or 5HT3/R in male rats with TMJ inflammation. Here we tested if these two 5HT/R subtypes contribute to Fos responses in VSP after MM injury. Formalin injection into MM produced Fos-like immunoreactivity (LI) in several areas of VSP and C2. Fos-LI was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-Vi/Vc) and Vc/C2 transition regions. The number of Fos-LI induced by MM injury was increased in these areas in CFA-evoked TMJ-inflamed rats for 7 days compared to naive rats. We tested if local 5HT2A or 5HT3/R antagonist affects Fos expression in both groups. The number of Fos-LI in the Vc/C2 but not vl-Vi/Vc region was reduced when drugs were injected locally prior to formalin injection in TMJ-inflamed rats. These data suggest that peripheral 5HT2A and 5HT3/Rs play critical roles in mediating MM nociception during TMJ inflammation.
Keiko Abe, Hidemasa Furue, Kohei Kga, Go Kato, Toshiharu Yasaka, Akihiro Tamae, Toshihiko Katafuchi, Megumu Yoshimura Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University, Japan
We examined the postsynaptic effects of 5-HT on substantia gelatinosa (SG) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. In ∼60% of SG neurons examined, 5-HT induced an outward current. The outward current was mimicked and suppressed by a 5-HT 1A agonist and 5-HT 1A antagonist, respectively. In ∼10% of SG neurons, 5-HT evoked an inward current which was mimicked by a 5-HT 3 agonist. The outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. These findings suggest that 5-HT inhibits excitatory neurons and excites inhibitory neurons in the SG through activation of 5-HT 1A and 5-HT 3 receptors, respectively. The reciprocal postsynaptic actions of 5-HT on SG neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by 5-HT. We examined effects of levobupivacaine, ropivacaine, bupivacaine and R-bupivacaine on EPSCs in substantia gelatinosa (SG) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. In SG neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic A␦ and C fiber-evoked EPSCs. However, A fiber-evoked EPSCs were slightly inhibited in amplitude. On the other hand, bupivacaine equally suppressed those three fiber-evoked EPSCs. In DRG neurons, IC 50 of bupivacaine and R-bupivacaine were almost equal on A, A␦ and C neurons. However, IC 50 of levobupivacaine and ropivacaine on A␦ and C neurons were lower than that on A neurons. The present results suggest that pure S (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of AP conduction through A␦ and C fibers.
PS2P-G103 Nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn Hiroshi Ikeda, Kei Kusudo, Kazuyuki Murase Dept. Human & Artificial Intelligence Systems, Univ. Fukui, Fukui, Japan NO plays an important role in the induction of long-term potentiation (LTP) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. In this study, to elucidate the relationship of NO to LTP, we measured the spatiotemporal distribution of NO signal with the NO-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. In superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than 2 h after low-frequency conditioning stimulation (LFS). In the same slices that exhibited LTP, NO was produced and distributed in the superficial dorsal horn during LFS. LTP and production of NO were inhibited in the presence of NO synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (CO).
Research funds: KAKENHI to HI (17700306) and KM (16500262) and grants from Novartis Foundation and Promotion of Science and Sumitomo Foundation to HI
Tao Liu, Tsugumi Fujita, Akiko Koga, Masafumi Kosugi, Terumasa Nakatsuka, Eiichi Kumamoto Dept. Physiol., Facult. Med., Saga Univ., Saga, Japan
In order to know the effect of a PLA 2 activator melittin on inhibitory transmission in the substantia gelatinosa (SG; lamina II of Rexed), we applied the blind whole-cell patch-clamp technique to SG neurons in adult rat spinal cord slices. In about 90% of neurons examined, melittin (1 M) superfused for 3 min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at 0 mV in the presence of bicuculline. This action was visible about 2 min after the beginning of its superfusion and subsided within 8 min after washout. These melittin actions were reduced in extent by a PLA 2 inhibitor 4-bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (COX) and lipooxygenase (LOX). It is concluded that PLA 2 activation pre-and postsynaptically enhances glycinergic transmission in SG neurons, possibly not through metabolites of COX and LOX; this action would contribute to a modulation of nociceptive transmission.
Research funds: KAKENHI (17500275) PS2P-G105 Presynaptic P2Y 1 receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn Terumasa Nakatsuka, Shugo Koga, Tsugumi Fujita, Tao Liu, Masafumi Kosugi, Eiichi Kumamoto Department of Physiology, Faculty of Medicine, Saga University, Saga, Japan
Using whole-cell patch-clamp recordings, we examined whether the activation of P2Y receptors can modulate synaptic transmission in dorsal horn (DH) neurons of adult rat spinal cord slices. Bath applied 2-Methylthio ADP (2meSADP, 30 M), a P2Y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in about 20% of DH neurons recorded. Miniature IPSC in the presence of TTX was increased in frequency by 2meSADP with no change in the amplitude. The 2meSADP-induced increase in miniature IPSC frequency was attenuated in extent by MRS2179 (30 M), a selective P2Y 1 receptor antagonist. These results indicate that the activation of presynaptic P2Y 1 receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of DH neurons. Thus, spinal P2Y 1 receptors can be involved in an inhibitory effect on pain transmission.
Research funds: KAKENHI (17700370), The Japanese Health Sciences Foundation (KH21006) PS2P-G106 Presynaptic enhancement by proteinaseactivated receptor-1 agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons Tsugumi Fujita, Terumasa Nakatsuka, Akiko Koga, Tao Liu, Masafumi Kosugi, Eiichi Kumamoto Dept. Physiol., Facult. Med., Saga Univ., Saga, Japan
We have previously reported that proteinase-activated receptor (PAR)-1 but not PAR-2 agonist (each 1 M) enhances glutamatergic excitatory transmission in substantia gelatinosa (SG) neurons. The present study examined a detail of the PAR-1 mediated enhancement by applying the whole-cell patch-clamp technique to SG neurons in adult rat spinal cord slices. PAR-1 agonist (SFLLRN, 1 M) reversibly increased the frequency of spontaneous EPSC without a change in the amplitude and also in holding current at -70 mV. This facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of PAR-1 antagonist (YFLLRNP, 1 M). These results indicate that the activation of PAR-1s existing in nerve terminals in the SG results in an increase in the spontaneous release of L-glutamate from there. It is suggested that PAR-1 activation in glutamatergic neuron terminals in the SG may be involved in the modulation of nociceptive transmission from the periphery.
Research funds: KAKENHI (16700340) PS2P-G107 Effect of tramadol metabolite M1 on glutamatergic excitatory transmission in rat spinal dorsal horn neurons Akiko Koga, Tsugumi Fujita, Tao Liu, Terumasa Nakatsuka, Eiichi Kumamoto Dept. Physiol., Facult. Med., Saga Univ., Saga, Japan
In order to know the antinociceptive effect of tramadol, we examined the effect of M1, which is one of its metabolites, at 1 mM on glutamatergic excitatory transmission in substantia gelatinosa (SG) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. Bath-applied M1 reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (EPSCs) at −70 mV. This action was not seen in the presence of a -opioid receptor antagonist CTAP (1 M). M1 also reduced the peak amplitudes of EPSCs which were monosynaptically evoked at −70 mV by stimulating primary-afferent A␦-and/or C-fibers in a spinal cord slice with an attached dorsal root. We conclude that M1 inhibits the quantal release of l-glutamate from nerve terminals in the SG through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent A␦-fiber and C-fiber transmission. This effect of M1 would give a cellular basis for the antinociceptive effect of systemically-administered tramadol.
Narihito Iwashita, Natsu Koyama Department of Physiology, Shiga University of Medical Science, Otsu, Japan In our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. These results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. In order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of NMDA receptor antagonist MK-801 or non-NMDA receptor antagonist CNQX was evaluated in hindpaws of pentobarbital-anesthetized rats. Attenuation of skin temperature increase induced by simultaneous MK-801 injection with glutamate was larger than that of skin temperature increase induced by simultaneous CNQX injection with glutamate at the same concentration. On the other hand, inhibition of paw edema formation by CNQX was stronger than by MK-801. These data demonstrate that peripheral NMDA receptor predominantly contributes to vasodilatation, while peripheral AMPA/KA receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation.
PS2P-G109 Studies on pain control system (Rept. 57): Changes in phosphorylation of NR2B-contained NMDA receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption Kan Miyoshi, Minoru Narita, Michiko Narita, Tsutomu Suzuki Dept. Toxicol., Hoshi Univ. Sch. Pharm. Pharmaceut. Sci., Tokyo, Japan Chronic ethanol consumption produces a painful peripheral neuropathy. Mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for 14 weeks. Under these conditions, the immunoreactivities of phosphorylated-Ser-1303 NR2B (p-Ser-1303 NR2B) subunit and phosphorylated-conventional protein kinase C (p-cPKC) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-Tyr-1472 NR2B subunit immunoreactivity was not changed in this region. The hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective NR2B-containing NMDA receptor antagonist. These findings provide evidence for a substantial role of the phosphorylation of cPKC-dependent NR2B-contained NMDA receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats.
PS2P-G110 Prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala Yumi Izawa, Yoriko Kawakami Dept. Physiol. Tokyo Women's Medical University, Tokyo, Japan High frequency stimuli (HFS, 100 Hz, 20 A, 30 s) delivered to the basolateral nucleus of the amygdala (BL) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (PFC). We examined the receptor mechanism underlying this depression of PFC neuron activity. Extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat PFC. Inhibitory effects of HFS delivered to the BL on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mGluR) or NMDA receptor microinjected locally into the PFC. Dopamine depletion, produced by 6-OHDA injected into the substantia nigra, also reduced the inhibitory effects of HFS. The mGluR and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by HFS of the amygdala. Our results suggest that emotional condition modulates pain sensation.
PS2P-G111 The Nav1.3 sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury Bryan C. Hains, Stephen G. Waxman Yale University School of Medicine, USA Spinal cord injury (SCI) induces pain-related phenomena associated with the aberrant expression of Nav1.3, a rapidly repriming voltage-gated sodium channel. In this study we hypothesized that, following SCI, neurons in the thalamus undergo similar electrophysiological changes linked to Nav1.3. Four weeks post-SCI, Nav1.3 protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral RFs, and bursting. These properties persisted after interruption of ascending spinal barrage. Lumbar intrathecal administration of specific antisense oligodeoxynucleotides against Nav1.3 caused a significant reduction in Nav1.3 expression and reversed electrophysiological alterations. These results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after SCI.
Tomoki Fukuda 1 , Hiroyuki Ichikawa 2 , Ryuji Terayama 2 , Tomosada Sugimoto 2 1 Department of Oral Maxillofacial Rehabilitation, Okayama University, Okayama, Japan; 2 Department of Oral Function and Anatomy, Okayama University, Okayama, Japan IB4-sap is a neurotoxin designed for targeting primary nociceptors with IB4 binding sites on the cell surface. However, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. We, therefore, unilaterally injected IB4-sap (2.5 l of 0.067% solution for each ganglion) directly into the 4th and 5th lumbar (L4 and 5) dorsal root ganglia (DRGs). Three weeks later, the rats were killed and DRG sections were stained for IB4-binding. After counterstain, the cell body size of neurons were measured. IB4-sap reduced the total number of DRG neurons in L4 and 5 ganglia combined by 21% (11652 ± 2213 on untreated side versus 9208 ± 1609 on treated side). Small neurons (<1200 m 2 ) were reduced by 28% whereas large ones (≥1200 m 2 ) were not affected. IB4-binding neurons were mostly small (≥96%) and were reduced by 46%. The number of small neurons, that were not stained for IB4-binding, increased by 35% (1994 ± 286 versus 2700 ± 600).
Schuichi Koizumi 1 , Kaoru Nasu-Tada 1 , Makoto Tsuda 2 , Emiko Kunifusa 1,2 , Kazuhide Inoue 2 1 Div. Pharmacol., Natl. Inst. Hlth. Sci., Tokyo, Japan; 2 Dept. Mol. System Pharmacol., Grad. Sch. Pharmaceut. Sci., Kyushu Univ., Fukuoka, Japan Although microglial P2X4 receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. Here, we describe the influence of fibronectin on P2X4 receptor expression in microglia. Microglia that were cultured on fibronectin-coated dishes showed a marked increase in P2X4 receptor expression. Western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. Interestingly, intrathecal injection of ATP-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. Taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the P2X4 receptor in microglia, leading to neuropathic pain.
Research funds: MF16, KAKENHI (170230570)
Ryousuke Fujita, Hiroshi Ueda Div. Mol. Pharmacol. & Neurosci., Nagasaki Univ. Grad. Sch. of Biomed. Sci., Nagasaki, Japan
We have reported that intrathecally administered LPA or endogenous LPA generated upon sciatic nerve injury causes demyelination of dorsal root (DR), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (Nat. Med. 2004). However it remained whether LPA has direct actions on myelinated Schwann cells (SC). In the present study we examined the direct effects of LPA on DR fibers in ex vivo culture system. Scanning electron microscopy (SEM) study revealed that LPA caused a swelling and disruption of myelinated fibers at 24 h. In transmission EM analysis, the addition of LPA caused a disruption of myelin sheath of A␦-and A-fibers. On the other hand, it was found that C-fibers were separated to each other by SCs in naive fibers. Following the addition of LPA, C-fibers showed direct contacts and some of them were uncovered. All these effects were also observed either with or without DR ganglion. Thus, it is suggested that LPA has direct actions on myelinated and unmyelinated SCs to cause demyelination of A-fibers and to uncover C-fibers.
Research funds: KAKENHI 17109015 PS2P-G115 Lysophosphatidic acid (LPA) down-regulates myelin associated proteins in cultured dorsal root fibers Norikazu Kiguchi, Ryousuke Fujita, Hiroshi Ueda Div. Mol. Pharmacol. & Neurosci., Nagasaki Univ. Grad. Sch. Biomed. Sci. Nagasaki, Japan
We have reported that intrathecally administered LPA or endogenous LPA generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (Nat. Med. 2004). These treatments also caused a decrease in myelin protein and their gene expression levels.
Here we report the biochemical evidence underlying this demyelination in cultured fibers. The addition of LPA at 1 mM decreased the protein levels of myelin basic protein (MBP) at 24 h. This action was significantly inhibited by botulinum neurotoxin/C3 (BoNT/C3). On the other hand, LPA also caused a decrease in gene expression of various myelin proteins, such as MBP, PMP22, MAG, P0 in cultured fibers. The maximal decrease was observed all at as early as 3 h after the addition of LPA. BoNT/C3 and Y27632 abolished the LPAinduced down-regulation of MBP gene. All these findings suggest that the down-regulation of gene expression of myelin proteins is through RhoA-ROCK pathway underlying LPA-induced demyelination. Neuropathic pain arise from peripheral never injury. The purpose of this study was to explore behavioral characteristics and investigate the involvement of NMDA receptors and opioid receptors in the behavioural responses following spared nerve injury (SNI). The hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at 0 and 3, 7, 14, 21, 28 days after operation. Pre-emptive co-administration of MK-801 and morphine were tested. SNI produces mechanical and cold allodynia and heat hyperalgesia. Co-injection of morphine and MK-801 decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. The present data demonstrate that the SNI procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. Coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain.
Satoshi Deyama 1,2 , Naomi Akiyama 1 , Mikie Hirata 1 , Takayuki Nakagawa 2 , Shuji Kaneko 2 , Masabumi Minami 1 1 Dept. of Pharmacol., Grad. Sch. of Pharm. Sci., Hokkaido Univ., Sapporo, Japan; 2 Dept. of Mol. Pharmacol., Grad. Sch. of Pharm. Sci., Kyoto Univ., Kyoto, Japan
The bed nucleus of the stria terminalis (BST) is involved in the regulation of negative affective states such as anxiety and fear. In this study, we examined the role of the noradrenergic (NA) transmission within the BST in the negative affective component of pain in rats. We found that excitotoxic lesion of the BST attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (CPA) without reducing nociceptive behaviors. We showed that NA release within the BST was significantly elevated by these noxious stimuli. Intra-BST injection of a -adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced CPA without affecting nociceptive behaviors. These results suggest that visceral and somatic noxious stimuli-induced NA release within the BST contributes to the negative affective, but not sensory, component of pain.
Noriyuki Ozaki, Mariko Kawai, Yasuo Sugiura Department of Functional Anatomy and Neuroscience, Nagoya University, Graduate School of Medicine, Nagoya, Japan Neonatal maternal separation induces visceral hyperalgesia in colon. This study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in Long-Evans rats. Maternal separation was performed for 3 h per day between postnatal day 1 and 14. Visceral sensitivities were assessed in stomach and colon at 10 weeks of age by visceromotor responses induced by either gastric or colorectal distension. Somatic pain sensitivities were also assessed by von Frey filaments and radiant heat. In contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. No difference was found between control and separated rats in somatic pain sensitivities. These results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon.
Research funds: Grant-in-Aid for Scientific Research 16500216 PS2P-G119 Change by aging in muscular mechanical hyperalgesia after lenghtening contraction K. Mizumura, T. Taguchi, T. Matsuda, T. Nasu Res. Inst. Environ. Med., Nagoya Univ., Nagoya, Japan
Our previous experiments have shown that the mechanical threshold of the EDL muscle underwent lengthening contraction (LEC) lowered 1 to 3 days after exercise in rats (8 w old). C-Fos expression in the superficial dorsal horn increased in L4 spinal segment when the EDL muscle was compressed 2 days after exercise. From these results we have concluded that the muscle became hyperalgesic after LEC. In the present experiment, we examined whether this hyperalgesia after LEC changes along aging. Male SD rats 8, 80 (81-84) and 130 (131-133) w old were used. The basal mechanical threshold (Randall-Selitto method) of EDL muscle tended to be higher in 80 w old rats, but not significant. After LEC, the threshold started to decrease 1 day after LEC in all three age groups. It returned to the pre-LEC level 4 days after LEC in 8 and 80 w old rats only. Recovery of 130 w old rats delayed up to 7 days after LEC. Increased c-Fos expression in the superficial dorsal horn was observed in L4 as well as in L5 in 130 w old rats. These results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals.
Tong Liu, Hong P. Wei, Chun Y. Yuan, Ai K. Guo Institute of Neuroscience, Chinese Academy of Sciences, China
Drosophila can display complex courtship behavior. Male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. To up-regulate dopamine level, UAS-TH/TH-GAL4 males were used, which showed high level of dopamine and performed active male-male courtship behavior. This behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. The increased courtship behavior in UAS-TH/TH-GAL4 males is specific to male partners, because the males courted females normally. To down-regulate dopamine level, pale ts , TH temperature sensitive mutant was used. When raised at restrictive temperature, pale ts showed obvious attraction to wild type males. Our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner.
Athushi Yokoyama 1 , Masaharu Akita 2 1 Kanagawa Life-science Res., Japan; 2 Kamkura, Kanagawa, Japan
We have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. The advantages of whole embryo culture are to examine the direct effects of l-calnitin (LCAL) on embryo and also to find the non-teratogenic agent (d-calnitin:DCAL). As the testing agent, LCAL was examined in this study using the rat embryo cultured from day 11 to 13 of gestation. In treated embryos of LCAL, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. On the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with LCAL. In treated embryos of d-calnitin (DCAL), there parameter was not decreased. The observed malformation of LCAL was not observed in the embryos cultured with DCAL. These results might be due to the differences between LCAL and DCAL in the embryo toxicity.
Yoshihisa Uenoyama, Kenji Takase, Junya Hirata, Hiroko Tsukamura, Kei-ichiro Maeda Laboratory of Reproductive Science, Nagoya University, Nagoya, Japan
The mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (GnRH) secretion are poorly understood. Recently, metastin was found to stimulate GnRH secretion and mutations of its receptor are associated with lack of puberty. Effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. When Wistar-Imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days 25-39 of age, they did not show the first estrus before 52 days of age with mean age of 54.7 ± 2.0 day. In contrast, most of normal mouse IgG-treated controls showed the first estrus by 43 days of age with mean age of 42.8 ± 2.2 day. The age of vaginal opening was also delayed in the anti-metastin-treated rats. Thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. Central metastin may be involved in timing the onset of puberty in female rats.
Kenji Takase, Yoshihisa Uenoyama, Shunji Yamada, Hiroko Tsukamura, Kei-ichiro Maeda Lab. of Reproductive Science, Nagoya University, Aichi, Japan Metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (GnRH)/luteinizimg hormone (LH) secretion to time the onset of puberty. The present study aimed to determine expression of metastin, a novel KiSS-1 gene product, in the rat brain during peripubertal period. Wistar-Imamichi strain female rat shows vaginal opening on around days 29 of age (D29), and first estrus on around D34. Brain tissues were obtained on D21, 26, 31, 36 and 41. KiSS-1 mRNA expression in the arcuate nucleus-median eminence region (ARC-ME) and anteroventral periventricular nucleus (AVPV) increased significantly from D21 to 26 and was kept at a high level thereafter. GPR54 mRNA expression in the medial preoptic area increased significantly from D21 to 31. Metastin-immunoreactive cells were not found on D21 but were apparent in the ARC-ME on D26 onward. These results indicate that metastin expression increases in the ARC-ME and AVPV before vaginal opening, suggesting that metastin triggers the onset of GnRH/LH secretion in female rats.
Toshiyuki Saito 1 , Sei-etsu Fujiwara 1 , Kenjiro Konno 2 , Takashi Yamaguchi 3 , Tetsu Nemoto 4 , Etsuko Kasuya 1 , Ryosuke Sakumoto 1 1 Anim. Neurophysiol. Lab., Natl. Inst. Agrobiol. Sci., Tsukuba, Japan; 2 Inst. Exp. Anim. Res., Fac. Med., Gunma Univ., Maebashi, Japan; 3 Grad. Sch. Sci. & Eng., Yamagata Univ., Yonezawa, Japan; 4 Sch. Health Sci., Fac. Med., Kanazawa Univ., Kanazawa, Japan
In the present study, we recorded and examined local field potentials (LFPs) in the hippocampus of piglets performing an operant task by a radio-telemetry system. Under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. After recovery from surgical procedures, the piglets were moved to a training cage. In the LFPs, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. These waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward.
Yasuo Osawa Department of bioscience, Tokyo University of Agriculture, Tokyo, Japan Memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. From the view of treatment of phobia and post traumatic stress disease (PTSD) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. Importantly, previous studies using Pavlovian fear conditioning have shown that d-cycloserine, a NMDA receptor agonist, facilitates memory extinction. In this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. Indeed, we performed conditioned taste aversion (CTA) task, where the ingestion of a novel taste is paired with transient sickness. Our results indicated the injection of d-cycloserine before but not after the re-exposure to CS facilitates extinction of CTA.
PS2P-G126 Hippocampal neural responses during a conditional delayed stimulus-response task in awake mice Nobuhide Kitabayashi 1 , Teruko Uwano 1,3 , Anh Tran 2,3 , Eturou Hori 1,3 , Taketoshi Ono 2,3 , Hisao Nishijo 1 1 System Emotional Science, Univ. of Toyama, Japan; 2 Integrative Neurosci, Molecular and Integrative Emotional Neurosci., Univ. of Toyama, Japan; 3 CREST, Japan
To investigate a hippocampal (HF) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. A trial was initiated by one of two different conditioned tones. After a 2 s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. Of 85 HF neurons, 26 responded to the tones, the reinforcements, and during the delay. Some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. The results suggest a crucial role of the HF in representation of serial events in episodic memory in mice as well as in rats and primates. Further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory.
Naoko Inoue 1 , Atsu Aiba 2 , Kaoru Inokuchi 1 1 Mitsubishi Kagaku Inst. Life Sci. (MITILS), Tokyo, Japan; 2 Grad. Sch. Med., Kobe Univ., Kobe, Japan Vesl-1S/Homer-1a and Vesl-1L/Homer-1c are splicing isoforms encoded by the vesl-1 gene. Vesl proteins bind and regulate mGluR1/5, IP3 receptor, ryanodine receptor, and TRP channel at the postsynapse. The expression of vesl-1S is upregulated by tetanic stimulation that elicits L-LTP. Vesl-1S is thought to play a critical role in the conversion from short-term to long-term memory (LTM). In this study we generated vesl-1S gene-targeting mice (KO) and examined whether Vesl-1S plays a role in the LTM formation. Analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in KO. KO further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. In contrast, consolidation process of the extinction was normal in KO. These results demonstrate that the Vesl-1S protein plays critical roles in various processes of the LTM formation. We now examine the signaling pathways important for LTM formation that are altered in KO.
Hiroshi Ageta 1 , R. Migishima 1 , S. Kida 2 , K. Inokuchi 1,3 1 Mitsubishi Kagaku Inst. Life Sci (MITILS), Japan, 2 Tokyo Univ. Agricul., Japan; 3 CREST, JST, Japan Memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. Activin A mRNA increases following L-LTP induction in the hippocampus, suggesting that activin plays a role in the memory formation. Here, we generated Activin and Follistatin (antagonizes Activin function) transgenic mice in which the transgene expression was tightly regulated by DOX in a forebrain-specific manner (Tet OFF system). Transgene expression was turned OFF or ON within 3 d by (+/−) DOX. Contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. Furthermore, activin plays a role in the re-consolidation process. Thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. These mice are useful for the study of PTSD.
PS2P-H129 Sex differences in the effects of chronic estrogen treatment on fear conditioning in C57BL/6J mice Takaaki Ozawa, Mumeko Tsuda, Sonoko Ogawa Kansei, Behavioral and Brain Sciences, University of Tsukuba, Tsukuba, Japan It has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. Although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. In the present study, we investigated the effects of different doses of estrogen on fear conditioning (FC) learning in both sexes of mice. Gonadectomized C57BL/6J mice were implanted with a silastic capsule containing 0, 5, 10 or 20 g of estradiol benzoate. Since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to FC. Mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued FC responses. We found that estrogen facilitated both types of FC learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors.
PS2P-H130 Analysis of brain regions activated during memory consolidation in passive avoidance task Zhang Yue Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan Short-term memory (STM) is labile. To generate long-term memory (LTM), STM is stabilized through a process known as memory consolidation. Importantly, previous studies have shown that memory consolidation requires the function of transcription factor CREB whose activation induces c-fos expression. In this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. Indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. Consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. More interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task.
Hiroshi Nomura, Norio Matsuki Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
We have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. Rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested 48 h after reexposure. Ethanol-treated groups expressed longer freezing and the effect lasted for 2 weeks. Reactivation was necessary for the effect. The injection of ethanol itself did not induce a fearful response. As memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. Increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. Post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. Moreover, picrotoxin inhibited the memory enhancement by ethanol. These studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of GABA A receptors.
PS2P-H132 Analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory Nori Mamiya, Akinobu Suzuki, Satoshi Kida Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan
The retrieval of conditioned fear memory by conditioned stimulus (CS) initiates two processes; reconsolidation or extinction. We previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. To understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. We measured the levels of phospho-CREB inducing changes in neural plasticity following the re-exposure to CS. Short re-exposure to CS inducing reconsolidation increased in phospho-CREB in amygdala and hippocampus. In contrast, longer re-exposure inducing extinction increased in phospho-CREB in amygdala and prefrontal cortex. These results indicate that distinct brain areas are activated in response to short or long re-exposure to CS and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction.
PS2P-H133 Analysis of molecular mechanism for the destabilization of retrieved contextual fear memory Akinobu Suzuki, Satoshi Kida Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan Reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. To understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. We previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. In this study, we compared molecular signatures of reconoslidation and extinction using mice. Pharmacological experiments using antagonists for cannabinoid receptor 1 (CB1) and L-type voltage-gated calcium channels (LVGCCs) indicated that both CB1 and LVGCCs are required for memory extinction but not consolidation and reconsolidation. More interestingly, blockade of either CB1 or LVGCCs function prevents the disruption of the original memory by protein synthesis inhibition. These results suggest that CB1 and LVGCCs are required for not only memory extinction but also the destabilization of reactivated memory.
Hotaka Fukushima, Akinobu Suzuki, Satoshi Kida Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan Previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. To understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. This task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. Brief re-exposure to light box did not affect the stability of fear memory (stable phase). Further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). In contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). Additionally, using pharmacological blockade of CB1 and LVGCCs, we also found that CB1 is required for only memory extinction but that LVGCCs are required for memory extinction and reconsolidation.
Wakoto Matsuda 1 , Takahiro Furuta 1 , Kouichi Nakamura 1,2 , Takeshi Kaneko 1,2 PS2P-H136 Difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum Fumino Fujiyama 1 , Tomo Unzai 1 , Kouichi Nakamura 1,3 , Sakashi Nomura 2 , Takeshi Kaneko 1,3 1 Department of Morphological Brain Science, Kyoto University, Kyoto, Japan; 2 Department of Physical Therapy, Kyoto University, Kyoto, Japan; 3 CREST, Japan
The striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. In the present study, the differences in synaptology of these inputs between both compartments were examined. Axon terminals positive for vesicular glutamate transporter (VGluT)2, thalamostriatal inputs, were less dense in patch region, whereas VGluT1-positive corticostriatal inputs were evenly distributed. Quantitative analysis revealed 84% of VGluT2positive synapses in patch region were formed with spines, whereas 70% in matrix region were made with dendritic shafts. In contrast, the targets of VGluT1-positive inputs were mainly spines in both regions. Moreover, VGluT2-positive axospinous synapses in patch region were larger than VGluT1-positive ones. The present observation suggests that thalamostriatal connection is more plastic in patch region.
Research funds: KAKENHI (16500217, 17022024,16200025, 17022020 (0131),17650100) PS2P-H137 Single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum Tomo Unzai 1 , Fumino Fujiyama 1 , Takeshi Kaneko 1,2 1 Department of Morphological Brain Science, University of Kyoto, Kyoto, Japan; 2 CREST, Japan
The striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. Thalamic intralaminar nuclei are known to project exclusively to matrix compartment. On the other hand, it has not been clarified which thalamic nuclei project to patch compartment. In the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. Recombinant Sindbis virus expressing membrane-targeted green fluorescent protein (palGFP) was injected into the rat thalamus. A single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. The axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex.
Research funds: KAKENHI (16500217, 17022024, 16200025, 17022020(0131) , 17650100) PS2P-H138 Lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice Yuji Tsutsui 1 , Kayo Nishizawa 1 , Nobuyuki Kai 2 , Kazuto Kobayashi 2,3 1 Dept. of Psychology, Fukushima Univ., Japan; 2 Dept. Mol. Genet., Fukushima Medi. Univ., Japan; 3 CREST, JST, Kawaguchi, Japan Dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. The lever pressing experiment was conducted to examine the effects of 6-OHDA injections into the nucleus accumbens (Acb) in C57BL/6J mice. All mice were trained to press the lever for a food pellet using a fixed ratio 5 (FR5) schedule. The mice were injected with ascorbate vehicle or 6-OHDA into the Acb, and then tested post-surgically using the FR5 schedule again. The 6-OHDA-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. This suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. These results suggest that the Acb dopamine system is important for the initiation and control of the operant behaviors in rodents.
Hideshi Shibata Laboratory of Veterinary Anatomy, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan Retrosplenial area 29 is one of the important structures for spatial memory and behavior in the rat. To understand more fully the functional roles played by area 29, it is essential to clarify the neural circuitry subserving these functions. In the present study, we analyzed the organization of frontal cortical projections to area 29 in the rat, using retrograde transport of cholera toxin B subunit (CTb). CTb injections into area 29d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. CTb injections into area 29c labeled cells in similar cortical regions, except for the orbital cortex. CTb injections involving areas 29a and b labeled cells in the caudal part of the anterior cingulate cortex. The results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area 29, suggesting different functional roles played by each subdivision of area 29 in spatial memory and behavior.
Eiichi Jodo 1 , Yoshiaki Suzuki 2 , Tadahiro Katayama 1 , Ken-yo Hoshino 2 , Yukihiko Kayama 1 1 Dep. of Physiol., Fukushima Med. Univ., Fukushima, Japan; 2 Dep. of Neuropsy., Fukushima Med. Univ., Japan It has been shown previously that the dopaminergic neurons in the ventral tegmental area (VTA) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. Since the VTA receives dense projection from the medial prefrontal cortex (mPFC), such response selectivity of VTA neurons may in part be produced by inputs from the mPFC. However, few studies have compared the firing pattern between these two regions. Our present experiment was designed to make such a comparison in freely moving rats. Two different tones were sequentially presented, one of which (target, 30%) was paired with intracranial simulation of the reward area. The unit activity was recorded from the mPFC and/or the VTA. PFC and VTA neurons exhibited phasic excitation with the peak latency of about 0.1 s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward.
Masato Inoue, Akichika Mikami Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan
To investigate the neuronal mechanism in the ventrolateral prefrontal cortex (VLPFC) and inferotemporal cortex (IT) for holding information for object and their order of presentation, we examined single neuronal activities in the VLPFC and IT while monkeys were performing a serial probe reproduction task. In the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. In 19% out of 438 VLPFC neurons, the delay-period activity showed objectselectivity and order-selectivity. In only 6% out of 254 IT neurons, the delay-period activity showed object-selectivity and order-selectivity. The starting time of the order-selective activity was earlier in the VLPFC. These results suggest that the VLPFC plays a role in holding information for object and their order of presentation and the IT receives information for object and their order of presentation from the VLPFC.
Masao Yukie, Yasutaka Oosawa Department of Behavioral Physiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Japan Relational memory theory (Eichenbaum et al., 1994) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (A+ versus B−; B+ versus C−; C+ versus A−). Very recent monkey study (Alvarado et al., 2005) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. In our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (Yukie et al., 2005) . In the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of 2D visual stimuli presented in a WGTA. Our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of 75 sessions in phase 3, although they learned easily the four problems in phases 1 and 2. Our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory.
Yasuko Sugase-miyamoto 1 , Noriyuki Higo 1 , Munetaka Shidara 2 1 Neuroscience Research Inst., AIST, Tsukuba, Japan; 2 Grad. Sch. of Tsukuba Univ., Tsukuba, Japan A recent dopamine D2 receptor study using antisense cDNA showed that D2 receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. Neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area TE are related to physical attributes of the cue independently of the schedule states. To investigate the cellular substrate for D2mediated associative learning, we examined monkey temporal lobe immunohistochemically with a D2 receptor antipeptide antiserum. D2 receptor immunoreactivity was observed in the pyramidal cells in layers II-VI of the rhinal cortex and area TE. The signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. The signal in layers V-VI was stronger in area 36 of the perirhinal cortex than in area TEav. The differential localization between area 36 and TE suggests the differential roles of the two areas in associative learning process. By using axonal transport of Fast Blue, Diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (Rsp) in the macaque monkey. The Rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (EC-ECL), and projects back to the presubiculum and the EC-ECL. The supracallosal portion of the Rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the EC-ECL. The caudoventral portion of the Rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the EC-ECL. The two portions of the Rsp, thus, have access to different portions of the medial temporal lobe. These results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network.
Hideko Nakano 1 , Natsuko Yoshida 2 , Kiyohisa Natsume 3 1 Kyushu Kyoritsu University, Fukuoka, Japan; 2 Kyushu Institute of Technology, Fukuoka, Japan; 3 Kyushu Institute of Technology, Fukuoka, Japan EEG activity was examined in English rhythm acquisition of Japanese students who learn English as a Foreign Language (EFL). We measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. The result shows that the increase of theta power at F3 and F4 was the highest and suggests that the theta rhythm at F3 and F4 may have a relationship to the process of English rhythm acquisition. Moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. This finding was observed in three right handlers except a left handler and a right handler who had just returned after 6-month English study experience in Australia. These results suggest that the change of theta power at frontal areas may be more closely related to the Japanese EFL learners' English rhythm acquisition.
Research funds: KAKENHI (1565081) PS2P-H146 Neural correlates of music retrieval: An eventrelated fMRI study using sparse temporal sampling Takamitsu Watanabe 1 , Sho Yagishita 1 , Hideyuki Kikyo 1,2 1 Department of Physiology, The University of Tokyo School of Medicine, Tokyo, Japan; 2 Department of Molecular Neuroimaging, National Institute of Radiological Sciences, Chiba, Japan
We investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. Written informed consent was obtained from all the subjects in accordance with the Declaration of Helsinki, and the experimental procedure was approved by the Institutional Review Board of the University of Tokyo School of Medicine. A 1.5 T scanner system was used (TE = 50 ms; TR = 14 s; acquisition time = 3.0 s). We demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. In addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory.
In this fMRI study, we determined the neural correlates of the intellectual excitement. Sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. After the fMRI, each subject self-evaluates subjective "intellectual excitement" of each sentence. Positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. Significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. It suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge.
Hyeonjeong Jeong 1 , Motoaki Sugiura 3 , Yuko Sassa 2 , Keisuke Wakusawa 2,4 , Kaoru Horie 1,5 , Shigeru Sato 1,5 , Ryuta Kawashima 2,5 1 GSICS, Tohoku University, Sendai, Japan; 2 NICHe, Tohoku University, Japan; 3 Miyagi University of Education, Sendai, Japan; 4 Department of Pediatrics, School of Medicine, Tohoku University, Japan; 5 The LBC Research Center, Tohoku University, Japan A foreign language word is learned and retrieved either in daily situations (Situation) or written text (Text), and memory transfer is required when the learning and retrieval modes are different. In this experiment, normal Japanese subjects learned Korean words in the Situation and Text modes in video clips. During a subsequent fMRI session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (Match), and the rest was presented in a different mode (Mismatch). Comparison of the Mismatch with Match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. The results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different.
Georgina E. Cruz 1 , Christie L. Sahley 2 , Kenneth J. Muller 1 1 Physio. & Biophys., Univ. of Miami, Miami, FL, USA; 2 Biol. Sci., Purdue Univ., West Lafayette, IN, USA In some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. The S-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. Its firing increases with sensitization although few additional S-cells initiate impulses during the reflex. We tested the hypothesis that the initial burst of impulses from the S-cell in the stimulated segment suppresses initiations in adjacent segments. Hyperpolarizing the central S-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent S-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. A computational model of S-cell refractoriness further supported the idea of interaction among S-cells during sensitization.
Research funds: NIH, U.S.A.
PS2P-H150 Sensory/motor modules regulating the development of peer social relationship Mamiko Koshiba 1,2 , Shun Nakamura 2 1 JST, CREST, Japan; 2 NCNP Social intelligence is indispensable for animal's survival and could have evolved to language capability. Further, as a recent problem in Japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. In order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. The chick has a merit of being precocial and unique in higher animal with no need of parent-care. Comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. Meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. DNA-chip study along the different social context brought candidates of social genes.
Shogo Sakata 1 , Minoru Hattori 2 1 Department of Behavioral Sciences, Hiroshima University, Higashi-Hiroshima, Japan; 2 Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan Peak interval (PI) 30-s procedure is a very good method to investigate for timing. Six male Wistar rats were trained for five days a week in PI 30-s procedure over 30 days. The 3-s bin of lever press responses on probe trials showed a clear peak point. The temporal distributions had the peak time of regression curve fitting with the Gaussian function. The peak time corresponded to near the 30-s with reinforcement durations. Then nicotine was administrated to the rat by intraperitoneal injection before daily PI 30-s session. Results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. However the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. It suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses.
PS2P-I152 The effect of random practice schedule on arbitrary stimulus-response association learning Satoshi Tanaka 1,2 , Ritz Oshio 1,3 , Norihiro Sadato 1,4 , Manabu Honda 5,6 1 NIPS, Okazaki, Japan; 2 JSPS, Tokyo, Japan; 3 Nagoya Univ., Nagoya, Japan; 4 RISTEX, JST, Tokyo, Japan; 5 SORST, JST, Tokyo, Japan; 6 NCNP, Tokyo, Japan Previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. It remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. We examined the simultaneous learning of multiple arbitrary stimulus-response (S-R) associations under three different practice schedules: blocked, random and regularly ordered. Behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. The present result indicates that random practice schedule is effective also for S-R association learning, which are considered as a bridge between motor control and cognitive control.
PS2P-I153 SPORTS rats show increased level of BDNF in the cerebellum, possibly learning and memorizing well Masaki Morishima, Sayuri Hara, Yutaka Nakaya Dept. Nutrition and Metabolism, Univ. of Tokushima, Japan Previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase A was observed in SPORTS, a novel hyper-running rat on wheel. This study assessed whether SPORTS show increased BDNF levels and better learning and memory. Compared to control, both protein and mRNA levels of BDNF in cerebellum were significantly elevated in SPORTS even without wheel running, and slightly increased in hippocampus. In the cerebellum of SPORTS, TrkB/PI3K pathway was activated, whereas MAPK pathway was activated in the hippocampus. Locomotor activity assessed by the open field test showed that the SPORTS were significantly more active in center coat than control. In the passive avoidance test, SPORTS did not enter a dark area at next time indicating that SPORTS showed better passive avoidance learning. These results suggest that BDNF signaling of SPORTS were activated from TrkB to MAPK and PI3K in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory.
Research funds: KAKENHI (17500429) PS2P-I154 Selective manipulation of working memory through D1 and D2 receptors: computer simulation Shoji Tanaka, Hiroki Yata Dept. of Electrical & Electronics Eng., Tokyo, Japan Though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. To elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. The neurons in the model are leaky integrate-and-fire model with AMPA, NMDA, GABA, and leak conductances and have dopamine D1 and D2 receptors. The computer simulation with this model shows that D1 receptor activation mainly affect working memory activity itself, while D2 receptor activation affect the termination of working memory, being consistent with the experimental result. The simulation also mimics the hyper-and hypo-dopaminergic states. Under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of D1 and D2 receptors indicate efficacies of some these treatments for the restoration of working memory. In conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and GABA.
Kozo Sugioka, Tomiyoshi Setsu, Tatsuro Yamamoto, Toshio Terashima Div. Anat. & Dev. Neurobiol., Dept. Neurosci., Kobe Univ. Grad. Sch. Med., Kobe, Japan
We examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. Pregnant rat (Jcl:Wistar) was injected with saline or 25 mg/kg MAM on the 15th day of gestation. The activity of male and female offspring was measured for each 1 h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every 1 min interval for 1 h under 5 min dark/light alternative schedule during weaning and adult periods. Activity was measured using infra-red sensor in a home-cage placed in the experimental room. The MAM-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. Sex difference of behavioral alteration was evident during adult period in both groups. These behavioral disorders were discussed in relation to the MAM-treated rat showing abnormal hippocampus (disruption of the CA1 pyramidal layer and ectopic neuron mass).
PS2P-I156 Long-lasting tagging of functionally activated neurons in the mouse brain Naoki Matsuo, Leon Reijmers, Mark Mayford The Scripps Research Institute, La Jolla, CA, USA Immediate-early genes (IEGs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. However, conventional IEG approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. Here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. The mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tTA) and tetO promoter regulated feedback loop. When strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (Dox), c-fos promoter driven tTA initiates the tetO-linked expression of mutant tTA (tTA*) that is not inhibited by Dox. This tetO-linked gene expression is then maintained indefinitely by feedback activation via the tTA* even in the presence of Dox. Using this system, we have examined the expression of bicistronic tetO promoter driven tau-lacZ and EGFP-GluR1.
Hamid Gholamipour 1 , Shirin Babri 2 , Khameneh Saied 3 1 Department of Physiology, University of Tabriz, Tabriz, Iran; 2 University of Tabriz, Iran; 3 University of Tabriz, Iran Diabetes mellitus is one of the most prevalent diseases in the world. Because Hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in CA1region of hippocampus on memory formation. Fifty male rats were divided into five groups. (1) control (2) sham operation (3) test (4) diabetic/saline (5) diabetic/insulin. Groups 4 and 5 were made diabetic by treatment with STZ (50 mg/kg, i.p.). In all but the control group, two canula were stereotaxically implanted in CA1 region of hippocampus. Learning was tested and compared between groups through passive avoidance test. Results showed that in the test group the latency increased as compared to control and sham groups (p < 0.05). Compared to sham group diabetic/insulin group showed increased latency (p < 0.05) but no significant difference was found between diabetic/saline and diabetic/insulin groups. In conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic Sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. The Ca 2+ /calmodulin (CaM) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (LTM) formation. We hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. We tested this idea by studying the role of CaM kinase kinases (CaMKKs). Using mouse molecular genetics we found that CaMKK is required for spatial, but not contextual LTM. Consistent with the impaired spatial memory formation, CaMKK null mutants lacked spatial training-induced CREB activation and had impaired late LTP. In contrast to CaMKK, CaMKK␣ is required for contextual, but not for spatial LTM. Furthermore, female CaMKK mutants had normal spatial and contextual LTM. Thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation.
Akshay Anand, Sudesh Prabhakar, Monika Bhatia, C.P. Das Department of Neurology, Post Graduate Institute of Medical Education and Research, Chandigarh, India Background: Parkinson's disease has a prevalence rate of 19 per 100,000 in India. We studied the PARK 4 polymorphism in North Indian population and Parkin expression in early Parkinson's disease (n = 30) and sporadic Parkinson's disease (n = 30). Methods: PCR, SSCP, RFLP and direct sequencing analysis were used to screen mutations. Results: Our results revealed homozygous exonic mutations in exon-1, 3 and 6 in early PD and exon-1 and 12 in sporadic PD, heterozygous mutations in exon 4 and 9 in five early PD and one sporadic PD patient. Frequency of S/N polymorphism was significantly high suggesting that exchange of serine to asparagine at position 167 of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. Our FACS analysis of these samples indicates reduced parkin expression correlating with severity of mutations. Conclusions: We conclude that high frequency of Parkin 4 mutations in PD population in India affect Parkin expression resulting in PD.
Wanida Tripanichkul 1 , Kittisak Sripanichkulchai 2 , David Finkelstein 3 1 Faculty of Medicine, Srinakharinwirot University, Thailand; 2 Faculty of Medicine, Khon Kaen University, Thailand; 3 University of Melbourne, Australia
Emerging data suggests beneficial effect of estrogen for Parkinson's disease (PD), yet the exact mechanisms implicated remain obscured. Activated glia observed in MPTP mouse model and in PD may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. Estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. To determine whether estrogen-elicited neuroprotection in PD is mediated through glia, adult male C57Bl/6 mice were pretreated with 17beta-estradiol (E2), injected with MPTP on the day 6 and brains were collected on day 11. E2 pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by MPTP. The neuroprotective effect of E2 was coincident with an attenuation of a glial response within the SNpc and striatum. These findings propose that E2 neuroprotection in MPTP mouse model may mediate through reactive glia inhibition.
PS2P-I163 Effect of angiotensin-converting enzyme inhibitor perindopril in MPTP-treated mice; immunohistochemistry and in vivo electron spin resonance (ESR) study Rumiko Kurosaki 1 , Fumihiko Yoshino 2 , Masaichi Chang-il Lee 2 1 Dept. of Food Sci. and Nutrition, Showa Women's Univ., Tokyo, Japan; 2 Clin. Care and Med. Div. of Pharmaco., Kanagawa Dental College, Japan
We investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after MPTP treatment. Administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after MPTP treatment. We have reported that TH, GFAP, PV, nNOS and Cu, Zn SOD positive cells in the substantia nigra was changed after MPTP treatment in our immunohistochemical study. The administration of perindopril significantly attenuated MPTP induced changes of these immunopositive nigral cells. We could measure increased oxidative stress in the brain of MPTP and perindopril treatment mice using by in vivo ESR technique. Our results provide further evidence that the ACE inhibitor perindopril may offer a novel therapeutic strategy for Parkinsonǐs disease.
Research funds: KAKENHI (17700577) PS2P-I164 Recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs Shigehiro Miyachi 1 , Kaori Sawada 1 , Haruo Okado 1 , Atsushi Nambu 2 , Masahiko Takada 1 1 Tokyo Met. Inst. Neurosci., Fuchu, Tokyo, Japan; 2 Natl. Inst. for Physiological Sci., Okazaki, Japan
There is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. Based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. An adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. Two to three weeks later, the parkinsonism-inducing drug MPTP was systemically administered several times. Parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of MPTP exceeded threshold for their bilateral onset.
Toru Yasuda, Hideki Mochizuki, Yoshikuni Mizuno Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan
Using the serotype-2 rAAV vectors, we have recently reported the protective effect of Parkin on the ␣-synuclein (␣S)-induced nigral dopaminergic neurodegeneration in a rat model. Here we investigated the neuronal specificity of ␣S toxicity and the effect of Parkin co-expression in a primate model. Another serotype (type-1) of rAAV (rAAV1) carrying αS cDNA (rAAV1-␣S), and a cocktail of rAAV1-␣S and rAAV1 carrying parkin cDNA were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral GABAergic and nigrostriatal dopaminergic neurons. The injection of rAAV1-␣S alone caused a decrease of TH-immunoreactivity in the striatum, while there was no effect on GABAergic neurons. In the presence of overexpressed Parkin, ␣S seemed to be less accumulated and/or phosphorylated at Ser129 residue in GABAergic neurons. These suggest that the ␣S toxicity is not expressed in non-dopaminergic neurons but the ␣S-ablating effect of Parkin is exerted in all neurons introduced in primates.
Tomokazu Oshima, Yohsuke Narabayashi Narabayashi Memorial Laboratory of Neurology, Neurological Clinic, Tokyo, Japan Rigidity in aged parkinsonians is often intractable against surgeries. We investigated how their rigidity scored by UPDRS was related with -band local field potentials (-waves) of the surgical targets in thalamic ventrolateral nucleus (VL) and posteroventral pallidal internal segment (PVP). Forty patients aged 70-80s gave informed consent for thalamotomy and/or pallidotomy. We divided the patients into groups with rigidity of 0.5-1 (I), 1.5-2 (II), 2.5-3 (III), and 3.5-4 (IV). The waves were rated with total periods (%) of 13-27 Hz wavelets in 3-s sample records. Rigidity was re-scored after the surgeries. The VL -waves were rated 65-70% in groups I-III with a slightly increasing tendency for increasing rigidity, but declined to about 55% in group IV. The PVP -waves were 60-80%, but with a decreasing tendency for increasing rigidity. The surgeries alleviated rigidity in all the groups, but were least effective in group IV with least VL and PVP -waves. The results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway.
Yoshihisa Tachibana 1,2 , Hirokazu Iwamuro 1,3 , Masahiko Takada 4 , Atsushi Nambu 1,2 1 Div. Syst. Neurophysiol., Natl. Inst. Physiol. Sci., Okazaki, Japan; 2 SOKENDAI; 3 Dept. Neurosurg., Univ. Tokyo, Tokyo, Japan; 4 Dept. Syst. Neurosci., Tokyo Met. Inst. Neurosci., Fuchu, Tokyo, Japan
To approach a new therapy for Parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (GPe/GPi) of MPTP-treated parkinsonian monkeys (Macaca cyclopus). Compared with the normal state, spontaneous oscillatory discharges were so often observed in the GPe/GPi and the subthalamic nucleus (STN) of the parkinsonian monkeys. Microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded GPe/GPi neurons reduced their abnormal oscillations. These results suggest that glutamatergic excitatory input from the STN contributes to the oscillatory activity of GPe/GPi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. One of the pathological features of Parkinsonǐs disease (PD) is loss of dopaminergic neurons in the substantia nigra pars comapacta (SNpc). And it has been known that ␣-synuclein is involved in the neuronal loss. During the dopaminergic neuronal loss, activated microglia were centered in SNpc. We hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. We demonstrated that ␣-synuclein induced the CD44 expression on microglia and also enhanced the MT1-MMP expression to shed off CD44 at the cell surface and degrade surrounding ECM to open the migratory way. A53T mutant ␣-synuclein showed greater level of CD44 shedding and cell migration. Extracellular treated ␣-synuclein also increased CD44 and MT1-MMP expressions dose-dependently. Among the multiple signaling pathways, ERK pathway was involved in ␣-synuclein induced cell migration. These induced cell migration were also confirmed in human PD patients.
Research funds: National Creative Research Initiative Grant (2004 Grant ( -2006 PS2P-J169 Serotonergic fibers are involved in the conversion of L-DOPA to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats Ryohachi Arai 1 , Hiromasa Yamada 1 , Yoshinari Aimi 1 , Ikuko Nagatsu 2 1 Department of Anatomy, Shiga University of Medical Science, Otsu, Japan; 2 Fujita Health University School of Medicine, Japan Dopaminergic neurons in the substantia nigra pars compacta (SNC) project their axons to the striatum (ST) and their dendrites to the substantia nigra pars reticulata (SNR). Dopamine released from these axons and dendrites is important in the regulation of motor activity. In Parkinson's disease, dopaminergic neurons in the SNC degenerate. L-DOPA is the most effective drug for this disease. We hypothesize that, in Parkinson's disease, a part of administered L-DOPA is converted to dopamine in serotonergic fibers of the ST and SNR. Here we produced parkinsonian model rats by the unilateral injection of 6hydroxydopamine into the SNC, and found that serotonergic fibers in the ST and SNR were immunohistochemically positive for dopamine after L-DOPA administration in the rats. Therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of L-DOPA for Parkinson's disease. In MPTP-induced PD monkey, reactive microglia are observed around neurons in nigra several years after MPTP treatment and may be related to the progression of PD. To evaluate if reactive microglia in striatum and/or nigra of MPTP-induced PD mice are present for a long time after MPTP administration, like PD monkey. Iba 1-and Tb4distribution in microglia were immunohistochemically investigated at 0 h and 7 days after twice MPTP-treatments (one treatment comprised of 4 intraperitoneal injections of 20 mg/kg MPTP at 2 h interval) to C57BL/6 and BALB/c at 6 months (Mo) interval. The recognizable change of Iba 1-and Tb4-distibution in microglia of both mice strains was observed even 6 Mo after the first treatment. The twice MPTP treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. These results suggest that reactive microglia are present for a long time after the treatment by MPTP and must play a role in the chronic progression of PD.
PS2P-J171 Activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with MPTP Hirohide Sawada 1 , Ryohei Hishida 2 , Yoko Hirata 3 , Kenji Ono 4 , Hiromi Suzuki 4 , Shin-ichi Muramatsu 2 , Imaharu Nakano 2 , Kunihiro Tsuchida 1 , Toshiharu Nagatsu 1,4 , Makoto Sawada 4 1 School of Medicine, Fujita Health University, Aichi, Japan; 2 Division of Neurology, Jichi Medical University, Japan; 3 Department of Biomolecular Science, Gifu University, Japan; 4 Research Institute of Environmental Medicine, Nagoya University, Japan
Microglia play an important role in inflammatory process of Parkinson's disease. We examined the effects between neonatal and aged microglia activated with LPS on the nigro-striatal dopamine (DA) neurons in mice treated with MPTP. By MPTP administration to neonatal mice, the number of DA neurons in the substantia nigra was significantly decreased, whereas that in mice treated with LPS and MPTP was recovered. On the contrary, the number of DA neurons of the 60 week-old mice treated with MPTP was significantly decreased with LPS treatment. These results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. Hyposmia is one of the most characteristic symptoms of Parkinson's disease (PD). It may occur even before the motor symptoms start. In the olfactory bulb (OB), dopaminergic cells were present at glomerular layer. Furthermore, it has been reported that OB contains neural stem cells. Thus, OB has attracted attention because of its unique regenerative potential. In the present study, we established isolation of neurosphere forming cells (NSFCs) derived from adult mice OB, and examined proliferation potential in OB after dopaminergic neuronal loss induced by MPTP, a selective toxin for dopaminergic neurons, utilized frequently as PD model. The number of neurospheres derived from adult OB was not decreased with MPTP administration, rather significantly increased. We also evaluated NSFCs differentiation into neural subtypes. The isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative CNS disease like PD.
PS2P-J173 Phosphorylation of ERp57 in adult rat brain with neonatal 6-OHDA treatment Qinghua Li, Yasuyoshi Watanabe Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan Dopaminergic neuron degeneration occurs in sporadic Parkinson's disease (PD), but the mechanism of sporadic PD is not clarified. We prepared neonatal dopamine depleted rats, by i.c.v. injection of 6-OHDA at 3(P3) and 6 days after birth, to investigate the mechanism of dopaminergic neuron degeneration. At P56, tyrosine hydroxylase (TH) immunostaining cells were significantly reduced in the substantia nigra, and TH immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic PD. By two-dimensional electrophoresis we found that a certain protein was phosphorylated in the 6-OHDA lesioned rats at P56, and it was identified as disulfide-isomerase A3 precursor (ERp57) a kind of molecular chaperone of the endoplasmic reticulum (ER) by MALDI-TOF MS. The result suggests that the phosphorylation of ERp57 may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic PD.
Katsunori Nishi Department of Neurology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan Regrowth of survived dopaminergic (DA) neurons after the administration of PSI, a potent proteasome inhibitor, was examined in vitro. Dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. PSI (20 or 25 nM, 48 h) was applied to cultures at 7 days in vitro and succeeding changes of DA neurons were investigated up to 35 days. More than 95% of DA neurons reduced in number after the administration of PSI, and a few truncated DA neurons, devoid of neurites, being observed. Non-DA neurons were less severely affected at these concentrations of PSI. Regrowth of DA neurites was observed approximately 2 weeks after the administration of PSI and continued during the observation period. In most of the regrowing DA neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. Regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. In conclusion, PSI-damaged DA neuron has strong regrowth potential in vitro.
PS2P-J175 Specific expression of proapoptotic factor PAG608 on motor neurons in spinal cords of L-DOPAtreated parkinsonian models Ikuko Miyazaki, Masako Shimizu, Francisco J. Diaz-Corrales, Maria F. Esraba-Alba, Masato Asanuma Dept. of Brain Sci., Okayama Univ. Grad. Sch. of Med., Dent. and Pharmaceut. Sci., Japan
We previously identified a proapoptotic gene, p53-activated gene 608 (PAG608), as a DOPA-induced gene in the striatum of L-DOPA-treated parkinsonian models, which increased p53 expression to promote apoptosis by its nuclear translocation. Last year, we also reported specific induction of PAG608 in the internal capsule of L-DOPAtreated and constitutive expression in the SMI-32-immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. In the present study, we examined distribution of PAG608 in the spinal cords of L-DOPA-injected parkinsonian rats by immunohistochemistry. L-DOPA treatment showed inducing tendency of PAG608 expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. The expression of PAG608 in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia.
PS2P-J176 An approach to the generation of AR-JP mouse model: Crossbreeding of Pael-R transgenic mice with parkin knockout mice Hua-Qin Wang 1,2 , Yuzuru Imai 2 , Haruhisa Inoue 1,2 , Ayane Kataoka 2 , Sachiko Iita 2 , Nobuyuki Nukina 2 , Ryosuke Takahashi 1,2 1 Neurology, University of Kyoto, Kyoto, Japan; 2 BSI, RIKEN, Saitama, Japan
Since loss of parkin E3 activity appears to be causal of AR-JP, accumulation of potentially toxic parkin substrates should result in degeneration of DA neurons. However, parkin knockout mice show no different DA neuronal loss even at old ages, presumably due to relative short lifespan of mice. Pael-R is one of the best characterized parkin substrates. We generated Pael-R transgenic mice and crossbred it with parkin knockout mice. Pael-R transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. However, when Pael-R transgenic mice were crossbred with parkin knockout mice, the DA neuronal loss was induced in a Pael-R gene dosage-dependant manner. These results strongly support that Pael-R accumulation substantially contributes to dopaminergic neurodegeneration in AR-JP. Parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. After dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of Parkinson's disease. Previous study (Lai et al., 2004) have found that application of an antisense oligodeoxynucleotide specific for NR1 have successfully knockdown the expression of NR1 gene expression in the striatum of 6-hydroxydopamine-lesioned rats. In the present study, modulation of gene expression of NR1 was re-addressed using a small interfering RNA (siRNA) specific for NR1. In PC12 cells, reductions of NR1 proteins after a single application of NR1 siRNA were found by western blot experiments. And after one single application of NR1 siRNA in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. Slight reductions in the levels of NR1 immunofluorescence were found in the striatum after the siRNA treatments. Lai et al., 2004. Neurochem. Int. 45, 11-22. Research funds: Faculty Research Grant, FRG/04-05/II-27, Hong Kong Baptist University PS2P-J178 Homocysteine and Parkinson's disease: Effects of acute intranigral administration on dopaminergic system G. Chandra, K.P. Mohanakumar Indian Institute of Chemical Biology, Kolkata, India Homocysteine (Hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. Parkinsonǐs disease (PD) patients receiving long-term L-DOPA therapy are reported to have elevated plasma Hcy levels. We studied whether Hcy is neurotoxic to the nigrostriatal dopamine (DA)-ergic system in SD rats. Animals infused unilaterally in substantia nigra pers compacta (SNpc) with Hcy (0.25-1 mol in 1 l) showed dose dependent loss of DA and its metabolites, in the ipsilateral striatum on 19th day. Animals with 1 mol Hcy exhibited significant motor disabilities and spontaneous and DA-ergic drug-induced turning behaviors. In these animals a clear loss of neurons was visible in SNpc, which were shown to be DAergic by tyrosine hydroxylase immunoreactivity. Intra-raphe infusion of Hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. These results indicate the toxic potential of Hcy to the DA-ergic system and suggest that chronic L-DOPA therapy in PD patients may further deteriorate the disease.
PS2P-J179 Ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥PKC found in SCA14 Takahiro Seki 1 , Takayuki Shimahara 1 , Naoko Adachi 2 , Naoaki Saito 2 , Norio Sakai 1 1 Dept. Mol. Pharmacol. Neurosci., Grad. Sch. Biomed. Sci., Hiroshima Univ., Hiroshima, Japan; 2 Lab. Mol. Pharmacol., Biosig. Res. Ctr., Kobe Univ., Kobe, Japan We have previously demonstrated that several mutant protein kinase C gamma (␥PKC), found in several families of spinocerebellar ataxia type 14 (SCA14), are susceptible to cytoplasmic aggregation and cause cell death in CHO cells, indicating that this property is involved in the etiology of SCA14. However, the relationship between the aggregate formation of mutant ␥PKC and cell death remains unclear. Accumulating evidences indicate that the impairment of the ubiquitin proteasome system (UPS) is related to the pathogenesis of many neurodegenerative disorders. Therefore, we examined whether the aggregate formation of mutant ␥PKC affects UPS function. The immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥PKC. Decreased proteasome activities were also observed in cells having aggregated mutant ␥PKC. These results indicate that the aggregation of mutant ␥PKC exert cytotoxic effect via the impairment of UPS. It is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. Recently, however, another type of stress, endoplasmic reticulum (ER) stress has also been reported to be associated with such diseases. ER stress is characterized by accumulation of unfolded proteins in the ER that is caused by inhibition of protein modification, disturbance of Ca 2+ homeostasis or oxygen deprivation. We recently reported that targeting disruption of Herp, a novel ER stress-related gene, caused F9 cells vulnerable to ER stress. Using these cells, we developed a screening system for molecules that suppress ER stress. Approximately 300 compounds have been screened, and we found some molecules that protect human neuroblastoma cells against ER stress and oxidative stress. We speculate that this system could provide novel therapeutic targets to the ER stress and oxidative stressrelated diseases.
Toshiyuki Araki 1 , Yo Sasaki 2 1 Department of Peripheral Nervous System Research, National Institute of Neuroscience, NCNP, Tokyo, Japan; 2 Washington University School of Medicine, St. Louis, Missouri, USA Axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. We previously reported that increased nicotinamide adenine dinucleotide (NAD) production by the overexpression of nicotinamide mononucleotide adenylyltransferase1 (Nmnat1) or exogenously applied NAD can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. The mammalian NAD biosynthesis is mediated by at least 6 different kinds of enzymes and each enzyme converts different substrate to NAD or its precursors. Here we investigated whether overexpression of these enzymes or exogenous application of NAD precursors protects neurites from degeneration through increased supply of NAD. Cocaine is considered to affect spine morphology and the composition of postsynaptic density (PSD) of medium spiny neurons in nucleus accumbens (NAc). We examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in PSD fraction of rat NAc at different time points. Total PSD protein yield was decreased at 10 min, but next increased at 2 h and returned to basal at 6 h after cocaine challenge. Actin showed a similar pattern but was maintained at high level at 6 h. Both PSD-95 and GluR1 were increased between 2 h and 6 h like actin. By contrast, some proteins such as drebrin were decreased after the peak at 2 h. Interestingly, the 20 S proteasome subunit demonstrated a dramatic upregulation at 2 h. These data suggest that the composition of PSD proteins is regulated by proteasome activity as well as actin cycling. It is possible that some proteins may be removed from PSD by proteasome following transient requirement for organizing PSD in the NAc of chronic cocaine-administrated animals.
PS2P-K184 Effects of MDMA and 5-MeO-DiPT on serotonin transporter and dopamine transporter Yosuke Yamauchi, Takaya Izumi, Takayuki Nakagawa, Shuji Kaneko Dept. Mol. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ., Kyoto, Japan By two electrode voltage-clamp recordings from Xenopus oocytes heterologously expressing serotonin transporter (SERT) or dopamine transporter (DAT), the effects of two addictive agents, 3,4methylenedioxymethamphetamine (MDMA) and 5-methoxy-N,Ndiisopropyltryptamine (5-MeO-DiPT), on SERT and DAT were examined. As previously reported, MDMA (0.3-10 M) dose-dependently induced transport-associated, inward current response in the SERTexpressing cells. Interestingly, MDMA-induced current response was also observed in DAT-expressing cells. On the other hand, 5-MeO-DiPT (0.3-300 nM) evoked an outward current response in SERTexpressing cells similarly to that of selective 5-HT reuptake inhibitors. No current response was observed when 5-MeO-DiPT was applied to DAT-expressing cells. These results suggest that MDMA is transported not only by SERT but also by DAT, and that 5-MeO-DiPT suppresses the spontaneous transport activity of SERT.
Junichi Kitanaka 1 , Nobue Kitanaka 1 , Tomohiro Tatsuta 1,2 , Yoshio Morita 2 , Motohiko Takemura 1 1 Department of Pharmacology, Hyogo College of Medicine, Nishinomiya, Japan; 2 Department of Neuropsychiatry, Hyogo College of Medicine, Nishinomiya, Japan
We examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. A single administration of morphine (30 mg/kg, i.p.) to male ICR mice induced a hyperlocomotion. The ANOVA analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. Clorgyline pretreatment itself did not affect the spontaneous locomotion. Clorgyline at a dose of 0.1 mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. Clorgyline at the doses of 1 and 10 mg/kg significantly inhibited dopamine and serotonin metabolism. These results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through MAO inhibition. Recent studies in our laboratory have shown that methamphetamine (METH)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. In this presentation, the effect of clorgyline pretreatment on METH reward was assessed by conditioned place preference (CPP) paradigm, using an apparatus developed with Supermex ® sensors. Although intact male ICR mice showed a significant CPP for METH (0.5 mg/kg, i.p.), pretreatment with subchronic clorgyline (0.1-10 mg/kg, s.c.) did not affect the magnitude of CPP. Pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. These results indicated that clorgyline pretreatment did not influence METH reward in mice.
of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice Motohiko Takemura 1 , Nobue Kitanaka 1 , Tomohiro Tatsuta 1,2 , Yoshio Morita 2 , Junichi Kitanaka 1 1 Department of Pharmacology, Hyogo College of Medicine, Nishinomiya, Japan; 2 Department of Neuropsychiatry, Hyogo College of Medicine, Nishinomiya, Japan
The effects of lobeline, an alkaloid constituent of Indian tobacco, on methamphetamine (METH)-induced stereotypy and monoamine metabolism were investigated in male ICR mice. Pretreatment with lobeline (3.0-30 mg/kg, i.p.) 15 min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. In saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. The range of lobeline doses examined except 30 mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum 20 min after drug challenge. These results suggested that the inhibitory effect of lobeline (3.0-10 mg/kg) on METH-induced stereotypy did not attribute to the change in the brain monoamine metabolism.
Kazuto Sakoori, Niall Murphy RIKEN BSI, Wako-shi, Japan
Previously we showed that endogenous nociceptin suppresses drug reward. Here, we examined the effect of blockade of NOP receptors on methamphetamine (METH) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. First, NOP receptor KO and WT mice were treated with 1 mg/kg METH and locomotor activity measured daily for 14 days. WT mice showed gradually increasing sensitivity to METH with repeated treatment of METH, whereas NOP receptor knockout mice did not. Next, 5 nmol UFP-101 (a NOP receptor antagonist) and 1 mg/kg METH were co-administrated to mice and locomotor activity measured daily for eight days. UFP-101 strongly suppressed locomotor activity. Thus, it was unclear if UFP-101 suppressed behavioral sensitization to METH during chronic drug treatment. However, when challenged with METH after four or more days without treatment, UFP-101 co-administrated mice showed a lower locomotor response. These results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. The influence of olanzapine (a D2dopamine receptor antagonist) on the morphine-induced conditioned place preference (CPP) in male and female mice was investigated in the present study. Subcutaneous (s.c.) injection of morphine (1-10 mg/kg, three drug sessions) induced place preference both in male and female mice. Intraperitoneal (i.p.) administration of olanzapine (0.5-5 mg/kg) induced place aversion (CPA) in female mice but not in male mice. Administration of olanzapine (1, 2.5 and 5 mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced CPP in male and female mice. However, olanzapine (5 mg/kg, i.p.) caused more than 80% mortality in female but not male mice. The effects of olanzapine were reversed by l-arginine (20 mg/kg, i.p.) pre-administration. In conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (NO) mechanism. Feed-forward associative learning (FFAL) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. When a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. FFAL theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. From this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. Another theoretical issue is the credit assignment problem of motor error. A motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. This problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. FFAL theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis.
PS2P-D191 Regulation of cAMP responsive element binding protein to stress in rat amygdala and hippocampal formation
The Department of Anatomy and Histology, Shanghai Medical School, Fudan University, China Amygdala (AM) and hippocampal formation (HF) are important structures relating with emotional learning and memory. Transcription factor, cAMP-responsive element binding protein (CREB) in AM and HF plays important roles in memory modulating processes. CREB is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. CREB is activated in response to a vast array of physiological stimuli and then becames phosphorylated CREB (pCREB). Neurophysiological and neuropharmacological studies said that CREB may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. But we cannot tell exactly via what kind of neurons in AM and HF CREB regulate these processes. We used the animal model, forced swimming (FS) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pCREB antibody. The distributing profiles and changing rules of pCREB immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. The neuronal types of pCREB immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pCREB, anti-Glu and anti-PV antibodies. The results were: (1) The number of pCREB immunoreactive neuclei and total amount of pCREB in the subnuclei of rat amygdala, Dentate gyrus (DG) and Cornu Ammonis 3(CA3) were increased after FS. The rule of this kind of changing was of region-and time-specific. (2) pCREB immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. These results suggested that pCREB in limbic system regulated the FS process and the regulation was finished via exciting neurons, glutamate neurons.
Hideto Takahashi 1,2 , Tomoaki Shirao 1 1 Dept of Neurobiol & Behav.; 2 ERCGSM, Gunma Univ. Grad. Sch. of Med., Maebashi, Japan Dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. Drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. Here we find that chronic blockade of AMPA receptors (AMPAR) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of NMDAR. Further, the analysis of fluorescence recovery after photobleaching for EGFP-drebrin A reveals that only 22.7 ± 3.0% of drebrin in the spine is stable, with a turnover time of 5.8 ± 0.4 min. Blockade of AMPAR by 20 M CNQX reduces the population of stable drebrin (8.9 ± 3.6%), and has no effect on a turnover time. On the other hand, blockade of NMDAR by 100 M AP5 has no effect on the population of stable drebrin, whereas shortens a turnover time (4.0 ± 0.3 min). These data suggest that AMPAR activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. Instead, NMDAR activities regulate spine-shaft shuttling of drebrin.
Itsuko Nihonmatsu 1 , Yoshito Saitoh 1 , Kaoru Inokuchi 1,2 1 Mitsubishi Kagaku Inst. Life Sci. (MITILS), Tokyo, Japan; 2 CREST, JST, Tokyo, Japan Dendritic protein synthesis requires dendritic localization of mRNAs in neurons. However, ultrastructural localization of these mRNAs have not been well described. Here we employed in situ electronmicroscopic technique to examine the precise localization of ␣CaMKII mRNA in dendrites. ␣CaMKII mRNA was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. We observed an increase in the ␣CaMKII mRNA signals at the synaptic layer undergone L-LTP in the hippocampal dentate gyrus in unanesthetized freely moving rats. The increase was transient and returned to the basal level at 1 h. The alteration in the ␣ CaMKII mRNA localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity.
Reiko Okubo-Suzuki 1,2 , Daisuke Okada 1 , Kaoru Inokuchi 1,2,3 1 Mitsubishi Kagaku Inst. Life Sci. (MITILS), Tokyo, Japan; 2 Yokohama Natl. Univ. Environment Information Sci., Kanagawa, Japan; 3 CREST, JST, Japan Late-phase long-term potentiation (L-LTP) depends on de novo protein synthesis. Synaptopodin (SYNPO), an F-actin-associated protein, increases in the activated synapses following L-LTP induction. Spine volume and F-actin content in the spines also increase during L-LTP. To reveal the roles SYNPO plays in the regulation of spine volume and F-actin content, we examined SYNPO-EGFP (SE) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. SE-overexpression did not alter spine volume, but the amount of SE in spines positively correlated with the spine volume. Pharmacological activation of the NMDA receptors increased both spine volume and SYNPO content in spines. Furthermore, experiments with PtK2 cells indicated that SYNPO stabilizes F-actin. These results suggest that SYNPO synthesized in soma and transported into the activated spines following L-LTP induction stabilizes spine F-actin that may lead to the maintenance of increased spine volume.
Mineo Matsumoto 1 , Mitsutoshi Setou 1,2 , Kaoru Inokuchi 1 1 Laboratory for Molecular Gerontology, MITILS, Japan; 2 Laboratory for Nano-structure Physiology, NIPS, Japan Subcellular localization of RNA is an efficient way to localize proteins to a specific region of a cell. A requirement for dendritic RNA localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. In spite of several attempts to identify these RNAs, the population of RNA species present in dendrites as a whole has not been well described.
Here we show the results of microarray analyses with RNAs isolated from RNA granule or synaptosome fractions prepared from the rat brain. These analyses revealed the complex nature of the dendritic RNA population, which included RNAs that were not expected to be in the dendrites. Neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. Our results suggest that the redistribution of dendritic RNAs is one of the mechanisms regulating local translation in response to synaptic inputs.
PS3A-A005 An activity that traps Vesl-1S protein into spines serves as synaptic tag Synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. Original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. Using live-imaging techniques, we measured entry of Vesl-1S-EGFP into spines (VE trapping) of rat hippocampal neurons in culture, and found that VE trapping activity serves as the synaptic tag in many criteria. VE trapping required synergistic activation of postsynaptic NO-PKG pathway and an activity abolished by TTX at 1 M, but not 50 nM. Because 50 nM TTX is supposed to suppress Na channels only postsynaptically, we concluded that VE trapping is a Hebbian-like process that requires both pre-and postsynaptic activities. However, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity.
PS3A-A006 Acute effects of dehydroepiandrosterone sulfate (DHEAS) on the synaptic transmission and plasticity in rat hippocampal slices Yuxia Xu 1 , Ling Chen 2 , Masahiro Sokabe 1,3,4 1 Dept. Physiol., Nagoya Univ., Grad. Sch. Med., Nagoya, Japan; 2 Dept. Physiol., Nanjing Med. Univ., Nanjing, China; 3 SORST Cell Mechanosening, JST, Nagoya, Japan; 4 Dept. Mol. Physiol., NIPS, Okazaki, Japan
The neurosteroid dehydroepiandrosterone sulfate (DHEAS) is known to improve memory and learning in mammals. Recently we report that chronic administration of DHEAS facilitates the induction of LTP in the rat hippocampus. To elucidate the underlying synaptic mechanism of the DHEAS effects, we examined in this study the acute effects of DHEAS on the synaptic transmission and plasticity at the CA1 region in rat hippocampal slices. An application of 0.1 DHEAS for 10 min to the slice augmented instantly the EPSP, which was terminated within 30 min. However, even 1 h after the drug application, a subthreshold tetanus could induce LTP without alteration of PPF. This facilitating effect of DHEAS on LTP induction was blocked by a coapplication of a NMDA receptor antagonist with DHEAS for 10 min, suggesting that the DHEAS effect involves a sustained modulation of the postsynaptic signaling mediated by NMDA receptor.
Xiaoniu Dai 1 , Ling Chen 2 , Masahiro Sokabe 1,3,4 1 Dept. Physiol., Nagoya Univ., Grad. Sch. Med., Nagoya, Japan; 2 Dept. Physiol., Nanjing Med. Univ., Nanjing, China; 3 SORST Cell Mechanosensing, JST, Nagoya, Japan; 4 Dept. Mol. Physiol., NIPS, Okazaki, Japan
To know whether 17-estradiol (E2) can protect CA1 neurons from functional deficit due to ischemia, adult male Wistar rats were subjected four-vessel occlusion (4VO) for 10 min, and the effect of E2 against this ischemic injury was examined. The electrophysiological properties of CA3-CA1 synapses were examined by a real-time optical recording method 7 days after ischemia. The ischemic brain showed a decreased synaptic transmission and an impairment of LTP induction but no alteration in paired-pulse facilitation. Administration of E2 (1 mg/kg) 3 h before 4VO was able to protect CA1 neurons from these ischemic synaptic dysfunctions. The estrogen receptor-␣ selective agonist PPT (2 mg/kg) produced a similar protective effect, but the estrogen receptor- agonist DPN (8 mg/kg) did not. Above results suggest that E2 can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia.
PS3A-A008 Non-genomic rapid effects of estradiol on hippocampal synapses: Multi-electrode dish analysis Kohei Nakajima 1 , Mari Ogiue-Ikeda 1 , Yuki Oishi 2 , Suguru Kawato 1,2 1 Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, University of Tokyo at Komaba, Tokyo, Japan; 2 Department of Physics, University of Tokyo, Tokyo, Japan Estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. Recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (ER␣) at synapses. Although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic ER␣, there are few electrophysiological evidence about it. Here we investigated effects of estradiol on LTD by using wild type, ER␣ hetero and ER hetero mouse hippocampal slices with a multi-electrode dish (MED, Panasonic). MED enabled us to measure EPSPs in CA1, CA3, and dentate gyrus simultaneously. Hippocampal slices were perfused with estradiol before NMDA-induced LTD. We found that estradiol enhanced LTD both in wild type and ER hetero mouse, but not in ER␣ hetero mouse. Our data suggested non-genomic rapid action of estradiol through synaptic ER␣.
Withdrawn PS3A-A010 Morphological changes of dendritic spines mediated by glucocorticoid receptor (GR) in rat hippocampus Yoshimasa Komatsuzaki 1 , Gen Murakami 2,3 , Tetsuya Kimoto 2,3 , Suguru Kawato 2,3 1 College of Humanities and Sciences, Nihon University, Tokyo, Japan; 2 Department of Biophysics and Life Sciences, University of Tokyo, Tokyo, Japan; 3 CREST, JST, Japan Modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. Dendritic spines are essential for memory storage processes. Here we investigated the effect of dexamethasone (DEX), a specific agonist of glucocorticoid receptor (GR), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of Lucifer Yellow-injected spines in slices. The application of 100 nM DEX induced rapid modulation of the density and morphology of dendritic spines in CA1 pyramidal neurons within 1 h. The total spine density increased from 0.88 spines/m to 1.36 spines/m. DEX significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. Because the presence of 10 M cycloheximide, an inhibitor of protein synthesis, did not suppress the DEX effect, these responses are probably non-genomic.
Hideki Tamura 1 , Yuji Ikegaya 2 , Sadao Shiosaka 1 1 Division of Structural Cell Biology, NAIST, Nara, Japan; 2 Laboratory of Chemical Pharmacology, University of Tokyo, Tokyo, Japan
The capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (I-O) conversion efficiency at the synapses. In the adult mouse hippocampus in vivo, we carefully compared the I-O relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. In extreme cases, long-term potentiation (LTP) and depression (LTD) coexist in the same synaptic pathway with LTP dominating over LTD at higher levels of presynaptic activity. These findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process.
Research funds: 21st Century COE Research PS3A-A012 Actin organizations within single dendritic spines in CA1 pyramidal neurons studies with two-photon photoactivation Naoki Honkura, Masanori Matsuzaki, Haruo Kasai Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Japan
The major cytoskeleton of dendritic spines is filamentous actin (Factin). We have here investigated sub-spine actin organizations using two-photon photoactivation of PA-GFP fused with -actin in rat CA1 pyramidal neurons. We found segregated and discontinuous organizations of two pools of F-actin, dynamic and stable pools, which turned over with time constants of 1.2 min and 17 min, respectively. Fractions of the stable F-actin pool were greater in larger spines, therefore, the entire F-actin pool was more stable in larger spines. We succeeded in visualizing a retrograde flow of F-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds (0.2-1.2 um/min) and lengths (0.2-0.7 um) of the F-actin flow were greater in spines with larger head volumes. Moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin A, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of F-actin.
Tomoharu Nakamori 1 , Katsushige Sato 2 , Kohichi Tanaka 1 , Hiroko Hamazaki 1 1 Mol. Neurosci., TMDU, Tokyo, Japan; 2 Physiology, TMDU, Tokyo, Japan
The visual Wulst (VW) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. To understand the function of the VW in the learning process of imprinting, we investigated the neuronal activity of VW region in chick brain. The slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. When chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at 1 or 4 days post-hatching (P1 or P4), and at P7. This corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until P4, but they lose this ability at P7. In addition, the light-exposed chick showed larger optical response than the dark-rearing one. The optical response in the VW was partly inhibited by the glutamate-and GABA-receptor antagonists. These results suggest that the glutamatergic as well as GABAergic neurons are active in the area including VW and that the neuronal activity of VW affects the learning ability for imprinting.
Withdrawn PS3A-B015 Effect of estrogen on hippocampus in male and female mice Takanori Sugawara 1 , Shinji Hayashi 1 , Victoria Luine 2 1 Graduated School of Integrated Sience, Yokohama City University, Yokohama, Japan; 2 Department of Psychology, Hunter College, City University of New York, New York, USA We examined structural difference in the hippocampal neurons with Golgi stain among the male, the female and the female treated with estrogen neonatally. The mice were gonadectomized and received 17 -estradiol (E2) or oil-vehicle injections at adult before Golgi impregnation. Spine densities 10 m of apical dendrites of the pyramidal neurons in the hippocampus CA1 region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. As a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by E2 treatment in adult. The present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. Thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period.
Jun-Ichi Goto 1,2 , Takafumi Inoue 2,3 , Akinori Kuruma 1 , Katsuhiko Mikoshiba 1,2,3 1 Lab. Developmental Neurobiology, Brain Science Inst., RIKEN, Saitama, Japan; 2 Div. Molecular Neurobiology, Inst. Medical Science, Univ. Tokyo, Tokyo, Japan; 3 Calcium Oscillation Project, ICORP-SORST, JST, Tokyo, Japan Changes in synaptic efficacy at the parallel fiber (PF)-Purkinje cell (PC) synapse are postulated to be a cellular basis for motor learning. Although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (STP) lasting for tens of minutes, have not been discussed to date. Here we report that this synapse shows an apparent STP reliably by a periodic burst pattern of homo synaptic stimulation. This STP is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic Ca 2+ reduction by BAPTA injection or in P/Q-type Ca channel knockout cerebella. This novel type of synaptic plasticity at the PF-PC synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse.
Aya Ishida, Wataru Kakegawa, Michisuke Yuzaki Department of Physiology, Keio University, Tokyo, Japan Mitogen-activated protein kinase (MAPK) cascade is thought to be essential for the synaptic plasticity and learning. In the hippocampus, three different MAPK subfamilies, including extracellular signalregulated kinase (ERK), p38 MAPK and c-Jun NH2-terminal protein kinase (JNK), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (LTP), longterm depression (LTD), and depotentiation after LTP, respectively. Although ERK was previously shown to play a role in cerebellar LTD in cultured Purkinje cells, the role of MAPKs has not been systemically studied. Here, we examined the effect of specific inhibitors of three different MAPKs on LTD by patch-clamp recordings from cerebellar slices. We found that U0126, a specific inhibitor for ERK activation, significantly inhibited LTD induction, whereas SB203580 and SP600125, antagonists for p38 MAPK and JNK, respectively, had no effect. Therefore, unlike hippocampal LTD, cerebellar LTD was dependent on ERK, suggesting involvement of different intracellular downstream pathways.
PS3A-B018 Regulation of AMPA receptor trafficking by AAA ATPases in cerebellar Purkinje cells: Are NSF and VCP playing complementary or antagonistic roles?
Thomas Launey 1 , Chou-Chi Li 2 , Yumiko Motoyama 1 , Junko Yamaoka 1 , Masao Ito 1 1 RIKEN Brain Sci. Inst., Japan; 2 National Cancer Institute, NIH, MA, USA The number of postsynaptic AMPA receptors (AMPAR) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. AAA ATPases influence several of these stages, the most extensively studied being NSF's contribution to AMPAR trafficking. In cerebellar Purkinje cell (PC), we show that Valosin Containing Protein (VCP), an ATPase with high homology to NSF, is bound to AMPA receptors in PC's dendritic compartment. Following GluR2 co-IP from molecular layer, VCP was detected by MS/MS and by monoclonal anti-VCP. Pull-down assay showed a direct interaction between VCP and GluR2 C-term domain, requiring VCP N-term domain and both the NSF and PDZ binding domains of GluR2. GluR2 Phospho-Ser880 promotes VCP complex dissociation, suggesting a relation with synaptic plasticity. Further, Pep2m-related peptides, thought to interfere specifically with NSF-regulated AMPAR trafficking, also blocked the GluR2-VCP interaction.
Yuichi Kitagawa 1,2 , Shin-ya Kawaguchi 1,2 , Tomoo Hirano 1,2 1 Dept. Biophys., Grad. Sch. Sci., Kyoto Univ., Kyoto, Japan; 2 CREST, JST, Kawaguchi, Japan At inhibitory synapses on a cerebellar Purkinje neuron (PN), postsynaptic depolarization induces long-lasting potentiation of the GABA A receptor (GABA A R) responsiveness (Rebound Potentiation: RP). Previous studies have clarified the molecular mechanism regulating RP induction. Whether RP is induced or not is determined by the balance of activities of protein kinases (CaMKII and PKA) and phosphatases (PP-1 and calcineurin). To understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. Computer simulation reproduced the bistable states of GABA A R phospholyration according to stimulation patterns, which apparently corresponded to whether RP was induced or not. We further studied the systematic property of the molecular network, and obtained several experimental predictions. These possibilities were evaluated by experiments such as immunocytochemistry using cultured PNs.
PS3A-B020 Long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning Yuki Haruta, Yachun Huang, Neal Hessler Vocal Behavior Mechanisms RIKEN Brain Science Institute, Japan
In order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. We previously reported that, in synapses of the song motor nucleus RA, participation of postsynaptic NMDA receptor NR2B subunits and presynaptic transmitter release both decrease from young birds to adults. Here, we tested whether synaptic function could be modified in a similar way by acute stimulation. After pairing slight postsynaptic depolarization with presynaptic stimulation, LTD was reliably induced at both HVC and LMAN inputs in juvenile birds from 35 to 42 days old. This depression required activation of postsynaptic NMDA receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. No LTD could be induced in normal birds over 60 days old, when song learning is nearly complete, but LTD remained possible in birds over 60 days old who had been isolated from song tutors, and thus retained the capacity for learning.
PS3A-B021 Involvement of Ca 2+ -permeable AMPAR in the repetitive-LTP induced synaptic enhancement (RISE) Yukiko Ueno, Keiko Tominaga-Yoshino, Akihiko Ogura Graduate School of Frontier Biosciences, University of Osaka, Osaka, Japan
We showed previously that 3 exposures to Glu of cultured rat hippocampal slices at 24 h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (RISE). We examined here whether the conversion of AMPAR subunits occurred during the development of RISE. Immunochemical staining for AMPAR subunits, GluR1 and GluR2, showed that the number of GluR1-positive puncta increased transiently after the repeated Glu exposures, whereas the number of GluR2-positive puncta increased gradually and persistently. JSTX (a Ca 2+ -permeable AMPAR blocker) suppressed fEPSP amplitude recorded at CA3-CA1 synapses by 20-40% in the period corresponding to the transient increase of GluR1-positive puncta. This transient increase should represent the delivery of Ca 2+ -permeable (GluR2-lacking/GluR1-including) AMPAR to synaptic sites. Furthermore, JSTX application at that period blocked the RISE production. These results suggest that the transient delivery of Ca 2+ -permeable AMPAR to synaptic sites is involved in the RISE production.
Yoshihiro Egashira, Tsunehiro Tanaka, Yuji Kamikubo, Yo Shinoda, Keiko Tominaga-Yoshino, Akihiko Ogura Osaka Univ. Grad. Sch. Frontier Biosciences, Toyonaka 560-0043, Japan
Long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. Using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after 3 inductions of LTP, not after its single induction. This synaptic enhancement required protein synthesis for its establishment. We recently found an apparently mirror-image phenomenon; 3 inductions of LTD led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. To know whether this synaptic decrement also requires protein synthesis, we induced LTD 3 times (24 h intervals) by applications of DHPG (a type I mGluR agonist), during or after which anisomycin (a protein translation blocker) was applied. We found that anisomycin did not block the induction of LTD but blocked the establishment of the long-lasting synaptic decrement.
Haruo Mizutani, Tetsuya Hori, Tomoyuki Takahashi Department of Neurophysiology, Graduate School of Medicine University of Tokyo, Tokyo, Japan Bath-application of 5-HT (10 M) attenuated the amplitude of evoked EPSCs and facilitated paired-pulse ratio without affecting the miniature EPSC amplitude, suggesting that its site of action is presynaptic. The 5-HT 1B receptor agonist CP93129 mimicked the presynaptic inhibitory effect of 5-HT. 5-HT 1B receptor antagonist NAS-181 reversed the 5-HT inhibitory effect, indicating that the 5-HT induced inhibitory effect occurs by mediating 5-HT 1B receptors. The presynaptic inhibitory effect of 5-HT became weaker as animals matured. In whole-cell recordings from calyceal presynaptic terminals, 5-HT attenuated voltage-dependent calcium currents, but had no effect on potassium currents. This 5-HT effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, BAPTA. These results suggest that 5-HT, upon activating 5-HT 1B receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse.
Takako Ohno-Shosaku 1 , Masato Ano 1 , Yuki Hashimotodani 2 , Tadasato Nagano 3 , Masanobu Kano 3 1 Dept. Impair. Study, Grad. Sch. Med. Sci., Kanazawa Univ., Kanazawa, Japan; 2 Dept. Neurophysiol., Grad. Sch. Med., Osaka Univ., Osaka, Japan; 3 Dept. Cell. Neurosci., Grad. Sch. Med., Osaka Univ., Osaka, Japan Retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. Endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of Gq-coupled receptors. Here we report that NMDA receptors can also contribute to generation of endocannabinoid signal. Inhibitory postsynaptic currents (IPSCs) were recorded in cultured hippocampal neurons prepared from newborn rats. Application of NMDA induced a transient suppression of cannabinoid-sensitive IPSCs but not cannabinoid-insensitive IPSCs. The NMDA-induced suppression of IPSC was blocked by a cannabinoid receptor antagonist. These results indicate that activation of NMDA receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. The most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. A small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (NOS) positive. A dense distribution of nerve fibers immunoreactive to CGRP, SP, and NPY was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. In addition, ENK-, 5-HT-, and TH-immunoreactive varicose fibers were richly distributed throughout the sectional plane. To analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. For this purpose, we made an electron microscopic study using NADPH-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. Adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (A1, A2a, A2b and A3). Adenosine A1 and A3 receptors have been reported to inhibit high-threshold Ca channel currents in neurons. To investigate the interaction between adenosine A1 and A3 receptors in rat striatum neurons in culture, L-type Ca channel currents were recorded by whole-cell clamp method before and after administration of A1 agonist (CPA) and A3 agonist (2-Cl-IB-MECA). Ca currents were decreased after administration of low concentration of CPA and 2-CL-IB-MECA as reported previously. Although Ca currents were decreased by 2-Cl-IB-MECA in the presence of CPA, Ca currents applied with CPA were not decreased on cells in the presence of 2-Cl-IB-MECA. At administration of CPA and 2-Cl-IB-MECA on cells simultaneously, Ca currents were not decreased. These results suggested that adenosine A3 receptor may inhibit adenosine A1 receptor throughout a intracellular pathway in neurons.
PS3A-C028 Influence of extracellular GABA and taurine to GABA A receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation T. Furukawa 1 , J. Yamada 2 , K. Inoue 1 , Y. Yanagawa 3 , A. Fukuda 1, 2 1 Dept. Physiol., Hamamatsu Univ. Sch. Med., Japan; 2 Dept. Biol. Info. Process, Grad. Sch. Elec. Sci. & Tech., Shizuoka Univ., Hamamatsu, Japan; 3 Dept. Developmental and Integrative Neurosci., Gunma Univ. Sch. Med., Gunma, Japan It is well known that role of GABA A -R mediated actions is important for early CNS development. The radially migrating cells may affected by the actions. GABA content in the brain of GAD67-GFP knock-in mouse decrease compared with the wild type mice. Therefore, we investigate the influence of the circumferential GABA concentration to radially migrating cells. Furthermore, as it was known that GABA A -R is affected by taurine, the influence of taurine to radially migrating cells was also investigated. There was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. Evoked GABA A -R mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. Therefore, we have examined the influence of circumferential taurine to GABA A -R mediate actions.
Takashi Hayakawa 1 , Hiroyuki Hioki 1 , Kouichi Nakamura 1,2 , Hisashi Nakamura 1 , Takeshi Kaneko 1,2 1 Dept. Morphol. Brain Sci., Grad. Sch. Med., Kyoto Univ., Kyoto, Japan; 2 CREST, JST, Kawaguchi, Japan
We previously reported that almost all vesicular glutamate transporter 3(VGLUT3)-immunoreactive (ir) cells were also GABAir in neocortex and choline acetyl transferase (ChAT)-ir in caudate-putamen in rat. Although, in dorsal and median raphe nuclei, many VGLUT3-positive cells showed immunoreactivity for 5-hydroxytryptamine (5HT), a significant proportion (12.3%) of VGLUT3-postive cells was 5HT-negative. In this study, triple immunofluorescence staining was performed for VGlUT3, 5HT and one of the following proteins: neuronal nuclear antigen (NeuN), glial fibrillary acidic protein (GFAP), glutamic acid decarboxylase (GAD67) and tyrosine hydroxylase (TH). Our results showed that all of the VGLUT3-positive/5HT-negative cells were immunoreactive for NeuN but not for GFAP. Furthermore, we found that these VGLUT3positive/5HT-negative neurons didn't show any immunoreactivities for GAD67 nor TH, and thus it is indicated that there is a group of exclusively glutamatergic VGLUT3-positive neurons in these nuclei.
Research funds: KAKENHI 16200025, 17022020, 17650100 PS3A-C030 Cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: Modulation by serotonin M Victoria Puig 1 , Mika Ushimaru 1 , Yoshiyuki Kubota 1 , Akiya Watakabe 2 , Tetsuo Yamamori 2 , Yuchio Yanagawa 3 , Yasuo Kawaguchi 3 1 Div. Cerebral Circuitry, NIPS, Okazaki, Japan; 2 Div. Brain Biology, NIBB, Okazaki, Japan; 3 Dept. Genetic and Behavioral Neurosci., Gunma Univ. Graduate School of Med., Japan
We studied how cortico-striatal (CS) and fast-spiking (FS) cells are modulated by slow-wave-sleep (SWS) oscillations and by serotonin (5-HT). CS and FS cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a GFP in GABAergic interneurons. FS displayed a highsuccess excitation to striatal stimulation, suggesting a control of CS over FS. During SWS, both CS and FS fired during the up-states though with different patterns. The stimulation of the dorsal raphe promoted longer up-states. Moreover, 60% of the CS were inhibited by 5-HT through 5-HT 1A R and 6% were excited through 5-HT 2A R. However, 44% of the FS cells were inhibited and 28% excited. These results show that CS cells are more inhibited by 5-HT than FS. The expression of 5-HTR was confirmed by in situ hybridization.
Research funds: JSPS PE04061 and 15300110
Ryohei Tomioka, Kathleen Rockland Laboratory for Cortical Organization and Systematics, RIKEN Brain Science Institute, Saitama, Japan
In small mammals, GABAergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. Here, we report in monkey as well that some GABAergic neurons send long-distance projections. Identification was partly based on Golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas V4, TEO, or TEp. Aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer 3 but also in layer 5 and/or 6. In each of the 3 cases, about 50-100 GABAergiclike neurons were scored, with a preferential location anterior to the injection sites. In addition to their characteristic dendritic morphology, the neurons were identified as positive for GABAergic neuronal markers; namely, GAD67, somatostatin, or NOS. Thus, we conclude that GABAergic projection neurons are phylogenetically conserved; but more work is needed to determine (1) their other features, (2) possible species variability, (3) their functional significance. Supported by RIKEN BSI.
Withdrawn PS3A-C033 Regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons Allan Gulledge 1,2 , Susanna B. Park 2 , Greg J. Stuart 2 , Yasuo Kawaguchi 1 1 National Institute for Physiological Sciences, Japan; 2 Div. Neurosci., JCSMR, Australian National University, Canberra, Australia
We examined cholinergic modulation of pyramidal and nonpyramidal neurons in 3 neocortical areas (prefrontal, somatosensory, and visual cortex). Transient ACh exposure (100 M) inhibited layer 5 pyramidal neurons in all areas via activation of an SK-type potassium conductance. Pyramidal neurons in layers 2/3 were generally less responsive to ACh, but ACh inhibited layer 3 cells in visual cortex. Prefrontal layer 5 pyramidal neurons were more responsive to ACh than were layer 5 cells in other areas of cortex. Fast spiking (FS) nonpyramidal neurons were completely non-responsive to ACh, even at very high concentrations (5 mM). On the contrary, ACh generated fast, nicotinic receptor-mediated responses in 37% of non-FS interneurons (24 of 65 cells). Laminar or regional differences in ACh responses were not observed in nonpyramidal neurons. These data suggest that ACh may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons.
Toshikazu Kakizaki 1,2 , Kenzi Saito 1,3 , Yuchio Yanagawa 1,2 1 Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi, Japan; 2 SORST, JST, Kawaguchi, Japan; 3 SOKENDAI, Hayama, Japan A major inhibitory neurotransmitter GABA is synthesized by glutamate decarboxylase (GAD), and is accumulated into synaptic vesicles by vesicular GABA transporter (VGAT). Another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by VGAT, and be co-released with GABA. Several molecules related to GABAergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that GABAergic and glycinergic systems exert their activities outside the CNS. VGAT-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that GABA and/or glycine are involved in body wall formation. To further investigate whether GABA is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the GAD67-deficient mouse fetus.
PS3A-C035 GABA mediated glutamate release from developing cerebellar cortex and Ca sensitivity Sachiko Yoshida, Miyuki Ohshita, Masakazu Uematsu, Shoichiro Hirano, Shinya Tanaka, Naohiro Hozumi Toyohashi University of Technology, Toyohashi, Japan GABA (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. During cerebellar development, GABA-mediated responses, GABA excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. Many observations of GABA excitations using cultured cells have been reported, whereas few using acute slices. We recently reported the spatial nature of glutamate and GABA releases from acute slice with an enzyme-linked assay system and CCD imaging technology. In the present study, we evolved this measurement system to allow observations of spontaneous or GABA-mediated glutamate release from developing postnatal acute cerebellar slices. Glutamate was released spontaneously, but GABA-mediated glutamate release appeared from postnatal 4 to 6 day in EGL. Its release, especially from premigratory zone, was inhibited by Ni 2+ , but Cd 2+ couldn't. We suggest that GABA excitation induces granule cell migration.
PS3A-C036 GABAergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection Hiroyuki Hayashi 1 , Hiroaki Wake 2 , Junichi Nabekura 2 , Osamu Takahashi 1 1 Department of histology, Kanagawa Dental College, Yokosuka, Japan; 2 National Institute of Physiological Science, Okazaki, Japan It has been reported that GABAergic nerve terminals are seen in the trigeminal motor nucleus (Vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the Vm cell bodies. We recently found that the number of these GABAergic fibers projecting to Vm is markedly reduced in postnatal development. In this study, to elucidate the possibility that the re-arrangement of GABAergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the GABAergic circuits in the Vm. Two to eight weeks after unilateral surgical transection of the masseter nerve of rats, GABAlike immunoreactive (GABA-IR) varicosities were examined using immunofruorescence technique. The significant increase in number of GABA-IR varicosities were seen after eight weeks of the operation. This result suggest that GABAergic inputs may play one of important role for reorganization of afferent inputs in the Vm.
Akiko Arata 1 , Kunihiko Obata 2 , Jonathan Davies 3 , Mark Bellingham 3 , Peter G. Noakes 3 1 Lab. for Memory & Learning, RIKEN-BSI, Wako, Japan; 2 Obata Res. Unit, RIKEN-BSI, Wako, Japan; 3 Sch. Biomed. Sci., Univ. Queensland, Queensland, 4072, Australia During embryonic development, approximately half of the motoneurons (MNs) undergo programmed cell death. This process depends also on glycinergic and/or GABAergic synaptic activity, as suggested by increased MN number in gephyrin-deficient mice (Banks et al., 2005) . We investigated the involvement of GABA alone in the MN death using GAD67-deficient mice, in which cerebral GABA is reduced to less than 10% of the wild-type. MN numbers at embryonic day (E) 18 were counted by the method of Banks et al. Brainstemupper spinal cord blocks were prepared from E18 embryos and subjected to electrical recording from the C4 and C8 ventral roots and also GABA measurement. In GAD67-deficient embryos, increase in number of brachial MNs (139%) and decrease in both spontaneous discharges in the C4, C8 roots and GABA content (less than 20%) were observed, compared with those of the wild-type littermates. GABA might control cell death in developing network.
Abolghasem Esmaeili, Joe Lynch, Pankaj Sah Queensland Brain Institute, The University of Queensland, Australia
The amygdala has key role in processing emotional information. Distribution of GABA A receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. We examined the pharmacology of GABA A receptors by expressing different subunit combinations in HEK293 cells and comparing the pharmacology with specific GABAergic inputs in the amygdala. DMCM blocked the actions of GABA at expressed ␣11␥2 and ␣21␥2 combinations (75% reduction) but had no effect at ␣11␥1 or ␣21␥1. In slice recordings DMCM blocked IPSCs by 70% in the lateral amygdala and had variable effects in the central amygdala. Diazepam and zolpidem enhanced IPSCs in the lateral whereas the response in the central amygdala was either reduction or enhancement. Real time PCR and western blotting revealed differences in the distribution of GABA A receptor subunits between the lateral and central amygdala. We conclude that in the lateral amygdala all inputs have ␥2 subunits whereas in the central amygdala some inputs contain ␥1 while others contain ␥2 subunits.
Masayuki Kobayashi Department of Pharmacology, Nihon University School of Dentistry, Tokyo, Japan Noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. The present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. The suppressive effect by activation of ␣ 1 -adrenoceptors was mediated by protein kinase C, and excitatory effect by activation of -adrenoceptors was mediated by cAMP/protein kinase A cascade. Phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mEPSC amplitude and increased mIPSC frequency. Isoproterenol increased mEPSC frequency and decreased mIPSC amplitude. GABA-induced postsynaptic currents were suppressed by isoproterenol. These results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of GABA from presynaptic terminals. On the other hand, isoproterenol may facilitate glutamate release and suppress GABA A receptor-mediated postsynaptic currents.
PS3A-D040 Hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons Mamiko Tsugane 1 , Takashi Iwai 2 , Yasuo Nagai 1 , Junichiro Oka 2 , Hideo Kimura 1 1 Dept. Mol. Genetics, Nat'l. Inst. Neurosci., NCNP, Tokyo, Japan; 2 Lab. Pharmacol., Fac. Pharm. Sci., Tokyo Univ. Sci., Chiba, Japan Hydrogen sulfide (H 2 S), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. The aim of this study is to understand the mechanism of regulation on synaptic transmission by H 2 S. We examined the effect of H 2 S on spontaneous excitatory postsynaptic currents (sEPSC) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. Sodium sulfide (Na 2 S), a donor of H 2 S, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. The frequency and the amplitude of sEPSC were initially reduced by Na 2 S then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by Na 2 S. These observations suggest that H 2 S may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. Several studies show that activation of Locus coeruleus (LC) play an important role in the symptoms of opiate withdrawal. In this study the effects of LC inactivation on self-administration of Morphine and on morphine withdrawal syndrome in rats has been investigated. Male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. After 5 days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. LC was inactivated by (1 l) lidocaein (2%) 5 min before training. Animals were allowed to self administer morphine (1 mg/kg per inf.) ten consecutive daily 2-h session. During all morphine self administration session lever pressing was measured. Our results show that: (1) LC inactivation produced a significant decrease in the initiation of morphine self administration during all session. After the last test session morphine withdrawal symptom signs (MWS) precipitated by naloxone were measured. (2) Most of MWS were decreased by LC inactivation in comparison with morphine group. These results suggest that extracellular ATP plays a dual role in astrocytic Ca 2+ wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats.
The electrophysiological analysis of the rescue effect of 17 estradiol from glucocorticoid activity Yuki Oishi 1 , Suguru Kawato 2 1 Department of Physics, Graduate School of Science, University of Tokyo, Tokyo, Japan; 2 Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan It is well known that stress reduces several activity of brain. Especially, hippocampus is the largest target of stress. These phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. On the other hand, 17 estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as -amyloid, glutamate, glucocorticoids. In this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (LTP), which is the index of learning and memory. The results was that corticosterone (glucocorticoid of rat) acutely reduced LTP via glucocorticoid receptor. 17 estradiol rescued this reduction via estrogen receptor ␣ and . So we found that 17 estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely.
PS3A-D044 The hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment Jinko Konno, Ayuka Ina, Sachine Yoshida, Hideki Ohmomo, Fumihiro Shutoh, Setsuji Hisano Lab. Neuroendocrinol., Graduate Sch. Comprehensive Human Sci., Univ. Tsukuba, Ibaraki, Japan Effects of dexamethasone (DEX) on hypothalamic neuropeptide Y (NPY) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. Adult male Wistar rats received an injection of DEX (0.2 mg/100 g B.W., sc) or sesame oil (vehicle control) everyday for 9-10 days. The two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. NPY-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the DEX-treated rat than in controls. NPY hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. No difference was found between both control animals. These results indicate stimulatory effects of DEX on hypothalamic NPY production and suggest enhanced NPY influences on pituitary function.
Akiko Shingo, Idumi Yamashita, Shozo Kito Lab. of Neuroscience, Hyogo University, Hyogo, Japan
We examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces IGF-1 mRNA expression, upregulates estrogen receptors and facilitates ERE binding in these brain areas. Materials are OVXed and non-OVXed rats. Each group of rats were divided into the following groups. A: rats fed with phytoestrogen-free control diet, B: rats fed with diet with soy bean-derived estrogen and C: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of -estradiol. After feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. Expressions of mRNAs of IGF-1, estrogen receptors ␣ and , and ERE binding were analysed. As the results, it was revealed that isoflavones induced increased expression of mRNAs of IGF-1 and estrogen receptors in both OVXed and non-OVXed rats. Difference between estrogen receptor ␣ and  in responses to isoflavones were analysed. Isoflavones feeding increased ERE binding as much as chronic injections of estrogen did in the OVXed rats.
Research funds: Kampo Science Foundation, Japan PS3A-D046 Mechanism of central metabolic control by TGF-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline Teppei Fujikawa, Kazuo Inoue, Tohru Fushiki Division of Food Science and Biotechnology, Graduate School of Agriculture, University of Kyoto, Kyoto, Japan
We have previously reported that activated transforming growth factor-beta (TGF-beta) increase in the rat brain during exercise. Intracranial administration of TGF-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. These results suggest that activated TGF-beta in the rat brain participates in metabolic control of peripheral tissue by CNS. It is, however, not known how TGF-beta increases in specifically fat oxidation. Many investigations suggest that hypothalamus is essential for central metabolic control. In addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. In this study we measured concentration of extracellular noradrenaline (NA) in the hypothalamus by using microdialysis after injection of TGF-beta. Then, we measured respiratory exchange ratio and serum samples, after administration of TGF-beta in the rat with depletion of hypothalamic NA by injection of 6-hydroxydopamine.
PS3A-D047 The effect of brain-derived neurotrophic factor (BDNF) on neuropeptide Y (NPY) neurons in the mouse corpus callosum: An examination using organotypic brain slice culture Ryoichi Yoshimura, Kazuto Ito, Yasuhisa Endo Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan
The morphology of neuropeptide Y (NPY) neurons existing in the corpus callosum (CC) and the effects of brain-derived neurotrophic factor (BDNF) on the NPY neurons were examined by using organotypic slice culture system. BDNF treatment significantly increased the number of the NPY-immunopositive cell bodies and fibers in CC assessed with immunocytochemistry. Electron microscopy demonstrated that the NPY immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in CC nerve fibers. The sectional area of NPY-positive fibers was larger in the BDNF-treated culture than in the control culture. The number of nerve fibers adjacent to the NPY-positive fibers was also larger in the BDNF-treated culture than the control. These results suggest that NPY may play a key role in the neuronal regeneration, and BDNF takes part in the development of NPY neuron fibers as well as the increase of the number of NPY neurons in CC.
Reiji Semba 1 , Kimi Watanabe 1 , Munekazu Komada 2 1 Institute for Developmental Research, Aichi Human Service Center, Aichi, Japan; 2 Graduate School of Medicine, Kyoto University, Kyoto, Japan D-serine is hypothesized to be a glia-derived neurotransmitter activating the NMDA receptor because D-serine was reported to be formed and localized exclusively in astrocytes. However, we reported strong immunoreactivity of D-serine in some axons. To reveal which cells are producing D-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing D-serine from L-serine, was performed. Using antibodies against NeuN, a neuronal marker, GFAP, an astrocyte marker, and CNPase, an oligodendrocyte marker, type of the cells containing the mRNA was examined. Coincidentally with our immunohistochemical study of D-serine, strong signals for serine racemase mRNA were found in some neurons while weak signals were found in astrocytes. Present results suggest that D-serine will be a neurotransmitter activating the NMDA receptors produced in a specific type of neurons.
Takatoshi Hikida 1,2 , Asif K Mustafa 2 , Kenji Hashimoto 3 , Kumiko Fujii 2,4 , Kazuhisa Maeda 2,5 , Hiroshi Ujike 6 , Richard L. Huganir 2 , Solomon H. Snyder 2 , Akira Sawa 2 1 Dept. of Systems Biology, OBI, Suita, Japan; 2 Depts of Neurosci. & Psychiat, Johns Hopkins Univ. Med., Baltimore, Maryland, USA; 3 Chiba Univ. Forensic Mental Health, Chiba, Japan; 4 Dept. of Psychiat, Shiga Univ. Med. Sci., Shiga, Japan; 5 Div. of Neuropsychiat, Tottori Univ., Yonago, Japan; 6 Dept. of Neuropsychiat, Okayama Univ., Okayama, Japan Accumulating evidence from both genetic and clinical studies suggests a critical role of D-serine in schizophrenia (SZ). We identified and characterized PICK1 as a protein interactor of the D-serine synthesizing enzyme, serine racemase (SR). D-serine levels in the hippocampus and frontal cortex of PICK1 knockout mice were significantly lower than those of their wildtype littermates at age of p7, but not in adults, suggesting regulation of PICK1 on SR at developing stage. In case-control association study, we observed an association of the PICK1 gene with SZ, which is more prominent in disorganized SZ. Our findings suggest that PICK1 contributes to SR activity, D-serine production, and NMDA neurotransmission in the pathophysiology of SZ.
PS3A-D050 Epileptiform activity is inhibited by taurine which can activate glycine and GABA A receptors in immature rat hippocampus Akihito Okabe 1,2 , Werner Kilb 2 , Ileana L. Hanganu 2 , Taizhe Qian 2 , Daiichiro Nakahara 3 , Atsuo Fukuda 1 , Heiko J. Luhmann 2 1 Dept. of Physiol., Hamamatsu, Japan; 2 Inst. of Physiol., Mainz, Germany; 3 Dept. of Psychol., Hamamatsu, Japan
Many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. The depolarizing GABAergic responses in immature neurons may contribute to higher epilepsy susceptibility. To investigate whether taurine, a neurotransmitter found in high concentrations in the immature CNS, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal CA3 region of an intact preparation. 5 mM taurine blocked epileptiform activity induced by Mg 2+ free ACSF and 20 M 4-AP. This taurine effect was prevented by the glycinergic antagonist strychnine and the GABA A antagonist gabazine. Inhibition of taurine uptake by GES also suppressed epileptiform activity in strychnine and gabazine sensitive manner. These results suggest that taurine mediates an inhibition in immature hippocampus via glycine and GABA A receptors that suppresses epileptiform activity.
PS3A-D051 Responses of PGE 2 in undifferentiated and differentiated NG108-15 cells Kayoko Matsushima 1 , Takashi Imanishi 1 , Akinori Kawaguchi 1 , Tetsuyuki Wada 1 , Shigeru Yoshida 2 , Seiji Ichida 1 1 School of Pharm. Sci., Kinki Univ., Osaka, Japan; 2 School of Pharm. Sci., Kinki Univ., Osaka, Japan; 3 School of Pharm. Sci., Kinki Univ., Osaka, Japan; 4 School of Pharm. Sci., Kinki Univ., Osaka, Japan; 5 School of Sci. & Eng., Kinki Univ., Osaka, Japan; 6 School of Pharm. Sci., Kinki Univ., Osaka, Japan
Our previous findings showed that 5-HT-and BK-induced [Ca 2+ ] i increases were enlarged in differentiated NG108-15 cells. For the next stage, we investigated the effect of PGE 2 , an inflammatory mediator for 5-HT and BK, on the cells. NG108-15 cells were loaded with fura-2/AM, and the change in [Ca 2+ ] i was monitored by an image processor. The results showed: (1) PGE 2 -induced response was decreased when NG108-15 cells were differentiated by Bt 2 cAMP, (2) 10 −5 M AH6809 and SC19220 irreversibly inhibited PGE 2 -induced response by about 50% and 26%, respectively, while 10 −5 M AH23848 and sulprostone had no effect, and (3) PGE 2 -induced response was abolished under Ca 2+free conditions in about 70% of both NG108-15 cells. These results indicate that the response to PGE 2 , via EP1 and EP2 receptors, significantly decreased during differentiation.
Mitsumasa Murano, Fumihito Saitow, Hidenori Suzuki Department of Pharmacology, Nippon Medical School, Tokyo, Japan
The most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (DCN) and by the electrical membrane properties of DCN neurons themselves. This study aimed at examining mechanisms underlying the serotonergic modulations of both the GABAergic transmission at the Purkinje-to-nuclear cell synapses and the membrane properties of DCN neurons using cerebellar slices prepared from 11-to 21-day-old rats. Bath application of serotonin (5-HT) decreased the amplitude of stimulation-evoked IPSCs in DCN neurons in a dose-dependent manner. Furthermore, slow inward currents ware observed in DCN neurons during 5-HT application. Under the current-clamp recording, 5-HT markedly depolarized and increased action potential discharges of DCN neurons. Taken together, these results suggest that 5-HT facilitates the voluntary activity in DCN neurons by both pre-and post-synaptic mechanisms.
PS3A-D053 Searching for endogenous ligands of trace amine receptors in mammals (1) Akira Komatsu 1 , Airi Yamaguchi 2 , Noriko Makikusa 2 , Osamu Koizumi 2 1 Dept. Physiol., Tokyo Women's Med. Univ., Sch. Med., Tokyo, Japan; 2 Neurosci. Lab., Fukuoka Women's Univ. Fukuoka, Japan
Trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. To search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (IHC). Monoamines, phenylethylamine (PEA), tyramine (TA) and histamine (HA), were conjugated to a hemocyanine, KLH, using an imidoester cross-linker, dimethyl suberimidate (DMS). Rabbits were immunized by the conjugated macromolecule. The obtained antibodies were assayed by ELISA and competitive ELISA technique to check their antibody titer and specificity respectively. The antibodies recognized specifically the monoamine-DMS part within the complex. For IHC, the rat brain was perfused by 2% DMS, post-fixed by 4% formaldehyde and then frozen-sectioned. The antibody against HA revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. The antibodies against PEA and TA failed to reveal immunoreactive neurons in the rat brain.
PS3A-D054 Effects of Mg 2+ on neural activity of cultured cortical neurons of the rat and mouse Yuriko Furukawa 1,2 , Nahoko Kasai 1 , Akiyoshi Shimada 1 , Keiichi Torimitsu 1,2 , Kunihiko Obata 3 , Yuchio Yanagawa 2,4 , Tadaharu Tsumoto 2,3 1 NTT Basic Research Laboratories, Kanagawa, Japan; 2 SORST/JST, Saitama, Japan; 3 Neuronal Circuit Mechanisms Research Group, Brain Science Institute, RIKEN, Saitama, Japan; 4 Dept. of Genetic and Behavioral Neurosci., Grad. Sch. of Med., Gunma University, Gunma, Japan It is well known that Mg 2+ plays an important role not only in energy metabolism, but also in neural information processing. However, the mechanism of such a role in CNS is not well understood. Previously we reported that neural activity and the intracellular Ca 2+ concentration are largely affected by Mg 2+ removal in cultured cortical neurons of the rat. Transient glutamate release was also detected. In the present study, we investigated effects of the Mg 2+ removal on neural activity in cultured cortical and hippocampal neurons. In particular, we measured the intra-and extracellular Mg 2+ concentration and their actions on neural activity using a Mg 2+ indicator, KMG-20-AM together with Fluo4-AM. We observed different effects of the Mg 2+ removal on GABAergic and non-GABAergic neurons by using GAD67-GFP knock-in mice.
Research funds: JST/SORST PS3A-E055 Transient zinc-positive terminations in the developing rat somatosensory cortical system Noritaka Ichinohe, Daniel Potapov, Kathleen S. Rockland Lab. for Cortical Organization and Systematics, BSI, RIKEN, USA Synaptic zinc (Zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. During development, transiently high levels of synaptic Zn occur in both sensory and nonsensory cortical areas. By injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of Zn + thalamocortical neurons from P7-P13. Zn + cortical neurons were also labeled, intrinsic and extrinsic, from P5. Unlike in the adult, these were in layer 5, instead of layers 2, 3, and 6. At P9, neurons occurred in layers 2, 3, and 5 and, in some areas, layer 4. At P15, Zn + neurons first appeared in layer 6; and at P22, there is the adult lamination. As whisking and exploratory behavior commences in the second postnatal week, these transient Zn + terminations may play a role in experience-dependent adjustments in cortical circuitry.
Research funds: BSI, RIKEN and KAKENHI No. 17024064 PS3A-E056 Systematic comparison of the structure of the serotonin immunoreactive neurons between insect species Masaaki Iwano 1,3 , Ryohei Kanzaki 2 , Kei Ito 1,3 1 Center for Bioinform., IMCB, Univ. of Tokyo, Tokyo, Japan; 2 Dept. of Mechano-Inform., Grad. Sch. of Inform, Sci. and Tech., Univ. of Tokyo, Tokyo, Japan; 3 BIRD, JST, Saitama, Japan
In the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (SIRNs) is known to be preserved remarkably during evolution. Systematic comparison of the invertebrate SIRNs has not been performed, on the other hand. In the current study we analyzed the morphology of the SIRNs in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. In spite of the large variation in the size and cell numbers of the brain, the number and distribution of the SIRNs were highly consistent between species. For example, we observed either one or two pairs of bilateral SIRNs with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. Variation was greater in the antennal lobe, the insect primary olfactory center, where SIRNs project either ipsil-or contra-laterally depending on the species.
Maki Kagohashi 1,2 , Taizo Nakazato 2 , Shigeru Kitazawa 2 1 Neurol, Juntendo Univ., Tokyo, Japan; 2 Physiol, Juntendo Univ., Tokyo, Japan
In vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. Nakazato, 2005) . However, task freedom was restricted by cables connecting the head and the measurement system. To overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (Kagohashi et al., JNS2005). The system consisted of a wireless transmitter with a potentiostat and a signal receiver. In the present study, we reduced the size and weight and measured dopamine (DA) currents in vivo with the wireless system mounted on the back of the rat. A single-step voltage pulse (100 to 250 mV for DA; 300 to 450 mV for 5HT) was applied at 4 Hz through a carbon electrode that was chronically implanted in the striatum. After administration of L-DOPA, DA currents showed a gradual increase in good agreement with the data measured with conventional systems. The present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction).
Research funds: Scientific Research on Priority Areas (Mobiligence)
Hiroyuki Yamazaki, Tomoaki Shirao Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Japan Dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. In the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. Yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. In neurons, this protein was localized primarily in nucleus and dendritic spines. Hence, we named it Spikar for its unique intracellular localization in spine and karyoplasm. We studied the role of Spikar in spine formation. Hippocampal neurons were transfected with shRNA expression vector for Spikar at several developmental stages. In early stage, Spikar knock down (KD) did not affect the density of dendritic protrusions that were mostly filopodia. In contrast, Spikar KD reduced spine density at the stage of synapse formation. These results suggest that Spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation.
PS3A-E059 Time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation Toshiyuki Mizui 1,4 , Yuko Sekino 2,3 , Tomoaki Sirao 1 1 Dept. of Neurobiol. & Behav., Gunma Univ. Grad. Sch. of Med., Maebashi, Japan; 2 Div. of Neural Network, Inst. Med. Sci. Univ. of Tokyo, Tokyo; 3 CREST, JST, Kawaguchi, Japan; 4 JSPS, Japan
We have shown that NMDA receptor activation induced translocation of drebrin, with retaining its binding to F-actin, from dendritic spines to their parent dendrites. In the present study, we analyzed the time course of GFP-tagged drebrin A (GFP-DA) dynamics after glutamate receptor activation. We prepared primary hippocampal cultured neurons, transfected them with GFP-drebrin A expression vector using microinjection methods at 14 days in vitro (DIV), and analyzed the dynamic localization of GFP-DA at 16 DIV. Glutamate stimulation started GFP-DA translocating within 10 s and completed in 2 min. After washout of glutamate, GFP-DA gradually re-accumulated in the spine, and the fluorescence intensity of GFP-DA is fully recovered in 10 min. These data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines.
Research funds: Grant-in-Aid for JSPS Fellows PS3A-E060 Distribution of the SRF co-activator MAL in developing mouse brain Mitsuru Ishikawa 1 , Jun Shiota 1 , Hiroyuki Tsutsumishita 1 , Hiroyuki Sakagami 2 , Masaaki Tsuda 1 , Akiko Tabuchi 1 1 Dept. Biol. Chem., Fac. Pharm. Sci., Univ. Toyama, Toyama, Japan; 2 Dept. Cell Biol., Tohoku Univ., Grad. Sch. Medicine, Sendai, Japan
The SRF co-activator MAL (megakaryocytic acute leukemia) plays an important role in controlling SRF-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. Recent studies with conditional deletion of srf gene demonstrated that SRF was required for inducing genes such as egr-1, c-fos,-actin but also for neuronal migration and plasticity. In this study, we investigated the expression of MAL in developing mouse brain and the role of MAL for dendritic morphology. The in situ hybridization analysis revealed that MAL mRNA was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. Staining of MAL displayed cytoplasmic localization at cell bodies and apical dendrites. Furthermore, dominant negative MAL mutants and RNAi led to a reduction of dendritic number, as well as a decrease of SRF transcription. These findings indicate that MAL is involved in the formation or the stability of dendrites.
Research funds: KAKENHI (17790055) to A.T.
Shoko Shimizu 1 , Shinsuke Matsuzaki 1 , Tsuyoshi Hattori 1 , Ko Miyoshi 2 , Masaya Tohyama 1 1 Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Japan; 2 Department of Brain Science, Graduate School of Medicine and Dentistry, Okayama University, Japan
Disrupted-in-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11) (q42.1;q14.3) translocation segregating with schizophrenia and affective disorders in a Scottish family. kendrin was identified as a protein which interacts with DISC1 at centrosome and residues 446-533 of DISC1 (kendrin-binding region: KBR) were essential for the interaction with kendrin. In this study, we show that C-terminal of DISC1 downstream of KBR is indispensable structure for KBR to interact with kendrin and also essential for DISC1 to target to the centrosome. Furthermore, we have shown that inhibition of the DISC1-kendrin interaction perturbs the tubulin network formation. These results suggest that the C-terminal region of the DISC1 is important to the DISC1-kendrin interaction and that a truncated form of DISC1 lacking the C-terminal downstream of the translocation breakpoint might affect the microtubule organization.
Tatsuro Kumada, Yasuhiko Nakanishi, Atsushi Fukuda Department of Physiology, Hamamatsu University School of Medicine Migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. The morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as Ca 2+ or Cl − . Although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. To clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and Ca 2+ and Cl − imaging. We found that the relatively low density A172 glioma cells actively moved on the substrate. The movement has the correlation with intracellular Ca 2+ oscillation in the cells. The relationship between cell movement and intracellular ion levels is further studied.
PS3A-E063 Involvement of Ca 2+ influx in the unpolarized non-vesicular release of FGF-1
Hayato Matsunaga, Hiroshi Ueda Division of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Little is known of molecular basis mechanisms for the ER-Golgiindependent or non-vesicular release of FGF-1 lacking a conventional signal peptide sequence. We found that FGF-1 is co-released with S100A13, a Ca 2+ binding protein from cultured rat astrocytes upon the serum-deprivation stress. Here, we report that FGF-1 is co-released with S100A13 from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. The interaction between FGF-1 and S100A13 required Ca 2+ . The overexpression of S100A13 88-98 mutant lacking an ability of interaction with FGF-1 inhibited the their release, suggesting that S100A13 is a cargo molecule. The release of FGF-1 upon either stimulation was abolished by voltage-dependent N-type Ca 2+ channel blocker. These findings suggest that Ca 2+ influx may be involved in the unpolarized non-vesicular release of FGF-1. In neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. PC12 cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that 5-HT has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([Ca 2+ ] i ) in PC12 cells. However, it is unclear how [Ca 2+ ] i regulates neurite outgrowth via actin cytoskeleton. In this study, we investigated effects of [Ca 2+ ] i on actin dynamics in PC12 cells transfected with YFP-actin. Filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, A23187. Treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with CaMK inhibitor did not. These effects could contribute to 5-HT induced enhancement of neurite elongation. The actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. On stimulation of PC12 cells by NGF, proteins that directly interact with F-actin such as actinin rapidly translocate to the F-actin-rich cytoskeleton. CLP36 is a PDZ-LIM protein which was originally identified as an actinin-interacting protein in skeletal muscles. Here, we show that CLP36 is endogenously expressed in PC12 cells and plays an important role in actin dynamics during NGF-induced neurite outgrowth. Immunofluorescent studies showed that CLP36 is accumulated in irregular cell surface and membrane extrusion soon after NGF-stimulation, where colocalized with actin filaments. We next performed RNAi experiments to explore the role of CLP36 in actin dynamics in growth cones and found that knockdown of CLP36 expression lead to the suppression of NGF-mediated neurite outgrowth. In addition, we revealed using CLP36 deletion mutants that both of PDZ and LIM domains are necessary for the proper function of CLP36.
PS3A-E066 Screening of genes expressed preferentially in migrating GABAergic neurons of developing cerebral cortex Toshiya Kimura 1 , Tsuyoshi Kobayashi 1 , Yuchio Yanagawa 2 , Kunihiko Obata 3 , Fujio Murakami 1,4 1 Grad. Sch. of Frontier Biosci., Osaka Univ., Osaka, Japan; 2 Grad. Sch. of Medicine, Gunma Univ., Maebashi, Japan; 3 BSI, RIKEN, Wako, Japan; 4 SORST, JST, Japan Neuronal migration plays a critical role in constructing brain architecture organization. However, molecular mechanisms underlying this process still remain elusive. In an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. Subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. Among the 2208 clones tested, two genes, neuronatin and seizure related gene 6 (SEZ-6) attracted our attention. They were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. Postnatally, mRNA signals were hardly detectable. These results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons.
Yan Zhu 1,2 , Tomoko Matsumoto 1,2 , Sakae Mikami 3 , Takashi Nagasawa 3 , Fujio Murakami 1,2 1 Grad. Sch. of Frontier Biosci., Osaka Univ., Japan; 2 SORST, JST, Japan; 3 Inst for Frontier Med. Sci., Kyoto Univ., Japan Long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. The migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. We address these issues using the hindbrain precerebellar system as model system. Precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. We therefore studied the role of pia-secreted chemokine SDF-1 and its receptor CXCR4 in the precerebellar migration. We show that CXCR4 is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. In CXCR4 and SDF-1 knock out mice, migrating precerebellar neurons are less confined to the pial surface. More strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster.
PS3A-E068 Involvement of an immunoglobulin superfamily molecule, NEPH2/mKirre in the migration of precerebellar neurons Kazuhiko Nishida 1,2 , Kazuhide Nakayama 1 , Saori Yoshimura 1,2 , Fujio Murakami 1,2 1 Grad. Sch. of Frontier Biosci., Osaka Univ., Osaka, Japan; 2 SORST, JST, Saitama, Japan Neural cell migration plays a crucial role in central nervous system development. In this study, we analyze the involvement of NEPH family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (PCNs). Postmitotic PCNs derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (Kawauchi, D., Taniguchi, H., Watanabe, H., Saito, T., and Murakami, F., Development, in press ). In situ hybridization analysis showed that among NEPH family members including NEPH1, NEPH2/mKirre, and NEPH3, only NEPH2/mKirre was strongly expressed in PCNs. Expression of NEPH2/mKirre was detected from E13.5 when PCNs migrate tangentially. The expression level became weaker at P1, when PCNs stop the radial migration, raising the possibility that NEPH2/mKirre might be involved in the migration of PCNs. We are currently analyzing the function of NEPH2/mKirre in the migration of PCNs.
Hiroki Umeshima 1,2 , Toshio Ohshima 3 , Tomoo Hirano 2 , Mineko Kengaku 1 1 Lab. for Neural Cell Polarity, RIKEN BSI, Wako, Japan; 2 Department of Biophysics, Kyoto University, Kyoto, Japan; 3 Lab. for Developmental Neurobiology, RIKEN, BSI, Wako, Japan During lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. We analyzed the molecular mechanisms regulating migration of granule cells. Using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of CDK5 (CDK5-DN) disrupted the morphology of granule cells during radial migration. Recently, centrosome positioning is thought to be one of the important factors for neuronal migration. Double-labeling of the centrosome and the whole-cell images by transfecting Centrin2-GFP and RFP enabled us to record dynamic movement of the centrosome during radial migration. We found that the motion kinetics of the centrosome was disrupted by CDK5-DN. Based on these results, we will discuss the role of centrosome during neuronal migration.
Keisuke Ito 1,2 , Takahiko Kawasaki 1,2 , Tatsumi Hirata 1,2 1 Division of Brain Function, National Institute of Genetics, Mishima, Shizuoka, Japan; 2 Department of Genetics, School of Bioscience, SOKENDAI Newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. We have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. Among various drugs, we found that a protein kinase inhibitor, K252a has the most interesting effect on the lot cell migration. During the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but K252a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. We also found that K252a has a similar effect on cerebellar granule cells. These phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. We are now analyzing possible targets of K252a, aiming for dissection of these phenomena.
The conserved Ser/Thr kinase UNC51 functions with UNC-76 to regulate axonal transport in Drosophila Hiroaki Mochizuki 1 , Hirofumi Toda 1,3 , Emiko Suzuki 2 , Joseph Gindhart 4 , Toshifumi Tomoda 3 , Katsuo Furukubo-Tokunaga 1 1 Grad. School Life and Envir. Sci., Univ. Tsukuba, Tsukuba, Japan; 2 Gene Net. Lab., Natl. Inst. Genet. Mishima; 3 Beckman Res. Inst., City of Hope, CA, USA; 4 Dep. Biol., Univ. Richmond, VA, USA Neural network develops through regulated guidance of axons and interconnection among them. Despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. We have identified the Drosophila homolog of unc51, which encodes a Ser/Thr kinase and is required for axonal formation in C. elegans and mouse. We found that UNC51 is essential for neural development in Drosophila. Loss of function of Drosophila unc51 results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. We also found that unc51 genetically interacts with Unc-76, an evolutionarily conserved cytoplasmic protein that binds to Kinesin Heavy Chain. In unc51 mutants, UNC-76 was separated from Synaptotagmin vesicles. These results suggest that UNC51 coordinates kinesin-cargo interaction via UNC-76 to regulate dynamic axonal transport.
PS3A-E072 Change in microtubule polarity during the conversion of dendrites into axons Kensuke Hayashi 1 , Daisuke Takahashi 2 1 Life Science Inst. Sophia University, Tokyo, Japan; 2 Waseda University, Tokyo, Japan Axons and dendrites of neurons differ in the polarity of their microtubules. The mechanism for the difference, however, is not well understood. We found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. In this study, we examined whether microtubule polarity changes during the conversion. In dendrites of neurons before culture, microtubule polarity was nonuniform. After 24 h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. This indicates that microtubules with their minus-ends distal disappeared during the culture. Microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. However, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. Our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes.
Research funds: KAKENHI (16500193) and KAKENHI on Priority Areas (16027207) PS3A-E073 Generation and analysis of region-specific Rac1-deficient mice Hidetoshi Kassai 1 , Masahiro Fukaya 2 , Eriko Miura 2 , Mizuho Sakahara 1 , Masahiko Watanabe 1,2 , Atsu Aiba 1 1 Div. Cell Biol., Kobe Univ. Grad. Sch. Med., Kobe, Japan; 2 Div. Physiol. Sci., Hokkaido Univ. Grad. Sch. Med., Japan Rac1 is a member of the Rho family of small GTPases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. Nevertheless, physiological role of Rac1 in the CNS is poorly understood because of the embryonic lethality of Rac1 knockout mice. In this study, we generated and analyzed region-specific Rac1-deficient mice (Emx1-Rac1 KO mice) by the Cre-loxP system, in which a promoter for Emx1 homeobox gene induces expression of Cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. Emx1-Rac1 KO mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. Furthermore, Emx1-Rac1 KO mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. These results suggest that Rac1 regulates neuronal cell migration and axonal growth in cerebral cortex. Previously we reported overexpression of MAP1B containing Nterminal 126 amino acids promoted neuronal death. To reveal the mechanism of MAP1B N-terminal induced neuronal death, we searched for the proteins that interact with N-terminal of MAP1B by two-hybrid system. Alpha-tubulin was found to interact with MAP1B N-terminal and their in vitro interaction was proved with pull-down assay. The interaction of tubulin and MAP1B N-terminal has not yet been reported. Beta-tubulin was also found to interact with MAP1B N-terminal. When  tubulin was divided in 2 fragment at between amino acid 213 and 214, there was no interaction between  tubulin fragments and MAP1B N-terminal. Interaction needs the continuous region over aa 213 and 214. There were much proportion of round formed COS7 cells in N-terminal containing MAP1B transfected cells than in N-terminal lacking MAP1B transfected cells. There might be some interference in interaction between MAP1B and tubulin in cells express MAP1B containing N-terminal.
Otone Endo 1,2 , Masaaki Mizuno 3 , Yasukazu Kajita 2 , Jun Yoshida 2 1 Department of Neurosurgery, JA Kainan Hospital, Aichi, Japan; 2 Department of Neurosurgery, Nagoya University, Nagoya, Japan; 3 Department of Molecular Neurosurgery, Nagoya University, Nagoya, Japan Primate ES cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human ES cells might show wide similarity to cynomolgus ones. We refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate ES cells. Our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of Parkinsonism. Differentiation of other types of neurons and development of fully functional neural network must be established.
Shigeki Ohta 1 , Masae Yaguchi 1 , Yumi Matsuzaki 2 , Yoshiaki Toyama 3 , Yutaka Kawakami 4 , Hideyuki Okano 2 , Masahiro Toda 1,5 1 Neuroimmunology Research Group, Keio Univ., Tokyo, Japan; 2 Physiology, Keio Univ., Tokyo, Japan; 3 Orthopaedic Surgery, Keio Univ., Tokyo, Japan; 4 Institute for Advanced Medical Research, Keio Univ., Tokyo, Japan; 5 Neurosurgery, Keio Univ., Tokyo, Japan
We have shown that mouse dendritic cells (DCs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (NSPCs) in vitro. Implantation of DCs into injured mouse spinal cord could improve the motor function through activation of endogenous NSPCs in vivo. In this study, to identify an effective DC subtype for the treatment of spinal cord injury (SCI), we analyzed the effects of different mouse DC subtypes on the proliferation of NSPCs in vitro. Among mouse splenic CD11c + DCs, CD8␣ + DCs increased the number of neurospheres most effectively in vitro. Furthermore, a significant functional recovery after mouse SCI was induced by implantation of CD8␣ + DCs compared to CD11c + DCs. These results suggest that CD8␣ + DCs can be an effective subtype of mouse DCs for the treatment of SCI.
PS3A-E077 von Hippel-Lindau protein regulate the neurogenesis in skin-derived precursor cells Atsuhiko Kubo 1 , Hiroshi Kanno 1 , Takaakira Yokoyama 1 , Shuichi Nakano 2 , Naoki Sugimoto 2 , Nahoko Kobayashi 3 , Tetsuhiko Yoshida 3 , Isao Yamamoto 1 1 Dept. of Neurosurgery, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 2 FIBER and Dept. of Chemistry, Konan University, Kobe, Japan; 3 Toagosei Co., Ltd. Corporate research laboratory, Nagoya, Japan Skin-derived precursors (SKPs), multipotent somatic stem cells, are preferred cell source for autologus CNS cell replacement therapy. They are proliferated by the mitogens of EGF and bFGF. To investigate the effects of von Hippel-Lidau (VHL) protein in the neural cell fate commitment, SKPs were inoculated with HSV vector expressing VHL protein. SKPs showed promotion of neurogenesis and inhibition of gliogenesis. To detect the intrinsic factors that control lineage commitment, VHL peptides fused with the protein transduction domain (PTD) were synthesized. The PTD-VHL peptides showed rapid cell internalization in nearly 100%, and peptide with the elongin C binding site (residues 157-171) showed a high ability of inducing neuronal differentiation by interacting with JAK/STAT pathway. These findings are important in its application to the CNS cell grafting.
PS3A-E078 The effect of Pueraria mirifica on ERK1/2 and S-100 following sciatic nerve injury in rats Pornpen Chaiworakul, Supin Chompoopong Department of Anatomy, Mahidol University, Bangkok, Thailand
To investigate the effects of Pueraria mirifica (PM) compared with genistein (G) and estrogen (E2) on the expression of ERK1/2 and S-100 following sciatic nerve crush and transection in rats. Protein levels of pERK1/2 and S-100 in distal segments of nerve at day 7 were determined by Western blot analysis. It was demonstrated that PM and G treatments, similar to E2, caused a significant decrease in the expression of pERK1/2 levels in both nerve crush and transection injuries. However, transected nerves showed high and sustained levels of ERK1/2 phosphorylation. Following treatments, levels of S-100 were significantly decreased both in crushed and transected nerves with respect to control group at p < 0.05. This estrogenic effect was blocked by ICI182,780. Because of their structural similarity to E2, PM may have therapeutic potential in nerve injuries which as previously reported to enhance SFI following sciatic nerve crush in rats after day 7. This study suggested that PM as well as G could enhance nerve regeneration like E2 by interfering with the injury-induced ERK signaling pathway.
Yasuhiro Kato 1 , Takafumi Suzuki 2 , Kunihiko Mabuchi 2 1 Department of Advanced Interdisciplinary Studies, Graduate School of Engineering, The University of Tokyo, Japan; 2 Department of Information Physics and Computing, Graduate School of Information Science and Technology, The University of Tokyo, Japan MEMS technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. There is, however, no suitable probe for long-term neural recording and stimulation. One main reason is the death of brain tissues damaged by the probe insertion and implantation. Thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. The skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. The probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (NGF) to improve the stiffness for the probe insertion, and deliver NGF for an optimal period to promote regrowth of damaged neural tissues around the probe. Damage-induced neuronal endopeptidase (DINE) is a newly identified nerve regeneration-associated molecule. It encodes neuronspecific membrane-spanning metalloprotease and belongs to NEP/ECE family which degrades/processes neuropeptides. Although the precise mechanism of DINE including substrate is still unclear, DINE seems to play a protective role in damaged neurons. The most marked property of DINE is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. To clarify the transcriptional regulation of DINE after nerve injury, we analyzed 5 untranslated region of DINE gene. Previously, we found that LIF treatment and NGF deprivation additively increased DINE mRNA. In this study, promoter analysis showed that DINE promoter activity was cooperatively up-regulated by ATF-3 and Stat3, which were induced after nerve injury and activated at the downstream of LIF treatment and NGF deprivation. This combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration.
Tomohiro Miyashita, Takekazu Kubo, Masashi Fujitani, Katsuhiko Hata, Toshihide Yamashita Department of Neurobiology, Graduate School of Medicine, Chiba University, Chiba, Japan Wnt proteins are known as those concerning with formation of central nervous system. We tested whether they play a role in inhibition of axon regeneration after spinal cord injury. Cerebral granule neurons from P6-10 Wistar rats were cultured. Wnt proteins were added into the culture medium. Twenty-four hours after culture, neurite length of each neuron was measured. Immunohistochemistry was done employing anti-Wnts antibody and anti-Ryk (Wnt receptor) antibody. Anti-Ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. Locomotor behaviour was evaluated up to six weeks after injury. Immunohistochemistry showed that several Wnt proteins and Ryk were upregulated after spinal cord injury. Wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. And this effect was abolished by Y27632, a Rho-kinase inhibitor, and anti-Ryk antibody. Suppression of Wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury.
Akihito Takeda, Richard Goris, Kengo Funakoshi Department of Neuroanatomy, Yokohama City University Graduate School of Medicine, Yokohama, Japan
In contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. We examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. In the lesioned spinal cord, neurogenesis reached the maximum level 7 days after the hemisection. Glial cells positive for glial fibrillary acid protein (GFAP) temporarily increased at the lesion site one day after. Many GFAP positive cells expressed somatostatin. Serotonin (5HT) positive cells increased in number progressively from 1 day to 6 weeks after. Six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and 5HT cells surrounded the axons and glia. Thus, 5HT may promote these neural elements to invade the fibrotic scar.
Six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. These results suggest that the goldfish have ability to reestablish correct projections after the spinal injury.
Masao Koda 1 , Yukio Someya 2 , Ryo Kadota 2 , Chikato Mannoji 2 , Tomohiro Miyashita 2 , Atsushi Murata 3 , Masashi Yamazaki 2 1 Department of Orthopaedic Surgery, Togane Hospital, 2 Department of Ortopaedic Surgery, Graduate School of Medicine, Chiba University, Japan; 3 Division of Rehabilitation Medicine, Chiba University Hospital, Japan
Objective: Anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. Preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. Here we tested the effect of BDNF for anoikis of Schwann cell. Methods: (in vitro) Schwann cells were cultured from sciatic nerves of neonatal rats. Schwann cells were transferred to suspension culture. BDNF was added into the culture medium. Cell death was detected 24 h after suspension culture. (in vivo) Schwann cells were transplanted with or without BDNF treatment into contused rat spinal cord. Immunohistochemistry was performed to detect survival of grafted cells. The olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. However, evidence for active functional involvement of neural stem cells is still very limited. This study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. For this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. We will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits.
Koichi Kawada, Masanori Yonayama, Kiyokazu Ogita Dept. Pharmacol., Setsunan Univ., Osaka
The subventricular zone (SVZ) contains undifferentiated cells, which proliferate and generate the olfactory bulb (OB) interneurons. Throughout life, these cells leave the SVZ and migrate to the OB via the rostal migratory stream, where they differentiate. We have shown that trimethyltin (TMT) causes neuronal damage in the hippocampal dentate gyrus. In this study, we examined neuronal degeneration and regeneration in the OB after TMT treatment in mice. ddY mice were given TMT (2.8 mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded DNA (ssDNA), 5-bromo-2 -deoxyuridine-5 -monophosphate (BrdU), neuronal nuclei (NeuN) and nestin. Positive cells immunoreactive to ssDNA markedly increased in the OB on days 1 after TMT treatment. Positive cells immunoreactive to BrdU markedly increased in the OB on days 2 after TMT treatment. Double staining of BrdU and NeuN in the OB revealed that almost BrdU was not incorporated into mature neurons on day 2 after the treatment. These results suggest possible enhancement of neurogenesis in the OB following TMT treatment.
PS3A-F087 Early migration of human umbilical cord blood neural stem cells transplanted into rat brain Miroslaw Janowski 1 , Hanna Kozlowska 1 , Marcin Jurga 1 , Aleksandra Habich 1 , Elzbieta Wanacka 1 , Barbara Lukomska, Krystyna Domanska-Janik Department of NeuroRepair, Medical Research Center, Warsaw, Poland Many neurological disorders result from progressive cell loss or rapid cell damage. As Stem Cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. Recently, in our laboratory human umbilical cord blood neural stem cell line (HUCB NSC) was established. The aim of the study was to analyze the migratory potential of HUCB NSC transplanted into intact rat brain. HUCB NSC transfected with GFP gene was stereotactically transplanted (tx) into intact brain of CsA immunosuppressed adult Wistar rats. Cell detection was performed 24 h, 48 h, 72 h and 7 days after transplantation using Abs anti GFP, HLA class I and NuMa. Analysis of rat brains revealed viable GFP positive HUCB NSC cells migrating from tx site and dispersed through the host brain tissue 1, 2, 3 and 7 days after grafting. Immunohistochemical studies confirmed that these cells were of human origin: HLA class I or NuMa. In future we plan to study their lesion directed migratory potential. Heparan sulfate proteoglycans (HSPGs) are considered to play roles in CNS development, such as axonal guidance. However, little is known about the function of HSPGs during nerve regeneration. In this study, we examined the expression of EXT2, one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. The upregulation of EXT2 mRNA was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. We also examined the expression of mRNA for HSPG core protein. The mRNAs for glypican-1 and syndecan-1 were upregulated in injured motoneurons. These results indicate that the synthesis of HSPG is upregulated in injured motoneuron and HSPG might be involved in nerve regeneration.
Masami Watanabe 1 , Hiroe Sagawa 2 , Masahiro Ichikawa 3 , Yoshihito Tokita 1 1 Dept. Perinatol., Int. Dev. Res., Kasugai, Japan; 2 Dept. Ophthalmol., Nagoya Univ. Sch. Med., Nagoya, Japan; 3 Dept. Neurosurg., Nagoya Univ. Sch. Med., Nagoya, Japan
We examined whether Rho/ROCK inhibitor, Y39983, can make injured RGC axons regenerate into the crushed optic nerve (OpN) of cats. Methods: Culture; Retinal pieces were cultured in DMEM for 14d. After fixation, the neurites were stained with anti-TUJ1 antibody to obtain number and length of TUJ1 neurites. Crush; after an intravitreal injection of drug, the left OpN was crushed with thread. On day 12, WGA-HRP was injected into the vitreous. Sections of OpN were reacted for HRP with TMB reaction.
Masanori Yoneyama, Kiyokazu Ogita Dept. Pharmacol, Setsunan Univ., Osaka, Japan
In this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of 15-days-old embryonic mice. Neural progenitor cells were prepared from the hippocampus of 15-days-old embryonic mice by culturing in DMEM/F12 medium for 9 days in vitro (DIV). Marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bFGF and EGF, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. To evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (BSO) were exposed into cultured neural progenitor cells for a period of 0-9 DIV. Treatment with BSO resulted in a marked reduction in endogenous glutathione in the cells. MTT assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for 3-9 DIV. These results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus.
Michio Hashimoto, Eisuke Kawakita, Masanori Katakura, Osamu Shido Dept. of Environ. Physiol., Sch. of Med., Shimane Univ., Japan Docosahexaenoic acid (DHA), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. We examined the effect of DHA on neuronal differentiation of neural stem cells (NSCs) in vitro and in vivo. NSCs obtained from rat embryos were propagated as neurospheres and cultured with or without DHA for 7 days. DHA increased the number of Tuj1(+) neurons compared with the control, and the newborn neurons in the DHA group were morphologically more mature than in the control. DHA decreased the incorporation ratio of BrdU, the mitotic division marker, during the first 24 h period. Thus, DHA promotes the differentiation of NSCs into neurons by promoting cell cycle exit. Furthermore, dietary administration of DHA significantly increased the number of BrdU(+)/NeuN(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. These results demonstrate that DHA effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis.
Research funds: KAKENHI (17590205) PS3A-F092 Interaction among cues for visual depth motion perception Tomokazu Shimizu, Akitoshi Hanazawa Kyushu Institute of Technology, Fukuoka, Japan When an object surface approaches or leaves us, we perceive visual depth motion. Cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. We investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. For the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. When binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. For the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. When binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. When depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. When from the other cues, depth perception from binocular disparity was suppressed.
Research funds: COE-J19
Kazuyuki Takahashi, Akitoshi Hanazawa Kyushu Institute of Technology, Japan
In phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. To clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. Moving dots were presented in three apertures that were aligned horizontally. Before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. The dots moving in the same as or different from the instructed direction were presented in the side apertures. The performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. The performance kept high when the directional difference was up to ± 10 • and declined as the difference increased. The high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. The underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation.
PS3A-F094 Effect of spatial context on structure-frommotion perception Koshi Makino, Akitoshi Hanazawa Kyushu Institute of Technology, Japan
When viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating 3D cylinder. This phenomenon is called structure-from-motion (SFM). The direction of the rotation is ambiguous. We investigated the influence of spatial context on the perceived direction of the rotation. Three spatially separated stimuli were horizontally aligned. Subjects reported in which direction the central random-dot SFM cylinder rotated. They perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. This effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. The perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. These results suggest that the neural mechanism determining the rotation direction of bistable SFM is strongly influenced by the 3D structure of surrounding stimuli defined by binocular disparity.
PS3A-F095 Rotational motion aftereffect in positive direction for 3-dimensional random-dot pattern Masako Ono, Akitoshi Hanazawa Kyushu Institute of Technology, Kitakyushu, Japan When viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. This phenomenon is well known as motion aftereffect (MAE). This MAE can be perceived for 3-dimensional motion such as rotating cylinders. We found that adaptation to the rotation of a stereoscopic random-dot cylinder generate MAE like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive MAE). This positive MAE was strong when cylindrical random-dot was used as a stationary test stimulus. This aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. Although ordinary MAE declined in a few seconds, this positive MAE remained for a few minutes. This is a new phenomenon that is different from known 3dimensional MAE. This finding suggests that the visual system has a mechanism that detect 3-dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction.
Research funds: COE-J19
Takanori Uka, Ryo Sasaki Department of Physiology 1, Juntendo University School of Medicine, Tokyo, Japan Crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. As a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. Human subjects were required to report the direction of moving dots within a center patch (3 deg) of a center/surround display presented 10 degrees to the left of fixation, and to ignore the dots in the surround. Motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. Motion coherence of the surround was always 0 percent. For each of 11 subjects, we calculated direction discrimination thresholds (at 82% correct) at each surround size. Consistent with crowding, thresholds increased when surround size was 4.5 and 6 degrees, compared to those with no surround. Surprisingly, however, thresholds decreased when surround size was 9 and 12 degrees, relative to 6 degrees. Our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size.
PS3A-F097 Generation of receptive fields in higher visual areas based on V1 columnar structure: A model study Yoshitaka Toyoda, Yoshiyuki Shimizu, Izumi Ohzawa Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Neurons in higher cortical areas of the visual pathway, such as V2 and V4, respond to stimuli with complex shapes. How do these neurons integrate signals from V1? In particular, does the well-known columnar organization of V1 play a role in determining the shape selectivity of higher-order neurons? To explore these questions, we devised a feed-forward hierarchical model. In our model, higherorder neurons sum the activities of V1 neurons linearly according to a neural receptive field (NRF), a weighting function defined over the cortical surface. The manner a NRF sums over multiple columns determines its shape selectivity. Since there is no physiological data regarding possible forms for NRF, we have tested simple functional prototypes, Gaussians and Gabor functions. Reponses of these model neurons are examined using non-Cartesian gratings and other stimuli, and compared to published physiological data. About 15% of model neurons exhibit responses similar to those of V2 and V4 neurons. Odd-symmetric Gabor NRFs tend to generate more of these neurons.
Taihei Ninomiya, Takahisa M. Sanada, Izumi Ohzawa Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan When images with different spatial frequencies (SFs) are projected onto the two retinae, a 3-D surface slant is perceived (Blakemore, 1980) . The relationship between the binocular receptive fields (BRFs) and SF tuning properties indicate that the early cortical neurons can signal slant-in-depth (Sanada and Ohzawa 2006) . However, their measurements of BRF were conducted in the spatial domain, and the SF tunings were tested monocularly. In this study, interactions are examined directly in the SF domain between grating stimuli presented to the left and right eyes. Frequency-domain BRFs were measured by a reverse correlation technique. Both binocular and monocular SF profiles were obtained by this method. We predicted binocular SF (BSF) maps from monocular SF profiles, and compared the prediction and the actual BSF maps to assess the binocular interactions. With this method, neural response properties which previous studies couldn't access were revealed.
Research funds: MEXT(13041033), JSPS(13308048), COE21
PS3A-F099 Consistency of simple cell receptive fields: space and spatial frequency domain measurements Yuka Tabuchi 1 , Kota Sasaki 2 , Izumi Ohzawa 1,2 1 Grad. School of Frontier Biosci., Osaka Univ., Japan; 2 Grad. School of Eng. Sci., Osaka Univ., Japan Frequency-domain subspace reverse correlation and 2-d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (RF) of early visual cortical neurons. However, it is not known whether results from these methods are mutually consistent. To examine this issue, we compared an RF in the space domain measured by 2-d noise stimuli and an RF reconstructed from the response in the spatial frequency (SF) domain measured by flash grating stimuli of various orientation (OR), SF and spatial phase presented in rapid succession. We fitted these two RFs by Gabor functions, and examined the consistency of their parameters. All parameters including SF, OR, spatial phase, and size of the RF agreed well when an expansive nonlinearity is considered for each cell. The optimal SF obtained in the space domain increased over time to the same extent as that obtained in the SF domain. Therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity.
Research funds: MEXT(13041033), JSPS(13308048), COE21
PS3A-F100 Firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey Shunta Tate 1,2 , Hiroshi Tamura 1,3 , Ichiro Fujita 1,3 1 Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan; 2 JSPS, Japan; 3 CREST, JST, Japan Inferior temporal (IT) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. Cluster analysis indicated that IT cells were classified into five groups based on inter-spike interval (ISI) histograms of their spontaneous firing. The first two groups showed a single peak at a long or a short ISI in ISI histograms. The other three had multiple peaks, whose positions and relative heights varied among the groups. Principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. Stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. One of the single-peak groups was modulated by natural images more strongly than the other groups. The results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. Supported by JSPS and CREST.
Research funds: KAKENHI 16-07757 PS3A-F101 Spatial-frequency dependency of receptive field size and surround suppression in LGN and V1
Hironobu Osaki 1 , Tomoyuki Naito 2 , Osamu Sadakane 2 , Masahiro Okamoto 3 , Hiromichi Sato 2,3 1 Med. Sch., Osaka Univ.; 2 Grad. Sch. Med., Osaka Univ.; 3 Grad. Sch. Front. Biosci., Osaka Univ., Osaka, Japan
In the primary visual cortex (V1), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (SF). We investigated how SF of stimulus affects on stimulus-size tuning property of responses of neurons in V1 (n = 93) and lateral geniculate nucleus (LGN) (n = 63) in anesthetized cats. First, we found that V1 neurons exhibited shifts of their SF tuning from high to low according to a change in stimulus size from small to large. Second, we measured stimulus-area summation curve of responses and found that a higher SF stimulus caused a reduction of the receptive field (RF) size and an increase of the surround suppression. Similar results were obtained for LGN neurons implying that the relationship between SF and area summation properties observed in V1 has its origin in LGN. These results suggest that the SF tuning of RF surround is broader than that of RF center and this center-surround mechanism reduces redundancy in visual information processing.
Hiroyuki Nakamura 1 , Akichika Mikami 2 , Kazuo Itoh 1 1 Department of Morphological Neuroscience, Gifu University Graduate School of Medicine, Gifu, Japan; 2 Department of Behavioral and Brain Sciences, Section of Neurophysiology, Primate Research Institute, Kyoto University, Inuyama, Japan An extrastriate visual area V3A is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. To demonstrate the cortico-cortical connections of V3A, we injected a bi-directional tracer biotinylated dextran amine into the V3A. Our results indicated that the V3A has connections with the occipital, parietal and temporal cortices. The V3A may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. In addition to these connections, we found that V3A has commissural connections with the V3, the V3A, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area V2. These commissural connections may convey ipsilateral visual information near the vertical meridian representations.
PS3A-G103 Activity of neurons in the isthmo-optic nucleus and its relationship with head movements Hiroshi Ohno, Hiroyuki Uchiyama Department of Information and Computer Science, Faculty of Engineering, Kagoshima University, Kagoshima, Japan
Retinopetal neurons in the isthmo-optic nucleus (ION) send their axons to the contralateral retina in birds. The centrifugal visual projection is thought to be involved in attentional modulation of retinal output. We recorded activity of neurons in the ION in awake, headunrestrained Japanese quails using an implanted electrode assembly. Head movements were videotaped with a high-speed video camera (100 fps), and were also monitored with a 2D or 3D accelerometer. We found two distinct types of activity pattern: phasic and tonic. The majority of neurons in the ION discharge in a phasic manner. Phasic and tonic cells are also different one from another in relation to head movements. Phasic cells show phasic elevation of activity 10-40 ms after end of head movements, while tonic cells show tonic suppression during head movements. We will discuss the activity profiles of neurons in the ION in terms of their possible role in visually guided behaviors.
PS3A-G104 Timing of face specificity in Fusiform Gyrus responses to stimuli in different parts of the visual field Yuka Okazaki 1,2 , Arman Abrahamyan 3 , Catherine Stevens 3 , Andreas A. Ioannides 1,2 1 Brain Science Institute, RIKEN, Saitama, Japan; 2 Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Fukuoka, Japan; 3 School of Psychology, University of Western Sydney, Sydney, Australia Neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (FuG). Event Related Potential (ERP) and Magnetoencephalography (MEG) studies have shown that such the responses specificity to faces occurs approximately 170 ms (N170) after stimulus onset by comparing with the other objects. In the present study, we examined whether these and earlier FuG activities, which have been already identified by our team (within 100 ms), were selective for face. We achieved this by analyzing MEG data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. We found robust statistically significant activities for faces in FuG about 100 ms after stimulus onset which depended on the stimulus location in the visual field.
Narihisa Matsumoto 1 , Shoutaro Akaho 1 , Kenji Fujikumi 2 , Yasuko Sugase-Miyamoto 1 , Masato Okada 3 1 AIST, Ibaraki, Japan; 2 ISM, Tokyo, Japan; 3 University of Tokyo, Chiba, Japan
To understand the temporal aspects of information encoded at a population level in the inferior-temporal (IT) cortex, we applied a cluster analysis method to the responses of 45 neurons. Each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. Population activity vectors of 45 neurons for 38 visual stimuli were clustered by a mixture of Gaussian model. We estimated the number of clusters by using variational Bayes algorithm. We assumed that the probability of the number of clusters depended on the one at one time step before. In the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). In the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. Moreover, the number of clusters changed smoothly over time. These results suggest that the responses of IT neurons represent different levels of categorical signals separated along the time axis.
PS3A-G106 Relationship between color and shape selectivity in area TEO of the monkey Masaharu Yasuda 1,2 , Hidehiko Komatsu 1,2 1 National Institute for Physiological Science, Okazaki, Japan; 2 Sokendai, Okazkaki, Japan Visual objects typically consist of multiple features such as color, shape, texture etc. It is reported that neurons selective for these object features exist in the inferior temporal (IT) cortex of the monkey and some of them are selective for more than one of these features. However, little is known about the relationship between the selectivity for different features. Last year, we have reported that there exist many neurons in the posterior part of IT cortex (area TEO) that are selective for both color and shape. To study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted SVD (singular value decomposition) analysis. We found that some TEO neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). These results suggest a possibility that color and shape informations interact at cellular level in this area.
PS3A-G107 Neural correlates of stimulus shape detection in monkey inferior temporal cortex Taijiro Doi Lab. Cogn., Neurosci., Osaka Univ., Japan
We searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (IT) cortex while a monkey performed a 3-choice shape detection task. The monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. When there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. Trial-totrial variation of firing rates of many IT neurons correlated with the monkey's seen versus not-seen choices. The mean choice probability (CP) of IT neurons was 0.58, a value significantly larger than the chance level. Neurons with stronger visual responses exhibited larger CPs. We also searched for temporal firing patterns within the spike train from a single neuron or across 2-3 simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. The results indicate a link between the firing rates of IT neurons with conscious perception of stimulus shape.
Research funds: MEXT grant (17022025) PS3A-G108 Behavioral visual performance of the zebrafish mutant, eclipse Yuko Nishiwaki 1 , Atsuko Komori 1 , Tomonori Manabe 2 , Toshihiko Hosoya 2 , Hiroshi Sagara 3 , Emiko Suzuki 3 , Hitoshi Okamoto 2 , Ichiro Masai 1 1 Masai Initiative Research Unit, RIKEN, Wako, Japan; 2 RIKEN BSI, Wako, Japan; 3 IMS, University of Tokyo, Minato-ku, Japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. In the last meeting, we reported that the els gene encodes the ␣ subunit of cGMP phosphodiesterase (PDE6C), which functions in phototransduction in cone photoreceptors. Since genetic mutations of PDE6C have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of PDE6C. Here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. Our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until 9 day-post-fertilization. However, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. These data suggest that the level of PDE6C activity is important for the sensitivity of vision.
PS3A-G109 Localisation of two markers of oxidative phosphorylation in the ageing human retina: An immunohistochemical study Tapas Nag, Shashi Wadhwa AIIMS, India
The enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. We examined the distribution of two markers of oxidative phosphorylation (NADH-ubiquinol oxidoreductase and cytochrome C oxidase) in the human retina at different ages. Eyeballs of donors (Age: 50-94 years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for NADH-ubiquinol oxidoreductase (complex I) and cytochrome C oxidase (complex IV; Molecular probe, USA). Complex I-immunoreactivity (IR) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from 50 to 80 years of age, and showed a decline and lack of IR in older retinas (85-94 years). Complex IV-IR was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from 50 to 91 years of age, and absent at 94 years of age. Thus, complex I and IV-IR decline with age, with the former showing an earlier reduction in its IR. The data signify a reduced mitochondrial activity in the retina with ageing.
Research funds: AIIMS PS3A-G110 Temporal characteristics of neural activity related to target detection during visual search Tomoe Hayakawa 1 , Norio Fujimaki 1 , Toshihide Imaruoka 2 1 NICT, Kobe, Japan; 2 KIT, Kanazawa, Japan MEG and fMRI experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fMRI-constrained MEG-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. Activity around the CaS consisted of a prominent and a subsequent smaller but still obvious peak (117, 215 ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. Activity around the pFuG had a prominent peak and subsequent small activity (125, 237 ms), whereas the target's presence or not had no influence on either activity. The activity of the right intraparietal sulcus (IPS) was significantly larger than that for the left IPS at latencies around 196 ms irrespective of the target's presence or not. The results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the CaS was related to the target segregation from its surroundings.
Kaoru Amano 1,2 , Derek Arnold 3 , Alan Johnston 4 , Tsunehiro Takeda 1 1 Univ. Tokyo, Chiba, Japan; 2 NTT CS Lab., Kanagawa, Japan; 3 Univ. Sydney, Sydney, Australia; 4 UCL, London, UK When a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as MISC (Arnold & Johnston, 2003) . In order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (MEG) were compared with the perceived rate of illusory jitter measured psychophysically. The result showed that the perceived rate was around 10 Hz and matched with the alpha frequency of MEG. As 10 Hz MEG responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical 10 Hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. These results support our hypothesis that MISC is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties.
PS3A-G112 The internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the Drosophila central brain Kazunori Shinomiya 1,2 , Kei Ito 1,2,3 1 Center for Bioinform., IMCB, Univ. of Tokyo, Tokyo, Japan; 2 Dept. Comput. Biol., Grad. Sch. Frontier Sci., Univ. of Tokyo, Kashiwa, Japan; 3 BIRD, JST Visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. Many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). The vlpr is therefore expected to be one of the major higher-order visual centers. The neural circuits in this area, however, remain essentially unknown. Our study is to reveal the detailed internal structure of the vlpr, for the first time, using the Drosophila brain as a model system.
We have identified 12 discrete glomerulus-like structures (GLs) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. We analyzed the detailed internal structure of these GLs by visualizing single cells in each visual pathway using the combination of the GAL4 enhancer-trap and the FLP-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals.
PS3A-G113 Dynamic reorganization of orientation maps in a late phase of the sensitive period Kazunori O'Hashi 1,2 , Toshiki Tani 2 , Shigeru Tanaka 1,2 1 Graduate School of Life Science & Systems Engineering, Kyushu Institute of Technology, Japan; 2 Laboratory for Visual Neurocomputing, Brain Science Institute, RIKEN, Japan
We have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. In this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. We observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of 5 weeks. We also found that when the goggles were removed after 1 or 2 weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. These results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure.
Manavu Tohmi, Seij Komagata, Yamato Kubota, Masaharu Kudoh, Katsuei Shibuki Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan Fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (Kalatsky and Stryker, 2003) . In the present study, we used Fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. Periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. The Fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. Retinotopic maps were constructed based on these components. Vascular artifacts could be removed when the stimulus frequency was higher than 0.3 Hz, since fluorescence signals but not vascular responses could follow up to these frequencies. Combination of flavoprotein fluorescence imaging and Fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex.
Yoshitake Kohei, Manavu Tohmi, Masaharu Kudoh, Katsuei Shibuki Dept. Neurophysiol., Brain Res. Inst, Niigata Univ., Nigata, Japan
We have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. Another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. However, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. In the present study, we designed a new prism goggle for mice. This goggle was attached on the skull of mice during the critical period. The neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. Preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. However, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice.
PS3A-G116 Retinotopy-based morphing of brain activity Hiroshi Ban, Hiroki Yamamoto, Jun Saiki Graduate School of Human & Environmental Studies, Kyoto University, Kyoto, Japan
The topographic visual field map is a fundamental property of the primate early visual cortex. We propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (Maeda et al., 2003. Neurosci. Res.) . The procedure was as follows. First, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. Second, individual cortical surfaces were reconstructed. Third, the transformation between the position in the visual field and that on the cortical surface was established. Finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. This retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures.
Masahiro Yamada 1 , Yasuhiro Enami 1 , Hiroshi Jouhou 2 , Takehiko Saito 3 , Kaj Djupsund 4 1 Tokyo Metropol. Univ., Hino, Tokyo; 2 Astellas Pharma. Inc., Osaka, Japan; 3 SUNY Upstate Med. Univ., Center for Vision and Ophthal., NY, USA; 4 Univ. Kuopio, Dept. Neurobiol., Kuopio, Finland ON-OFF type amacrine cells are intensely connected with each other by gap junctions (GJs), forming a syncytium with a wide receptive field. We studied effects of external pH (pH 0 ) on the control of cell functions. Photoresponses of the cells were recorded intracellularly. Slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. By lowering pH 0 only 0.2 units from the baseline of 7.60, we found a remarkable reduction of the conduction velocity by 20-30%, an increase of the length constant and a hyperpolarisation of the resting potential. Based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both GJ and plasmamembrane conductances of the cell. Thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at GJs.
PS3A-G118 Analysis of the band-pass filtering of the retinal rod by the ionic current model
It is known that the rod network behaves like a band-pass filter. It was found that the time to peak of the response was shorter in rods further away from a slit of light. The band-pass filtering behavior has been attributed to an inductance element, I h , or I K(Ca) . However, biophysical mechanism underlying the band-pass filter is not fully understood. To analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. The model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (I Ca , I Kv , I K(Ca) , I h , I Cl(Ca) ), calcium system and gap junctions between rods. In simulation, the band-pass properties of the rod was analyzed. It was found that single rod itself behaves as a band-pass filter. The mechanism underlying the band-pass filter was examined by changing model parameters. The result suggests that I K(Ca) , I Cl(Ca) and I h are responsible for the bandpass filtering.
Research funds: KAKENHI (17500195) PS3A-G119 Stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex Go Uchida, Mitsuhiro Fukuda, Manabu Tanifuji BSI, RIKEN, Wako, Japan
In inferior temporal (IT) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (SAs) of 30% (17 of 57) of neuron pairs (inter-neuronal distance >500 m) are significantly correlated. In the present study, to investigate how the correlated SAs relate to functional structure in IT cortex, we measured stimulus selectivity for each neuron of the 57 pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to 28 visual stimuli. This analysis revealed that the pairs with correlated SAs tended to show more similar selectivity than the pairs lacking correlated SAs. In addition, model analysis showed that in 71% (12/17) of the pairs the correlation of SAs reflect synchronous transition between two activity states: periods with high and low mean firing rates. These results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant IT neurons showing similar stimulus selectivity.
Toshiyuki Ishii 1,2 , Toshihiko Hosoya 1 1 BSI, RIKEN, Japan; 2 Dept. Biomolecular Science, Toho Univ., Japan Understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. It is often assumed that the firing rate is the sole carrier of information. However, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. The vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. Here we show that temporal patterns of spikes within these bursts carry visual information. When three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. This suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. We also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over 200 ms. We propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds.
Kazuhiro Shimonomura, Takayuki Kushima, Tetsuya Yagi Osaka University, Osaka, Japan
Purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (V1). We have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and FPGA circuits. The silicon retina has a concentric center-surround Laplacian-Gaussian-like receptive field. The output image of the silicon retina is transferred to the simple cell chips. The simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in V1. This architecture mimics the feed-forward model proposed by Hubel and Wiesel, and computes physically a two-dimensional Gabor-like receptive field. The FPGA circuits compute complex cell responses based on the disparity energy model. The system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of V1 neurons.
Masayoshi Tsuruoka 1 , Masako Maeda 1 , Bunsho Hayashi 1 , Ikuko Nagasawa 2 , Tomio Inoue 1 1 Dept. Physiol. Showa Univ. Sch. Dent. Tokyo, Japan; 2 Dept. Anestesiol. Showa, Univ. Sch. Dent. Tokyo, Japan
The present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. Under halothane anesthesia, the T13-L2 dorsal roots were cut bilaterally to eliminate thoracolumbar influences. An electromyogram (EMG) of the external abdominal oblique muscle evoked by colorectal distention was measured. Colorectal distention (80 mmHg) was produced by inflating a balloon inside the descending colon and rectum. EMG activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil (5%, 1 ml). The increased EMG activity significantly reduced following bilateral L6-S3 ventral rhizotomies. A baseline EMG did not significantly alter when the L6-S3 ventral roots were cut bilaterally prior to inflammation. Following the development of inflammation, there was less of an increase in EMG activities. These results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation.
PS3A-G123 Hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography Using positron emission tomography (PET), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. Activation within right dorsolateral prefrontal cortex (DLPFC) and right inferior parietal cortex (BA40) was significantly greater (P < 0.001, uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. On the other hand, activation within right medial frontal cortex (mPFC) was significantly greater (P < 0.001, uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. This is the first evidence with PET for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. These results suggest a role of DLPFC and mPFC in the cognitive control of the interoception. The participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. Bladder cooling reflex (BCR) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (DO). Recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (TRP). We detected cold sensitive receptor TRPM8 in the urothelial cell by immunofluorescence in an animal model for BOO. Intravesical administration of TRPM8 agonist (L-menthol: 0.06-6 mM) in freely moving rats, increased the micturition pressure (MP) in either normal (n = 7) or BOO rat (n = 8). The micturition interval (MI) did not change in normal rat, but decreased in BOO that have DO. The results suggest that BCR is enhanced in BOO by increasing TRPM8 on the urothelium cell of the urinary bladder.
PS3A-G125 Caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys Shin-ichi Ito 1 , A.D. Craig 2 1 Dept. Physiol, Shimane Univ. Sch. Med., Izumo, Japan; 2 Atkinson Res. Lab., Barrow Neurol Inst, Phoenix, USA We investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (Pf) (Ito & Craig, J Neurophysiol 2005) . Evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. The injection sites were all located in the ventrolateral part of caudal Pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. Labeled terminals were found in the caudate nucleus (Cd) in all cases. Terminal patches extended longitudinally in the head of Cd, concentrated in its ventral aspect. Dense terminal patches also occurred throughout the tail of Cd. These results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration.
Research funds: NIH grant NS40413 PS3A-G126 Ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, Tilapia Masami Yoshimoto, Naoyuki Yamamoto, Chun-Ying Yang, Hironobu Ito, Hitoshi Ozawa Department of Anatomy and Neurobiology, Nippon Medical School, Tokyo, Japan General visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. In teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. However, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. The medial inferior lobe (mIL), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost Tilapia. Hence, telencephalic connections of the mIL were studied in this study. Tracer injection experiments into the mIL revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., Vs, Vd, and Vv), and the dorsal telencephalon (i.e., Dm, rDc, and Dl). These findings suggest that the mIL corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus).
Tatsushi Onaka, Yuki Takayanagi Department of Physiology, Jichi Medical University, Tochigi, Japan Administration of prolactin releasing peptide (PrRP) decreases food intake. We have previously shown that an icv injection of anti-PrRP antibodies increases food intake. Neurones producing PrRP are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. It is thus possible that PrRP may mediate satiety signals in the brain. Here we examined effects of anti-PrRP antibodies upon total amounts of food intake and meal patterns. An icv injection of anti-PrRP antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. These data suggest that PrRP may play an important role in the short-term control of food intake and are consistent with a hypothesis that PrRP is a satiety signal within the brain.
Research funds: Grant-in-Aid for Scientific Research (C) PS3A-H128 Fasting induced Long-chain fatty acid receptor GPR120 expression in the anterior pituitary of mouse Ryutaro Moriyama, Shingo Imoto, Shinya Shano, Nobuyuki Fukushima Department of Life Science, Kinki University, Higashiosaka, Japan G-protein-coupled receptor 120 (GPR120) is known as a receptor for unsaturated long-chain fatty acids. The present study investigated the effect of 48 h fasting on GPR120 expression in several regions of male mouse by real-time quantitative PCR, in situ hybridization and immunohistochemical method. GPR120 mRNA expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. 48 h fasting induced GPR120 mRNA expression increase in the anterior pituitary, lung and rectum. In the anterior pituitary, GPR120-like immunoreactive cells were only observed in fasting animals. These results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via GPR120 at least fasting period.
PS3A-H129 Ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions Kinuyo Iwata, Mika Kinoshita, Hiroaki Sato, Hiroko Tsukamura, Keiichiro Maeda Laboratory of Reproductive Science, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. We have previously revealed that 3hydroxybutylate (3HB), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls (4V) in the rat brain to regulate reproductive functions and feeding behavior. The present study was aims to determine if the ependymocytes located on the wall of 4V respond to the change in 3HB. Change in the intracellular calcium concentration ([Ca 2+ ] i ) in vitro was measured in dispersed ependymocytes taken from the 4V in rats. The present results showed that the [Ca 2+ ] i increased in response to 3HB, but the increase was blocked by ␣-Cyano-4-hydroxycinnamic acid, which is a monocarboxylate transporter 1 (MCT1) inhibitor. Immunohistochemistry showed that MCT1-immunoreactivities were located on the 4V ependymocytes. These results indicate that the ependymocytes may sense 3HB through a MCT1-dependent mechanism.
Research funds: KAKENHI 14360177 PS3A-H130 Comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice Tomoko Ishizuka, Kouta Hatano, Atsushi Yamatodani Dept. Med. Sci. and Technol, Grad. Sch. Allied Hlth Sci., Fac Med., Osaka Univ., Osaka, Japan
Leptin is a satiety factor which is produced by the white adipose tissue. Peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. Circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. We previously reported that leptin decreases food intake via the activation of the histaminergic system. In the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (DIO) mice. Leptin (1.3 mg/kg, ip) reduced food intake in normal mice but not in DIO mice, suggesting that DIO mice have resistance for exogenous leptin like obese humans. The same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in DIO mice. These results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant DIO mice.
Tomoya Kitayama, Yuri Onitsuka, Katsuya Morita, Toshihiro Dohi Department of Dental Pharmacology, Hiroshima University, Hiroshima, Japan Parkinson disease (PD) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. Symptoms of PD include disorder of aspiration and mastication, and dysphagia. In this study, rats injected with 6-hydroxydopamine (6-OHDA) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at 4 weeks after 6-OHDA without affect on number of neuron in solitary tract. These rats were transplanted with neural progenitor cells at 0 mm; anteroposterior, +3 mm; lateral and −5 and −7 mm; dorsoventral from bregma at 2 weeks after 6-OHDA injection. The treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. However, neural progenitor cells were not detected in substantia nigra. These results suggest that transplantation of neural progenitor cells may better 6-OHDA-induced eating disorders via protection of neurons.
Research funds: Grant-in-Aid for Young Scientists B 17791323
Most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). To understand biological bases of using such tools, we trained Japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: (1) retrieving the food with a rake (a motortool), (2) retrieving the hidden food with a mirror-attached rake, (3) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, (4) moving a mirror hung along the rail by hand to find the food, (5) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. Finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. Thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image.
Kaori Sawada 1,2 , Shigehiro Miyachi 2 , Michiko Imanishi 2 , Masato Taira 1 , Masahiko Takada 2 1 Div. Applied System Neurosci., Nihon Univ. Sch. Med., Tokyo, Japan; 2 Dept. System Neurosci., Tokyo Metropol. Inst. Neurosci., Tokyo, Japan
To investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area 9 (9m), dorsal area 46 (46d), and ventral area 46 (46v). The retrograde transsynaptic labeling was examined in the temporal lobe cortex 3 days after prefrontal injections when the second-order neurons were labeled. The labeled neurons were observed in the lateral and medial aspects of the temporal lobe. In the lateral temporal lobe, neuronal labeling from 9m, 46d, and 46v was arranged topographically in and around the superior temporal sulcus. The labeing in the medial temporal cortex was also topographically arranged, such that 9m, 46v, and 46d receive multisynaptic projections from the entorhinal cortex, area 36, and both, respectively. These results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex.
Research funds: CREST, Japan Science and Technology Agency PS3A-H137 New neural activities of reward anticipation and task errors H. Ogawa 1 , H. Ifuku 2 , T. Nakamura 3 , S. Hirata 4 1 Kumamoto Kinoh Hosp, Kumamoto, Japan; 2 Fac Educ, Kumamoto Univ., Kumamoto, Japan; 3 Nat Kikuchi Hosp, Kumamoto, Japan; 4 Dept. Psych, Kumamoto Univ. Hosp, Kumamoto, Japan Neural activities at reward phase were recorded from the primary (PGC: areas G, 3 & 1-2) and higher-order (HGC: PrCO & OFC) gustatory cortices of a monkey engaged in a taste discrimination GO/NOGO task. A lever had to be pressed after LED onset when NaCl was delivered, but not to water delivery. Reward was given ca 2 s after LED offset at correct trials. Relations between cues and responses were reversed. Of 169 reward-related neurons found, 88.7% showed ON type responses and the rest usual expectation responses. Three types of ON responses were noticed; C-type (n = 96) only at correct trial, I-type (n = 5) at around possible reward onset only at incorrect trials, and C-I type (n = 49) at both. Two classes of the C-I type were found; Class I increased discharges at correct trials but decreased them at incorrect, but Class II increased them at both. All 3 types were found in both cortices, but most Class I were found in PGC and most Class II in HGC. I-type and Class II C-I type may represent error signals and reward anticipation.
Hiroaki Ishida, Masahiko Inase, Akira Murata Department of Physiology, School of Medicine, Kinki University, Japan
In the macaque monkey, the ventral intraparietal area (area VIP) integrated visual-tactile information in the body centered reference frame. The receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. Recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. This means other¸s body image is mapped on own body image in the same neuron. In our experiments, we studied visual-tactile receptive field of the bimodal neuron in VIP, then recorded activity during observing the experimenter being touched. Some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. The results suggested that bimodal neurons in VIP may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation.
Daichi Hirai 1 , Takayuki Hosokawa 2 , Masato Inoue 1 , akichika Mikami 1 1 Section of Brain Sciences, Primate Research Institute, Kyoto University, Inuyama, Japan; 2 Department of Psychology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan Amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. However, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. To elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. We compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. We found amygdala neurons to code the relative preference of available outcomes in a given trial block.
PS3A-H140 Neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex Kyoko Matsuda, Toshiyuki Sawaguchi Lab. Cogn Neurobiol, Hokkaido Univ. Grad. Sch. Med., Sapporo, Japan
To expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. To examine a possible involvement of the lateral prefrontal cortex (LPFC) in such functions, we recorded neuronal activity from the LPFC of monkeys that performed an oculomotor task. In this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (Cue1), and centrally allocated one of the objects presented in Cue1 (Cue2). The frequency of which object was presented as Cue2, i.e., task context, was manipulated across blocks. We focused on Cue2 period and found that a subset of neurons showed object preference depending on current task context (Cc type) or previous task context (Pc type). Cc type and Pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. Thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the LPFC.
PS3A-H141 Anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling Takashi Mizuhiki 1 , Barry J. Richmond 3 , Munetaka Shidara 1,2 1 Grad. Sch. of Tsukuba Univ., Ibaraki, Japan; 2 Neurosci. RI., AIST, Tsukuba, Japan; 3 Lab. Neuropsychol., NIMH, Bethesda, USA The human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fMRI studies. To identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in 2 conditions: (1) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], (2) a cue indicates whether the current trial will be rewarded or not [certain condition]. In the uncertain condition 84/120 neurons responded in all trials. In the certain condition 50/84 neurons responded in the rewarded trials only. 48 of these 50 showed significant differences in firing rate between in the first trials after reward and other trials. These insular neuron responses seem related to reward expectancy and recent reward delivery. These neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks.
Research funds: KAKENHI (Priority Areas17022052), AIST
Masamichi Sakagami 1,3 , Kosuke Sawa 2 , Xiaochuan Pan 1,3 1 BSRC, Tamagawa University, Tokyo, Japan; 2 Senshu University, Kanagawa, Japan; 3 PRESTO, JST, Japan Reward prediction behavior based on integration of associative information was investigated. Monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. At first, they learned two sequences of stimuli: A1-B1-C1 and A2-B2-C2. After monkeys could acquire the sequences, new pairs of stimuli (i.e., D1 and D2, E1 and E2, etc) were introduced to associated with B1 or B2 (D1-B1, D2-B2, etc). The asymmetric reward rule was instructed by pairing C (C1 or C2) with the reward. After this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. Monkeys could show reward predictive behavior for not only A1 and A2, which were associated with C1 and C2 in trained sequences, but also new pairs of stimuli, which were not directly associated either with C or reward. These results suggested that monkeys could use reward predicting information by integration of association among trained sequences, C-reward association, and new stimuli.
Research funds: KAKENHI (17022037), HSFP, PRESTO, JST PS3A-H143 Reward predicting activity of prefrontal neuron based on group of stimuli Xiaochuan Pan 1,2 , Kosuke Sawa 1,3 , Masamichi Sakagami 1,2 1 BSRC, Research Institute, Tamagawa University, Japan; 2 PRESTO, JST, Japan; 3 Department of Psychology, Senshu University, Japan Ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. The main purpose of this study is to examine the PFC neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. Monkeys performed a sequential association task with symmetric reward. At first, they learned two sequences of stimuli: A1-B1-C1 and A2-B2-C2. The asymmetric reward rule was instructed by pairing C (C1 or C2) with the reward block by block. Monkeys were also trained with two different orders of stimuli (B-C-A and C-A-B). Out of 202 neurons from the lateral PFC, 31% showed reward-related activity in the first cue period. And one third of them (SR type) predicted reward only when a preferred stimulus was presented as a first cue. Interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. The results suggest that about 10% of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. Attention evoked by novel stimuli is important for behavioral adaptation to new environment. However, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. We trained two monkeys on a Pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (LPFC and MPFC). In a block of the Pavlovian task (Pv block), a visual stimulus (CS) was paired with a liquid reward and the trial repeated 3 times. In a following block of the instrumental task, the monkey searched a correct action to obtain the CS as positive feedback. The CS was alternated every 4 Pv blocks. In many LPFC cells, responses to the CS were enhanced immediately after the change of CS, while such enhancement was less popular in MPFC. This result suggests that LPFC more contributes to coding of stimulus-novelty than does MPFC. When an outcome of action is uncertain, a top-down attention is directed to the coming outcome. To clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (MPFC and LPFC). In a Pavlovian block (Pv block), a visual stimulus was paired with a liquid reward. In a following instrumental block (Inst block), the monkey searched a correct action based on the visual feedback. The same visual stimulus as the one presented in the preceding Pv block followed a correct action, whereas another visual stimulus followed a wrong action. When the monkey made more than 3 consecutive correct trials, a new Pv block started. Both MPFC and LPFC cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in MPFC than in LPFC. These results suggest that the top-down attention first occurs in MPFC and propagates to LPFC in individual trials.
PS3A-H146 Neuronal activity in the presupplementary motor area during a bimanual sequential motor task Toshi Nakajima 1 , Hajime Mushiake 1 , Jun Tanji 2 1 Department of Physiology, Tohoku University School of Medicine, Sendai, Japan; 2 Brain Science Research Center, Tamagawa University, Machida, Japan
To investigate the involvement of the pre-supplementary motor area (pre-SMA) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. In this report, we focus on neuronal activity during a period when the monkey was preparing to start the 2-sequence movements in a memorized order. We made regression analysis of neuronal activity in this period. We found that neuronal activity in the pre-SMA rarely reflected muscle activity. Instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. We also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. We would demonstrate typical examples of pre-SMA neurons and discuss their functional implications.
PS3A-H147 Neuronal activity in the putamen and CM thalamus during response bias and its complementary process Yukiko Hori, Takafumi Minamimoto, Minoru Kimura Dept. of Physiol., Kyoto Prefect Univ. Med., Japan
We showed previously that CM thalamus participates specifically in complementary process to response bias (Minamimoto et al. 2005) .
To study the roles of the putamen and CM in response bias and it complementary processes, we recorded activity of CM and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded GO-NOGO button press task. Instruction of GO or NOGO activated CM neurons (n = 73) preferentially when the instruction was associated with small reward. The instructions activated 3 groups of putamen neurons preferring small reward-(n = 19), large reward-action (n = 3) and both types of action (n = 26). Onset latencies of these putamen neurons and RTs in large-reward-GO trials were shorter than those in small-reward-GO trials by 30-50 and 100-150 ms, respectively. Putamen neuron activation lead that of CM neurons by 50-70 ms. These results suggested that the putamen plays a major roles in both response bias and its complementary process while CM participates in the complementary process in concert with the putamen.
Research funds: KAKENHI (17022032) PS3A-H148 Encoding expected total rewards and their errors through a series of action choices by dopamine neurons Naoyuki Matsumoto, Kazuki Enomoto, Minoru Kimura Dept. Physiol. Kyoto Pref Univ. Med., Japan
To examine how dopamine (DA) neurons represent reward expectation and its error through a series of action choices, we recorded activity of DA neurons in two Japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. There are trials of first (T1), second (T2) and third (T3) choices with reward probabilities of about 30, 50 and 80%, respectively. Monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (R1, 95%). Most DA neurons (66/85) responded to the start cue of each trial and reinforcer beep after the choices. Magnitude of the start cue responses progressively increased from T1 to T2 and to T3 trials, then decreased in R1 trial. In another task with two repetition trials (R1 and R2, 96%), magnitude of start-cue responses decreased gradually from T3 to R1 and to R2 trials. Thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors.
Research funds: KAKENHI (17022032) PS3A-H149 Striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards Hiroshi Yamada 1,2 , Hitoshi Inokawa 1 , Minoru Kimura 1 1 Dept. of Physiol. Kyoto Prefect Univ. Med., Kyoto, Japan; 2 JSPS, Japan
To study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded 146 striatal projection neurons from a monkey. After depressing a start button, the monkey chose 1 of 3 target buttons with correct rates at 1st, 2nd, 3rd and repetition trials of 33, 50, 89 and 96%, respectively. Correct choices were followed by reward water. Neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice (11/55) rather than to upcoming choice of target (2/55). During the target choice, another subset of neurons showed firings selective to choosing particular target (29/57) rather than to expected reward probability (12/57). After the target choices, another group of neurons fired related to expected reward (41/56) rather than to chosen action (19/56). Our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal.
Research funds: KAKENHI (17022032), JSPS Fellows PS3A-H150 Encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum Hitoshi Inokawa, Hiroshi Yamada, Minoru Kimura Department of Physiology, Kyoto Pref. Univ. of Med., Japan
To study the signals encoded by tonically active neurons (TANs) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of 169 TANs was recorded from the putamen and caudate nucleus of a Japanese monkey. After depressing a start button, the monkey chose 1 of 3 target buttons at average correct rates of 33 (first), 50 (second), 89 (third) and 96% (repetition choices). Correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. About a half of TANs (80/169) responded differentially to the high and low tone beep respectively. Number of responsive TANs and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. These results suggested that the TANs may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation.
Research funds: KAKENHI (17022032) PS3A-I151 Representation of value of action, action and its outcome in sub-populations of striate neurons Y. Ueda 1 , K. Samejima 2 , K. Doya 3 , M. Kimura 1 1 Dept. Physiol., Kyoto Pref. Univ. Med.; 2 Brain Sci. Res. Center, Tamagawa Univ.; 3 IRP, OIST
To know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of 236 striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. After a 1 s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of 10, 50 or 90%. A linear regression of neuronal discharge rates showed: 39 neurons encoded reward values of either action during delay period before GO signal, with most (64%) of them not having the action value signal in other task epochs. Another subset of neurons encoded action signal selectively during action selection after GO signal (n = 33), while other 37 neurons encoded presence or absence of reward at reinforcer epoch after the action selection. Neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. These findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection.
Research funds: KAKENHI (17022032) PS3A-I152 Delay period activity of the monkey striatum in duration discrimination task Atsushi Chiba, Ken-ichi Oshio, Masahiko Inase Dept. Physiol., Kinki Univ. Sch. Med., Osaka Sayama, Japan Neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. Two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. Durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. Striatal neurons phasically responded during the first cue (C1), first delay (D1), second cue (C2), second delay (D2), and response periods. Activity during the D1 and D2 periods was analyzed in this study. Firing rates during the D1 period linearly depended on C1 durations. On the other hand, D2 period activity depended on trial types (LS and SL), but not on the variety of C2 durations in each trial type. Our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations.
Research funds: KAKENHI (17021039) PS3A-I153 Neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity Satoshi Eifuku 1 , Ryoi Tamura 1 , Teruko Uwano 1 , Taketoshi Ono 2 1 Dept. Integrative Neurosci., Univ. Toyama, Toyama, Japan; 2 Dept. Molecular Integrative Emotional Neuroscience, Univ. Toyama, Toyama, Japan
To elucidate neuronal basis of face memory, neuronal activities in the area TEav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (I-APA task). In the I-APA task, monkeys were required to memorize 4 paired associates of patterns and facial identity. Each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. During the I-APA task, neuronal responses to a particular paired associate were identified. Many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. This asymmetrical delay activity are indicative of the crucial role of the TEav area in face memory.
Research funds: KAKENHI (16500260 17021016) PS3A-I154 Reflexive social attention elicited by biological motion in monkeys and humans Yoshiya Mori 1 , Mikio Inagaki 1 , Wu Lisa 2 , Taijiro Doi 1 , Eishi Hirasaki 1 , Hiroo Kumakura 1 , Ichiro Fujita 1 1 Osaka Univ., Japan; 2 Massachusetts Institute of Technology, USA Determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. Here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. We examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (BM) stimulus. During the task, subjects responded to a target point after the disappearance of the BM stimulus and fixation point. The walking direction of the BM stimulus was not predictive of the target direction, and was irrelevant for performing the task. We found that the reaction times in congruent trials, where the walking direction of the BM stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. We believe the attention mechanisms driven by BM may be part of the intentionality inference system.
Research funds: Grants from 17022025 and Takeda Science Foundation PS3A-I155 Response properties of posterior parietal neurons during a multidimensional visual search task Tadashi Ogawa, Hidehiko Komatsu Natl. Inst. Physiol. Sci., Aichi, Japan
The posterior parietal cortex (PPC) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. Visual sensory information (e.g. stimulus features) might be integrated in PPC to form a saliency map that controls spatial attention. To examine this hypothesis, we recorded the neural activity from the lateral intraparietal (LIP) and 7a areas of monkeys performing a multidimensional visual search task. The monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. PPC neurons increased their activity when the receptive field stimulus became the target. Some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. The mixed existence of these two distinct types of activities suggests that PPC is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. Monkeys utilize visual information in social communication. To elucidate visual function to categorize sexes, (1) performance of visually guided sex discrimination task and (2) neuronal activity during the task in orbitofrontal cortex (OBF), the region could be related to sex recognition and vision processes, were investigated. Monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. The monkeys pressed the right bar for pictures of males and the left for females to get water reward. As a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. Extracellular recordings of neurons in OBF during the task showed that some cells responded to the pictures in a sexspecific manner. The present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. OBF could be involved in visual categorization of sex.
Research funds: KAKENHI (A) (16209006) (SA) and COE program in KIT from the MEXT PS3A-I157 Activities of bursting neurons during color discrimination task in the monkey prefrontal cortex Naoki Ishikawa 1 , Satoshi Katai 2 , Masanori Saruwatari 1 , Masato Inoue 1 , Akichika Mikami 1 1 Section of Brain Sciences, Primate Research Institute, Kyoto University, Inuyama, Japan; 2 Third department of Internal Medicine, Shinshu University, School of Medicine, Matsumoto, Japan
The neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. On the other hand, cerebral cortex consists of different types of neurons. In this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. And we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (FS), fast rhythmic bursting (FRB), and intrinsic bursting (IB) neurons (Katai et al. Neuro 2004) . Most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. These results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction.
Atsushi Matsumoto 1 , Tetsuya Iidaka 2 1 Department of Psychology, Nagoya University, Nagoya, Japan; 2 Department of Psychiatry, Nagoya University, Nagoya, Japan Several studies indicated that gamma band activity (GBA: 30-80 Hz) reflects the process to form mental representation of objects or information. We investigated whether the GBA is observed during subliminal visual word processing as well as supraliminal word processing. GBA were observed both in masked and unmasked condition. At the 400-600 ms time window, GBA was significantly higher in the word condition compared to the nonword condition in the unmasked condition. Similarly, in the masked condition, GBA of the word condition was significantly higher than that of the nonword condition at that time window. These results indicate that the unconscious lexical processing was reflected in the GBA at that time window. Furthermore, at the 600-700 ms time window, GBA induced by word was significantly higher than that induced by nonword. This effect was not observed in the masked condition. In addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously.
Wakayo Yamashita, Junichi Hayashi, Tomoki Murakami, Gang Wang Department of Bioengineering, Kagoshima University, Kagoshima, Japan
The purpose of this study was to investigate the dependency of view association learning on the separation of the views. Each stimulus set included 16 images (4 objects × 4 views). 4 novel objects were generated by deforming a prototype in four directions. For 30 deg-interval object sets, 4 views were obtained by rotating each object with the interval of 30 deg, 60 deg-interval set and 90 deg-interval set were with 60 deg and 90 deg interval respectively. Task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. The performance across 60 deg separated views was significantly higher in the trials with 30 deg-interval sets than those with 60 deg-interval sets. Similarly, the difference was also found in the performances across 90 deg separated views between those with 30 deg-interval sets and 90 deg-interval sets. The results suggest that the exposure of interpolated views significantly improved the association learning of the views.
PS3A-I160 Brain regional activity during attention task ( The Kana Pick-out Test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in Japan. However, few fMRI studies while demonstrating the test have reported. We therefore assessed the effect of brain regional activity with computerized Kana Pick-out Test projected on the screen with clicking a mouse button to pick kana out under fMRI running. Executing the test resulted in significant increases in BOLD signals in right prefrontal area, bilateral hippocampus and Broca's area. The results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of Kana Pick-out Test, suggesting that this test is useful in screening dementia.
PS3A-I161 Obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in Shiraz, Iran, 2005 Ashkan Mowla, Arash Mowla Shiraz University of Medical Sciences, Iran
Aim: The aim of this study is to evaluate OCD symptoms, Tic disorder, Body Dismorphic Disorder (BDD) and Trichotilomnia (TTM) among middle school students of Shiraz, Iran. Methods: 1682 middle school students were selected in a cluster random sampling from the four educational regions of Shiraz, Iran.Persion standardized MOCI was used to assess obsessional symptoms. For evaluating BDD, Tic disorder and TTM symptoms, a semi-structured interview was done according to DSM-IV-TR criteria. Results: Students with more obsessional symptoms were more girls and demonstrated more positive family history.They were more likely to be from lower socioeconomic class and with lower school average. They also showed more association with Body Dysmorphic disorder and Tic disorder. Conclusion: Girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. This study, like pervious ones, confirmed BDD symptoms and Tics to be more in individuals with OCD symptoms. It was seen that OCD symptoms would affect school performance.
PS3A-I162 siRNA-induced NR1 knockdown causes hypofunction of NMDA-R and cognitive deficit M. Saji 1,2 , T. Utida 2 , A. Ohnishi 2 , K. Noda 1 , M. Ogata 1 , H. Akita 1 , N. Suzuki 1,2 1 Physiol, Health Sci. Sch. Kitasato Univ., Sagamihara, Japan; 2 Brain Sci., Graduate Sch. Kitasato Univ., Sagamihara, Japan
Blockade of NMDA-R by antagonists causes psychomimetic effects, suggesting involvement of NMDA-R dysfunction in mental disorders like schizophrenia. However, the relationship between mental disorders and molecular abnormality has not been cleared. To identify the role of NMDA-R in brain function, we performed siRNAinduced knockdown of NMDA-NR1 using HVJ-envelope vectors. We confirmed that marked down-regulation (50%) of NR1 expression occurred only in the hippocampus among various brain regions 4-8 days after intra-ventricular injection of siRNA-vector complex. In the hippocampal slice from rats with the NR1 knockdown, the NR1 down-regulation prevented depressive effects of NMDA on fEPSPs, while the treatment did not affect LTP or LTD. In rats with the NR1 knockdown, the NR1 down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. These results suggest that hypofunction of hippocampal NMDA-R by siRNA-treatment causes a deficit of cognition.
Ken Hatanaka 1,2 , Hiroshi Ageta 2 , Ikuko Yao 2 , Kaoru Inokuchi 2 , Yutaka Kirino 1 , Mitsutoshi Setou 2,3 1 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan; 2 Mitsubishi Kagaku Institute for Life Sciences, Tokyo, Japan; 3 Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Okazaki, Japan Schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. To know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of UBL3 mRNA was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. UBL3 is a highly conserved protein, which has a ubiquitin-like domain (UBL domain) and CAAX motif which is a membrane localization signal. We found that UBL3 mRNA was expressed in the hippocampus, and Purkingie cells of the cerebellum. The putative molecular function of UBL3 wil be discussed.
Research funds: Grant-in-Aid for Young Scientists (B), PRESTO PS3A-I164 Decreased interneurons in the Pax6 mutant mouse limbic system Hasumi Haba 1 , Tadashi Nomura 1 , Yoshinobu Hara 1,2 , Noriko Osumi 1,2 1 Div. Dev. Neurosci., CTAAR, Tohoku Univ. Sch. Med., Sendai, Japan; 2 CREST, JST, Japan Core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. Recent studies have revealed abnormality in distribution of interneurons in these regions. We have previously found that Pax6 heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. In the present study, we thus analyzed distribution of interneurons in several regions of Pax6 heterozygous mutant mouse (Sey/+) brain. We focused on three subpopulations of interneurons: parvalbumin (PV)-, calretinin-, and somatostatin-posive interneurons. Immunohistochemical studies indicated marked decrease in PV-positive interneurons in two brain regions of Sey/+ mice, i.e., the olfactory bulb and the amygdala. Reduced number of PV-positive interneurons was observed in the Sey/+ amygdala at 8 weeks, but not at 4 weeks. Our results suggest that age-dependent decrease of PV-positive interneurons might underlie behavioral abnormalities in Sey/+ mice. Schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. Dysbindin (DTNBP1) is a susceptibility gene for schizophrenia. Genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. Recently, decreased expression levels of dysbindin mRNA and protein have been reported in postmortem brain in patients with schizophrenia. Thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. Sandy mouse showed decreased locomotor activity and time in the center in the open field test. And an acute treatment of atypical antipsychotic, olanzapine (0.2 mg/kg, i.p.), improved the decrease in time in the center. Moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. Our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment.
Research funds: 16790712, 17025055 PS3A-I166 Gene expression analysis of dysbindin mRNA in peripheral blood in schizophrenia Sachie Chiba 1,2 , Satoko Hattori 1 , Hiroaki Hori 1 , Tetsuo Nakabayashi 3 , Hiroshi Kunugi 1 , Ryota Hashimoto 1 1 Department of Mental Disorder Research, National Institute of Neuroscience; 2 Tokyo University of Agriculture and Technology Department of Biotechnology and Life Science, Koganei, Japan; 3 Musashi Hospital, NCNP, Kodaira, Japan Although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. As genetic evidence suggested that dysbindin (DTNBP1) is a susceptibility gene for schizophrenia, we measured dysbindin mRNA expression level in peripheral blood samples of 38 patients with schizophrenia and 38 age-sex matched healthy controls by a quantitative real time RT-PCR method. We quantified the expression levels of two major dysbindin transcripts among several known splicing variants. No significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. Further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia.
Research funds: 16790712, 17025055 PS3A-I167 Genetic variation in dysbindin influences memory and general cognitive ability Ryota Hashimoto 1 , Hiroko Noguchi 1 , Hiroaki Hori 1 , Tetsuo Nakabayashi 2 , Satoko Hattori 1 , Sachie Chiba 1 , Seiichi Harada 2 , Osamu Saito 2 , Hiroshi Kunugi 1 1 Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan; 2 Musashi Hospital, NCNP, Kodaira, Japan; 3 Tokyo University of Agriculture and Technology Department of Biotechnology and Life Science, Koganei, Japan Dysbindin (DTNBP1) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. We examined the possible association between genetic variants in the dysbindin gene and memory and IQ in 165 healthy volunteers and 72 patients with schizophrenia. Individuals who did not carry a protective haplotype had lower performance in several memory domains WMS-R, although this haplotype did not affect IQ measured by WAIS-R. A risk independent polymorphism for schizophrenia influences both memory and IQ in the opposite direction. These data suggest that dysbindin gene may have impact on the cognitive function such as memory and IQ and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia.
Research funds: 16790712, 17025055 PS3A-I168 Detection of 18F-DOPA signal in brainstem monoaminergic nuclei in schizophrenia Yuri Kitamura 1 , Nicola Bright 3 , Toshio Yanagida 1 , Masatoshi Takeda 2 , Paul Grasby 3 1 Department of physiology, Osaka University, Japan; 2 Department of Psychiatry, Osaka University, Japan; 3 Cyclotron Unit, Imperial College, Hammersmith Hospital, UK We used 18 F-Dopa PET to investigate presynaptic dopamine dysfunction in schizophrenic patients. The object of this study was to test that a schizophrenic cohort would show elevated AADC activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. All subjects and 18 F-Dopa scans were obtained from a database of scans published in McGowan et al. 2004 , Archives General Psychiatry. The 14 schizophrenic patients all met DSM-IV criteria on medication and 10 healthy volunteers were compared. We attempted to improve the quality of the 18 F-DOPA signal by implementing a FBF-realignment movement correction method. Significant increases in 18 F-DOPA uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > 0.02). Our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. In this study, we analyzed the P300 component of the visual eventrelated potential in 14 patients with schizophrenia and 14 healthy controls, and also performed LORETA analysis. The Ethics Committee of Kurume University approved this study. The P300 amplitude for the crying face was significantly smaller in patients than in controls. In controls, the P300 amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the P300 amplitude between the 2 faces. LORETA analysis demonstrated that there were significant differences in the activity in Brodmann area 10 between the 2 faces in controls, while in patients, there was no significant activity difference between the 2 faces. Stimulation with crying face induced higher activities in the 10 and right 21 areas in controls than in the patients. These results indicated that the cognitive function was influenced by affective stimulus.
PS3A-J170 Inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit Kensuke Nomura 1 , Shoji Tanaka 2 , Koki Yamashita 2 , Motoichiro Kato 1 , Haruo Kashima 1 1 Department of Neuropsychiatry, School of Medicine, Keio University; 2 Department of Electrical and Electronics Engineering, Sophia University A number of studies indicate that the prefrontal cortex (PFC) is intrinsically linked to working memory (WM) and that dopamine critically modulates WM activity.
According to the hypothesis proposed by Goldman-Rakic and her colleagues, we constructed an electrophysiological circuit model for WM which represents eight directions. The computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation.
A lot of studies suggested that increase of noise was related to schizophrenia, especially in WM disturbance. Our study indicates that noise inputs cause WM impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the PFC.
PS3A-J171 Pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex Ko Miyoshi, Ikuko Miyazaki, Masato Asanuma Department of Brain Science, Okayama University, Okayama, Japan
We previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of DISC1, a candidate gene for schizophrenia. In this study, we analyzed in vivo expression of pericentrin in the mouse embryo. In the developing cerebral cortex, pericentrin mRNA was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. The pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. Specific subtypes of receptors such as 5-HT6 are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. Dysregulation of GR has been thought to play an important role in the pathophysiology of mood disorders. Two isoforms of human GR-alpha and -beta arise from alternative splicing of the pre-mRNA primary transcripts. Previously, we evaluated these two isoforms mRNA level in the peripheral white blood cells of the patients with mood disorders. We found that the reduced GR-alpha mRNA level in the patients with both bipolar and major depressive disorders, while GR-beta mRNA level was not altered. These results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. To test this, we evaluated mRNA level of alternative splicing-related SR protein family, which regulate alternative splicing in several genes including GR, in the peripheral white blood cells of the patients with mood disorders. We did not find any differences in 7 of the 10 SR protein mRNAs level in the patients compared to healthy controls and now, we are examining other SR family mRNAs level.
PS3A-J173 Alteration of neocortical long-term depression following electroconvulsive shock Yoshifumi Ueta 1 , Ryo Yamamoto 1 , Shigeki Sugiura 2 , Kaoru Inokuchi 3 , Nobuo Kato 1 1 Dept. Integrat. Brain Sci., Grad. Sch. Med., Kyoto Univ.; 2 Nara Med. Univ.; 3 Mitsubishi Kagaku Inst. Life Sci.
Electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. There have been a few reports that studied effects of electroconvulsive shock (ECS) on long-term potentiation. However, its effects on long-term depression (LTD) have not been investigated to date. The present experiments examined roles of ECS in inducing LTD at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. Following ECS, LTD magnitude at layer II/III-to-VI pyramidal cell synapses was significantly reduced in comparison to no-ECS subjects. As described in recent microarray studies, Homer1a/Vesl-1S was identified as one of the most up-regulated molecules after ECS. We therefore injected Homer 1a protein by diffusion from patch pipettes. Homer 1a injection, as well as with ECS treatments, reduced LTD magnitude only at layer II/III-to-VI pyramidal cell synapses, implicating that Homer 1a may be a biological mediator of ECS effects.
Masanori Kasai 1 , Nozomi Miyagi 1 , Norio Kawashiro 2 , Daisuke Torizuka 2 1 Dept. of Chem. & BioSci., Faculty of Sci., Kagoshima Univ., Kagoshima, Japan; 2 Sanko shokuhin Co., Ltd., Tokyo, Japan It is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. To know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male Lewis rats received a suspension of complete Freund's adjuvant (1.0 mg), injected intradermally into the tail. Body weight, food intake and water intake were also measured. All rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the 11th day. The rats were sacrificed to measure the serum mineral contents (Zn, Na, Cl, P, Ca, K, Mg) on the 2nd, 7th, 14th, 21st, 28th and 35th days. The serum zinc level was decreased on all of the measurement and the average of serum zinc (75.1 ± 12.1 g/dl, n = 7) on the 35the day was significantly lower than that in intact rats (139.4 ± 11.9 g/dl, n = 7). This decrease of zinc was correlated with weight loss but not hind paw swelling. Other minerals did not show any significant changes throughout the measurement period.
PS3A-J175 Molecular cloning of a novel candidate for ethanol-responsive genes, YY1AP-related protein (YARP), in rat brain In order to elucidate the molecular mechanisms of EtOH action on the CNS, we investigated changes in gene expression in the adult rat brain after chronic EtOH treatment. By means of cDNA subtraction, we identified a candidate for EtOH-responsive genes in the hippocampus. cDNA cloning and sequence analysis revealed that this gene encodes a novel homolog of YY1AP (YY1-associated protein) and is well conserved in rats and humans. Homology search for functional domains predicted that the YARP polypeptide contains NLSs', a DNAbinding motif, and a chromatin decondensation domain, as well as YY1-binding and transactivation domains previously demonstrated in YY1AP. In the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of YARP and YY1 genes was demonstrated in the same neurons. Analogous to YY1AP as a co-activator of transcription factor YY1, it is postulated that YARP can regulate cerebral gene expression in response to EtOH treatment.
PS3A-J176 Excitotoxic degeneration of hypothalamic orexin neurons: Involvement of NR2B-containing NMDA receptors and rescue by GABA A receptor stimulation Hiroshi Katsuki, Shinsuke Kurosu, Toshiaki Kume, Akinori Akaike Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan Selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (QA), an endogenous NMDA receptor agonist. We report here that NR2B-selective NMDA antagonists ifenprodil (3 and 10 M) and Ro25-6981 (0.1 and 1 M) markedly inhibited degeneration of orexin neurons induced by 24 h application of NMDA (45 M) or QA (1.5 mM). We also show that stimulation of GABA A receptors by muscimol (10 and 30 M) or isoguvacine (30 and 100 M) potently inhibited QA cytotoxicity. In addition, the protective effect of GABA (100 M) plus a GABA uptake blocker nipecotic acid (1 mM) was abolished by a GABA A antagonist picrotoxin (100 M). Norepinephrine and serotonin did not provide a neuroprotective effect. Thus, GABAergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by NR2B-containing NMDA receptors.
Yoshika Kurokawa, Shinji Tsukahara, Hidekazu Fujimaki National Institute for Environmental Studies, Tsukuba, Japan
To evaluate neurotoxicological influence of volatile organic chemicals (VOCs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving VOC inhalation. We dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (RH795). We then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of 1 ms. We also examined optical signals in the hippocampus during toluene inhalation. As a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. On the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. We will discuss the usefulness of this technique for examination of the neurotoxicological influence of VOCs.
PS3A-J178 A simple method for fabricating electrodes array for multichannel neural recording -Investigation of the alignment of the array and the measurement system-Noriyuki Taniguchi 1 , Osamu Fukayama 2 , Takashi Sato 2 , Takafumi Suzuki 2 , Kunihiko Mabuchi 1,2 1 Dept. Biomed. Eng., Univ. Tokyo, Tokyo, Japan; 2 Dept. Info. Physi. Comp., Univ. Tokyo, Tokyo, Japan Various types of electrodes have been developed for use as Brain-Machine Interface (BMI) to record signals from neurons. Electrode arrays can be purchased from vendors. However, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. Thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. The electrodes were able to align for the experimental paradigms. The effectiveness of the arrangement of the array as a BMI device was investigated. The electrodes were implanted in the primary motor cortex of Wistar rats. We used a wheel-formed rat exercising kit to measure the walking speed of a rat. The neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. And we evaluated the estimation of the walking speed by multiple electrodes with different alignments.
PS3A-J179 On-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks Ikurou Suzuki 1 , Yasuhiko Jimbo 2 , Kenji Yasuda 1 1 Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan; 2 Department of Precision Engineering, Graduate School of Engineering, University of Tokyo, Tokyo, Japan
We have developed a single-cell-based on-chip 8 um-diameter multielectrode arrays with an agarose microchambers (AMC) for topographical control of the network patterns of living neurons. This system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. And sampling rates of measurement are 100 kHz in 64ch electrodes simultaneously. Using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the AMC array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. Moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. In the meeting we will present the results in detail and will discuss the potential of our method.
Yuichi Yamashita 1 , Tetsu Okumura 1 , Kazuo Okanoya 2 , Jun Tani 1 1 Lab. for Behavior & Dynamic Cognition, RIKEN-BSI, Japan; 2 Lab. for Biolinguistics, RIKEN-BSI, Japan How the brain generates and learns temporal sequences is a fundamental issue in neuroscience. The production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. Bengalese finches (BF) learn highly complex songs that have grammatical structure. The underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. To address this issue, we developed a neural network model of BF's songs that might explain how different regions of the brain work together. To test the model, we also conducted empirical experiments on the brains of BF. The model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. Moreover, comparison between the model and the empirical data on real birds shows similar trends. This work is a part of an integrated research project combining model simulations and empirical study. Please see also the empirical component of this project as reported by Okumura.
PS3A-K181 Local administrations of muscimol into the Nif alter song grammar of the Bengalese finches (BF) Tetsu Okumura 1 , Yuichi Yamashita 1 , Kazuo Okanoya 2 , Jun Tani 1 1 Behav & Dynamic Cognition, RIKEN-BSI, Saitama, Japan; 2 Biolinguistics, RIKEN-BSI, Japan Songs of passerines are learned behavior which used by males to attract females. Their songs consist of several song notes, and these notes are produced in a fixed temporal order. Among the passerines, however, BFs sing complex song which follows finite state syntax. The song control system of BF consists of a set of discrete nuclei including the HVC and Nif. Previous study showed that Nif lesioned BFs sung simpler songs, with less phrases to phrases branching. Therefore, Nif-HVC connection may play important role in generating song grammar. In this study, we perfused Nif with muscimol via microdialysis probes as a perturbation on Nif-HVC system. Following a local perfusion, song grammar was modified. Some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. Nif is also known as one of auditory relay nucleus to HVC. Part of the effects is possibly caused by disruption of auditory feedback. We also developed a neural network model of Nif-HVC system. Please refer Yamashitaǐs poster for details of this model.
The reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network Katsunari Shibata 1 , Shinya Ishii 1 , Munetaka Shidara 2 1 Dept. of E&E Engineering, Oita Univ., Oita, Japan; 2 Grad. Sch. of Comprehensive Human Sci., Univ. of Tsukuba, Tsukuba, Japan
In the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. It is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. Here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. The simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [1] S. Ishii, et al., "A model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", Neurocomputing, 2006 Behavior is adjusted by outcomes of actions. To examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (MPFC) of two monkeys performing a behavioral adjustment task. The monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (CS+) paired with a liquid reward and the other (CS−) that did not appear in a preceding Pavlovian conditioning. CS+ followed a correct action and CS− followed a wrong action. When the monkey made more than 3 consecutive correct trials, a new block of Pavlovian conditioning started. We calculated the prediction errors provided by CS+ and CS− on the basis of a reinforcement learning model of action selection. We found that the neuronal activity corresponds to the prediction error of value of the selected action. This result suggests that MPFC contributes to behavioral adjustment by providing prediction errors of action values.
Makoto Miyazaki 1 , Shinya Yamamoto 2 , Sunao Uchida 3 , Shigeru Kitazawa 4,5 1 Faculty of Hum Sci., Waseda Univ., Tokorozawa, Japan; 2 Neurosci. Res. Inst, AIST, Tsukuba, Japan; 3 Faculty of Sport Sci., Waseda Univ., Tokorozawa, Japan; 4 Dept. of Neurophysiol, Juntendo Univ. Grad. Sch. Med., Tokyo, Japan; 5 CREST, JST, Saitama, Japan Our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. To date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. In this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased Gaussian distributions (mean = ±80 ms, s.d. = 80 ms). Previous studies predict that the point of simultaneity would be shifted toward the peak of the Gaussian, i.e. toward the most frequent interval. However, the point of simultaneity was shifted away from the peak by about 50 ms. Our results disagree with the previous studies, but conforms to a contrasting prediction from a Bayesian integration theory.
Research funds: KAKENHI (15200031) PS3A-K185 Single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy Ichiro Shimoyama 1 , Fumiko Sato 2 , Ken Nakazawa 3 , Kenichi Ono 3 1 Chiba University, Japan; 2 Field of Home Economics, Faculty of Education, Chiba Univ., Japan; 3 Department of Integrative Neurophysiology, Graduate School of Med. Chiba Univ., Japan
To study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (OMM-3000, SHI-MADZU) over the frontal area (45 channels) for 7 volunteers (19-23 y). Thirty tasks were presented visually every 30 s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., How to cook curried rice? or How to fold paper into a turtle? etc). A comprehension-test was done just after the record. Easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between 2 tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. Grand averaged correlation coefficient was −0.4+/−0.45 between the dynamics of the oxy-and deoxy-hemoglobin. The correlation should be considered in discussing neural activation for NIRS. We thank SHIMADZU Corp. for providing the NIR Station.
Kazuya Ishibashi 1,2 , Kosuke Hamaguchi 3 , Masato Okada 1,2,3 1 Department of Complexity Science and Engineering, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan; 2 JST, Japan; 3 RIKEN BSI, Wako, Japan A synfire chain is one of the networks which generate stable synchronous pulse packets. Although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. By using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. We analyse the network dynamics with the Fokker-Planck equation. First, we analyze the activity of the network when we activated one memory pattern of the first layer. We show that the layered associative network has stable synfire state. Second, we investigate the activity when we activated 2 different memory patterns. Then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. We will report the details of those phenomena.
Research funds: KAKENHI (14084212) and (16500093) PS3A-K187 Auditory ERPs can be identified as corresponding stimuli by classifier with Naive Bayes method Akitoshi Ogawa 1,2 , Sachiko Koyama 1,2 , Takashi Omori 3 , Takashi Morotomi 4 1 Research Institue for Electronic Science, Hokkaido University, Sapporo, Japan; 2 Japan Science and Technology Agency, Saitama, Japan; 3 Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Japan; 4 Sakushin Gakuin University, Utsunomiya, Japan
In an attempt to reversely estimate the input stimulus from measured ERPs, we developed computational classifier using naive Bayes method. Correct classification rates could be index values of the ERP characteristic. In this study, we applied the classifier to identify auditory ERPs (N = 13). The ERPs were elicited by tones (500 Hz) with different durations (8, 16, 32, 64, 128, 256 ms) and gaps (8, 16, 32, 64, 128 , 256 ms) embedded in a continuous pure tone (500 Hz). To confirm the generality of the method, we used leave-one-out cross validation. ERPs of each subject were identified by the classifier which was constructed from the others' ERPs. As a result, the correct rates for 32 and 64 ms were high both for the tones (32 ms, 38%; 64 ms, 61%) and the gaps (32 ms, 46%; 64 ms, 46%).
PS3A-K188 Determination of channel parameters for construction of a neural model of Caenorhabditis elegans Kazumi Sakaa, Akane Andoh, Taro Ogurusu Laboratory of Bioscience, Faculty of Engineering, Iwate University, Iwate, Morioka, Japan
Caenorhabditis elegans (C. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. On the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of C. elegans has not been sufficient. We have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator GENESIS and parallel GENESIS. Using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. We report validity of obtained parameters and the possibility of the existence of unknown channel. Supported by grant from JSPS.
PS3A-K189 Theoretical consideration about NMDA current change and its effect on synaptic plasticity Shigeru Kubota, Tatsuo Kitajima Department of Bio-System Engineering, Yamagata University, Yonezawa, Japan It is well known that NMDAR plays an important role in learning and memory. Several experiments have shown that the property of NMDAR EPSC can change within a few weeks after birth, leading to the shortening of its decay time course. Since the calcium current through NMDAR is involved in LTP and LTD induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. Here we show by the biophysical compartmental model that the alteration of NMDAR property can modulate the calcium influx into the spine, which finally switches plasticity rule. We also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. Our results suggest that changing NMDAR time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit.
Manish Kumar Jain Department of Psychiatry, R.D. Gardi Medical College, India Introduction: I want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future.
Method: I keep the records with me since I join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers.
Results: It is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder.
Conclusions: There is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to 50% till now. Hepatic encephalopathy (HE) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (FHF). In the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (TAA), a hepatotoxin which induces FHF and thereon HE. Estimation of phospholipid fatty acid content in cerebral cortex membranes from TAA treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. Assesment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene, and 1-[4 (trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. The level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from TAA treated rats indicating prevalence of oxidative stress. Results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during TAA induced HE and their possible implications in the pathogenesis of this condition are also discussed.
Nagatoki Kinoshita, Shigenobu Yonemura Cellular Morphogenesis, CDB, RIKEN, Kobe, Japan Rho-GTPases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. However, little is known about their distributions and their physiological functions in vertebrates. Immunohistology of chick embryos revealed apical accumulation of Rho, Rac and Cdc42 in neural plate cells, especially in bending hinge points. After neural tube closure, the apical accumulation decreased. Coordinately, activities of Rho-GTPases and myosin II in neural plate cells were higher during neurulation than after neural tube closure. Inhibitions of actin filament formation, myosin II-mediated contraction or Rho-associated kinase activity affected neural tube formation. Inhibition of Rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. These results suggest that Rho-GTPases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. Furthermore, we are screening regulators and effecters of Rho-GTPases transiently expressed in neural plate cells during neurulation.
Setsuko Sahara, Dennis DM O'Leary MNL-O, The Salk Institute, USA Gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. Here we report mouse buttonhead homogoues, the Sp gene family, as candidates of downstream of those morphogens: Sp5 expression correlates with Wnts/Bmps in the cortical hem, Sp8 with Fgfs in the CoP, and Sp9 with Shh in the ventral midline and MGE. By using in utero electroporation, we show that Sp8 regulates anterior-posterior patterning of the cortex into areas by controlling distinct Fgfs that having opposing effects. Sp8 and Fgf8 exhibit reciprocal induction, indicating that Sp8 is a positive feedback regulator of Fgf8. Surprisingly, though, ectopic expression of both Sp8 and its dominant active form shift cortical areas in the opposite manner to Fgf8, suggesting that Sp8 activates additional targets that overcome Fgf8 function. Our results indicate that Fgf10 is an additional target of Sp8, showing effect on patterning similar to Sp8. These findings indicate that Sp8 balance the proper cortical arealization through Fgf8 and Fgf10.
Research funds: NIHR37NS31558 PS3P-C003 Fyn-FAK signal transduction is involved in the radial migration of late-generated neocortical neurons Eiko Nakahira 1 , Kotaro Hattori 1 , Takeshi Yagi 2 , Shigeki Yuasa 1 1 Dept. Ultrastructural Res., Nat. Inst. Neurosci., NCNP, Tokyo, Japan; 2 KOKORO Biology Group, FBS, Osaka Univ., Suita, Japan Fyn tyrosine kinase posphorylates focal adhesion kinase (FAK) that is involved in cell migration. Taking into account the defective formation of neocortical layers II-III in Fyn-deficient mice, Fyn-FAK signal transduction might be involved in the control of the migration of neocortical neurons. Accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. Late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. Fyn and FAK were colocalized in the migratory neurons, and fak siRNA transfer into neocortical primordium induced migration defect similar to that in Fyn deficiency. These findings strongly suggest that the coordination of Fyn and FAK is essential for the radial migration of late-generated neocortical neurons.
Noriyo Ishibashi 1 , Kazuko Keino-Masu 1 , Tatsuyuki Ohto 1 , Satoshi Kunita 2 , Satoru Takahashi 2 , Masayuki Masu 1 1 Dept. of Mol. Neurobiol., Grad. Sch. of Comprehensive Human Sci., Univ. of Tsukuba, Tsukuba, Japan; 2 Laboratory Animal Resource Center, Univ. of Tsukuba, Tsukuba, Japan Heparan sulfate (HS) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. These interactions are mediated by the specific HS structures generated by sulfation and epimerization. A recently identified extracellular sulfatase, SulfFP1, has been implicated in the regulation of growth factor/morphogen signaling through HS remodeling in vitro, but its physiological roles remain unknown. Here we generated knockout mice lacking the SulfFP1 gene, and examined the brain development. A previous study showed that the brain-specific disruption of the EXT1 gene, which encode a HS synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. In this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult SulfFP1-deficient mice. Heparan sulfate (HS) proteoglycans play a crucial role in mediating important signaling by Wnt, Hedgehog and FGF. Recently, novel sulfatases, SulfFP1/Sulfatase-1 and SulfFP2/Sulfatase-2, which have HS 6-O-endosulfatase activity have been isolated. Since these SulfFPs are detected in the extracellular space, SulfFPs are thought to regulate cell surface signaling through HS remodeling. In order to examine the function of SulfFP genes in zebrafish, we isolated zebrafish SulfFP1 and SulfFP2. Here we report the isolation and the characterization of the third homologue, SulfFP3. SulfFP3 has about 56% and 71% overall amino acid homology with SulfFP1 and SulfFP2, respectively. At 24 h postfertilization, SulfFP3 is expressed in the ventral region of spinal cord, whereas SulfFP1 is expressed only in the floor plate and SulfFP2 is expressed in the lateral floor plate and ventral regions of spinal cord. Detailed expression patterns of SulfFP3 will be presented.
Masahiko Ajiro, Kenichi Arai, Mika Maeda-Sato, Masuo Obinata, Wataru Shoji Dept. of Cell Biology, IDAC Tohoku Univ., Japan Collapsin response mediator proteins (CRMPs) are cytosolic proteins involved in neuronal differentiation and axonal guidance. A member of this family, CRMP2 was shown to mediate the repulsive effect of Sema3A on axons. CRMPs appear to play more complex roles in axonal differentiation, elongation and branching during development. Since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos.
At early axogenesis stage, zebrafish CRMPs are expressed in specific patterns. In trigeminal sensory ganglia, CRMP2, 3, 4, and 5 are highly expressed. Knocking down of these gene results in disorganization of the ganglia, separating into several clusters. However, their axonal patterns including direction, extension, and branching appears normal. Same defects were observed in the knockdown of neuropilins, receptor component for class 3 semaphorins. These results suggest that CRMPs may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals.
PS3P-C007 Developmental origin of diencephalic sensory relay nucley in teleosts Y. Ishikawa 1 , N. Yamamoto 2 , M. Yoshimoto 2 , T. Yasuda 1 , K. Maruyama 1 , T. Kage 1 , H. Takeda 3 , H. Ito 2 1 Nat. Inst. Rad. Sci., Chiba, Japan; 2 Nippon Med. Sch., Japan; 3 Tokyo Univ., Japan
We propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. It has been proposed that the relay system in teleosts is unique among vertebrates. Teleost relay nuclei, the preglomerular complex (PG), have been assumed to originate from the basal plate (posterior tuberculum, PT) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. Our results show, however, that many pax6-or dlx2-positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. Massive clusters of the migrated alar cells become localize in the mantle layer lateral to the PT neuroepithelium, from which the PG appear to differentiate. We therefore consider most neurons in the PG are be of alar, not basal origin. Thus, the teleost PG can be regarded as migrated alar nuclei. The organization of the diencephalic sensory relay system may have been conserved across vertebrates.
Hideyuki Dekimoto, Yoshihiro Oomiya, Satoshi Kikkawa, Toshio Terashima, Yu Katsuyama Department Anatomy and Developental Neurobiology, Kobe University Graduate School of Medicine Laminaiton is one of features unique to the brain of vertebrates. To understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. Since one of ETS family transcription factors, ER81 is expressed specifically in the layer V of the mouse neocortex, we selected this gene for the purpose of our study. Here we cloned zebrafish ER81 homologue (zfER81), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. Expression of zfER81 was observed in multiple sites of developing brain. The expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. ER81 expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. Based on these observations, evolution of the gene expression in the brain lamination will be discussed.
Hiroyuki Koizumi, Teruyuki Tanaka, Joseph G. Gleeson University of California, San Diego, USA doublecortin (DCX), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in X-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, RNAi-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. Dclk (doublecortin-like kinase) is one of the homologous genes of Dcx, encodes for protein with an N-terminus that is 70% identical to DCX, but also additional C-terminal protein kinase domain. Here, we report that the Dclk functions in a partially redundant pathway with Dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. Dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. RNAi-mediated knockdown of either gene results in similar migration defects. These results indicate the DCX microtubuleassociated protein family is required for proper neuronal migration and axonal wiring.
Hiraki Sakuta 1,2 , Hiroo Takahashi 1,2 , Takafumi Shintani 1,2 , Kazuma Etani 1 , Masaharu Noda 1,2 1 Div. of Mol. Neurobiol., NIBB, Okazaki, Japan; 2 CREST, JST, Japan
In the developing chick retina, the expression of BMP4 is relieved by that of BMP2 at around E5 with a change from a dorsal high to dorsotemporal high pattern, complementary to that of Ventroptin, a BMP antagonist. We previously demonstrated that misexpression of Ventroptin altered the retinotectal projection along both the DV and AP axes. Here, we show that topographic molecules along the DV axis, together with ephrinA2, are expressed in a double-gradient fashion from E6 on like Ventroptin and BMP2. When BMP2 expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. Moreover, in the BMP2 knockdown and ephrinA2-misexpressing embryos, the retinotectal projection is altered along the two axes. The expressional switching from BMP4 to BMP2 thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the DV axis toward the posterior side during retinal development.
Noriyuki Morita, Teiichi Furuichi Lab. for molecular neurogenesis, RIKEN-BSI, Wako, Japan
The mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. To elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. Not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". Parasagittally expressed genes were classified in comparison with the band pattern of En2, Wnt7b and Pcp2/L7 gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. Our accumulating data suggest that not only patterning genes like Engrailed and Wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes.
Yasufumi Tanaka, Tomiyoshi Setsu, Hideyuki Dekimoto, Yu Katsuyama, Toshio Terashima Kobe University Graduate School of Medicine, Japan
The Nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (PN) was reduced in the reeler compared with the normal counterpart. The injections of DiI and 4Di-10ASP into the left and right hemicerebellum, respectively, resulted in that only a few PN neurons were doubly labeled in the control, but in the reeler most of PN neurons were doubly labeled. The placements of solutions of DiI and 4Di-10ASP into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that DiI-labeled or 4Di-10ASP-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mRNA and Reelin were both expressed in the PN of the normal mice. These data elucidate that the Reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of PN neurons.
Kudoh Suguru 1,2 , Takahisa Taguchi 1 1 AIST, Ikeda, Japan; 2 PRESTO, JST
The spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a 2-dimensional electrode array. The map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. We confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. In addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. These results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing.
Research funds: PRESTO, JST PS3P-D014 Laterality of the human cerebral hemisphere Taiko Kitamura, Jinzo Yamada Department of Anatomy, Tokyo Medical University, Tokyo, Japan It has been reported that some functional predominance is located in the right or left hemisphere of the human brain. Especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. In this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. We measured the weight, the medial-lateral width (M-L), the anterior-posterior lenght (A-P), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. The weight of each hemisphere was roughly equal. The M-L was wider in the right side than the left side. The A-P was longer and the width of the medial surface was larger in the left side than in the right side. Because of the longer A-P and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. Such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere.
PS3P-D015 Synchrony-Induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns Takaaki Aoki 1 , Toshio Aoyagi 2 1 Department of Physics, Kyoto University, Kyoto, Japan; 2 Graduate School of Informatics, Kyoto University, Kyoto, Japan
Temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. However, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. In this modeling study, considering a network of Excitatory and Inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. Furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. This ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner.
Shozo Kito, Maiko Kitagawa, Akiko Shingo Lab. of Neurosci., Hyogo Univ., Kakogawa Japan
In our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased IGF-1 mRNA expressions. Nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. To clarify this problem, nicotine was intraperitoneally injected into 4 weekold Wistar strain rats in several doses followed by successive injections of BrdU for the next 4 days. Then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of BrdU/PSA-NCAM, BrdU/NeuN or BrdU/GFAP. As the results, numbers of both BrdU(+)/PSA-NCAM(+) cells and BrdU(+)/NeuN(+) cells were much decreased nicotine-dose dependently. On the other hand, as much as 1 mg/kg was needed for nicotine to exert its effect on the number of BrdU(+)/GFAP(+) cells. These results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats.
PS3P-D017 The establishment of the organotypic slice culture of postnatal rat forebrain involving eGFP-labeled neural progenitors Kaoru Sato 1 , James E. Goldman 2 1 Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan; 2 Department of Pathology, Columbia University, New York, USA After injecting eGFP-encoding retrovirus into P0 rat SVZ, sagital sections of forebrain were made at P3 and cultured for 6 days. The migration pattern of the eGFP-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. The expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. When slices were cultured with anti-␣3 integrin antibody, the migration of the neural progenitors inside SVZ was significantly enhanced along the rostrocaudal extent. These results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. Radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. To make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. It has been reported that tenascin-C (TN-C) is one of the markers for radial glia. Accordingly, diphtheria toxin (DT)gene and enhanced green fluorescence protein (EGFP)-gene both driven by TN-C gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. The numbers of EGFP-labeled cells in that TN-C gene promoter and subsequently DT gene are activated selectively decreased by this approach. Using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress.
Takayuki Manabe, Kouko Tatsumi, Eri Makinodan, Manabu Makinodan, Takahira Yamauchi Department of 2nd Anatomy, Nara Medical University, Kashihara, Nara, Japan It has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. In the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with BrdU in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. Significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the SVZ and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. Furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. These results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. If this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. Further characterization of the cells is underway.
PS3P-D020 Localization of neurotrophin receptors TrkA in PC12 cells: 3D reconstruction analysis of membrane proteins Tomoki Nishida 1 , Hiroshi Jinnai 2 , Tatuo Arii 3 , Akio Takaoka 4 , Ryoichi Yoshimura 1 , Yasuhisa Endo 1 1 Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan; 2 Department of Polymer Science and Engineering, Kyoto Institute of Technology, Kyoto, Japan; 3 National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan; 4 Osaka University, Mihogaoka, Ibaraki, Osaka, Japan It was previously reported that TrkA (NGF receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. We performed immunocytochemistry of TrkA and caveolin-1 in PC12 cells, analyzed by high-voltage electron microscopy, and reconstructed 3D structure of their subcellular distribution by IMOD. Our results indicated that localization of caveolin-1, known as an integral membrane protein of caveolae, was never found in the invagination structure in PC12 cells, but TrkA and caveolin-1 immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix.
Kensuke Shiomi, Kazuko Keino-Masu, Masayuki Masu Department of Molecular Neurobiology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
The Wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. Previously we identified Ccd1, a third-type of the DIX domain-possessing protein, as a positive regulator of the Wnt/-catenin pathway. Ccd1 mRNA was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of Ccd1 in the Wnt-mediated neuronal development. There are three subtypes of mouse Ccd1 gene products, Ccd1A, Ccd1B and Ccd1C, which are generated by different promotor usage. Mouse Ccd1A as well as zebrafish Ccd2A has a calponin homology domain which can mediate the interaction with the actin cytoskelton. We found that in the Ccdtransfected Hela cells, only the type A Ccd proteins co-localized with the actin filament. In order to examine the function of the type A Ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture.
Research funds: KAKENHI (15770137, 17300098) PS3P-D022 Analysis of a role of R-spondin2 on proliferation of the cortical neuroepithelium Yumiko Hatanaka 1 , Masahiro Yamaguchi 2 , Fujio Murakami 3 , Masayuki Masu 1 1 Grad. School of Comprehensive Human Sci., Univ. of Tsukuba, Japan; 2 Grad. School of Med., Univ. of Tokyo; 3 Grad. School of Frontier Biosci., Osaka Univ R-spondin2 (Rspo2) is a secreted activator of Wnt/-catenin signaling (Kazanskaya et al. 2004) . Rspo2 is expressed in the developing medial cerebral wall and transgenic mice expressing Rspo2 in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (Hatanaka et al. 2005) . Since Wnt3a has a role for expansion of caudomedial cortical progenitor cells (Lee et al. 2000) , these findings lead us to the idea that Rspo2 may synergistically promote proliferation of cortical neuroepithlial cells together with Wnt3a. To clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a -catenin/TCF reporter gene into these cells of embryonic day 11.5 mouse. An application of Wnt3a on these cells increased level of the reporter expression, and an addition of Rspo2 further increased its level. We are now monitoring incorporation of BrdU in neuroepithelial cells to know whether Wnt3a and Rspo2 directly promote their proliferation.
Tae Sun Kim, Hideki Hida, Tomoko Narita, Sachiyo Misumi, Hitoo Nisino Department of Neurophysiology & Brain Science, Nagoya City University Graduate School Medical Sciences, Nagoya, Japan
To investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (DA)-depleted striatum increase the number of DA neurons from ES-derived neural progenitor cells (NPCs), NPCs were treated with cytokine cocktail (IL-1, IL-11, LIF, GDNF) or lowered O2 (3.5%), followed by tyrosine hydroxylase (TH) immunostaining. Low oxygen increased total number of TH (+) cells (1.86-fold) as compared to normal O2. Cytokine cocktail significantly increased TH (+) cells (2.11-fold) compared to nontreated control. Treatment of LIF and IL-1 to NPCs exhibited major contribution in the effect of cytokine cocktail. Data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in DA-depleted striatum cause in the increase of DAergic neurons from ES-derived NPCs.
PS3P-D024 Structural basis for reelin signaling: Determination of receptor-binding site and its three-dimensional structure Norihisa Yasui 1 , Terukazu Nogi 1 , Mitsuharu Hattori 2 , Kenji Iwasaki 1,3 , Junichi Takagi 1 1 Research Center for Structural and Functional Proteomics, Inst for Protein Res., Osaka Univ., Suita, Japan; 2 Dept. of Biomed. Sci., Grad. Sch. of Pharm. Sci., Nagoya City Univ., Nagoya, Japan; 3 Core Research for Evolution and Technology (CREST) A large secreted glycoprotein reelin acts on target neurons through its receptors (ApoER2 and VLDLR), resulting in tyrosine phosphorylation of Dab1. In the present study, we have carried out structural and functional studies on the reelin signaling. First, we determined the structure of a single reelin repeat by X-ray crystallography. It had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. Moreover, electron micrographic 3D reconstruction of four-domain reelin fragment (i.e. R3-6) revealed an elongated rod-like structure. Next we determined minimum active unit within reelin. A fragment containing both the fifth and sixth reelin repeats (R5-6) was capable of binding to the receptor (ApoER2), and was also able to induce tyrosine phospholylation of Dab1 in primary neuronal culture.
PS3P-D025 Effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells Embryonic stem (ES) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by Neural Stem Sphere (NSS) method. Cultured with astrocyte-derived factor, colonies of ES cells give rise to NSSs. We analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. Quantitative real-time RT-PCR analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. These results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. We have previously established ES cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (NS/PCs) with the identity of early caudal neural tube. Taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (CMSC) has the activity to support the formation of neurospheres. This activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of NS/PCs. Moreover, RT-PCR analysis of regional identities of the CMSC treated neurospheres revealed elevated expression of pax3 and pax7 compared with those of untreated neurospheres, indicating that CMSC promotes dorsalization of NS/PCs or selective proliferation of dorsal NS/PCs. Elucidation of underlying mechanisms may provide important tools to derive early NS/PCs which can generate variety of projection neurons and be applicable to regenerative medicine.
Research funds: SORST JST PS3P-D027 Neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells Neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (J Neurosci Res 79:287, 2005) . Most neurotrophic factors are involved in neural cell proliferation and/or differentiation. However, the role of Neudesin in neural development remains to be elucidated. Neudesin mRNA was expressed in the neural precursor cells before the appearance of neurons. Therefore, roles of Neudesin in neural development were examined using the neural precursor cells. Neudesin significantly promoted neuronal differentiation. In addition, Neudesin transiently promoted neural cell proliferation early in the developmental process. The differentiation was mediated though activation of the PKA and PI-3K pathways. In contrast, the proliferation was mediated through the MAPK and PKA pathways. The expression profile and activity indicate that Neudesin plays unique roles in neural development.
PS3P-D028 FABP7 is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus Motoko Maekawa 1 , Miho Matsumata 2,3 , Yuji Owada 2 , Shigeki Yuasa 1 , Noriko Osumi 2,3 1 Natl. Inst. of Neurosci., NCNP, Tokyo, Japan; 2 Tohoku Univ. Sch. of Med., Sendai, Japan; 3 CREST, JST Pax6 transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. We have previously shown that Pax6 is necessary for keeping neural stem/progenitor cells in the hippocampus. In this study we have focused on a fatty-acid binding protein FABP7, a downstream of Pax6, regulating maintenance of embryonic neural stem/progenitor cells (Arai et al., 2005) . FABP7 was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (DG). 56% of FABP7-expressing cells co-expressed GFAP (a marker for early progenitors), and 33% of them co-expressed PSA-NCAM (a marker for late progenitors). FABP7 expression was also overlapped with Pax6, and expression of FABP7 was down-regulated in the DG of Pax6 deficient rats and mice. Finally, BrdU-labeling analysis revealed decreased cell proliferation in the DG of FABP7 knockout mice. Taking all together, it is concluded that FABP7 is required for maintenance of neural stem/progenitor cells in DG.
PS3P-D029 Involvement of the PSA-NCAM expressing cells in early development of the vascular system of the forebrain Momoko Miyakawa, Tatsunori Seki Department of Anatomy, Juntendo University School of Medicine, Tokyo, Japan Early development of the vascular system of the forebrain were studied in the chick embryo. Staining of vascular endothelial cells by FITCtomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day 4-7. Sections were examined under a confocal laser scanning microscope. Capillaries were found in the lateral pallium and seemed to grow from PSA-NCAM-positive outer zone to negative inner zone of the pallium. PSA-NCAM is thought to be expressed in the immature neurons. The rims of capillaries were immunoreactive with PSA-NCAM in both zones. Immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. By immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with PSA-NCAM. PSA-NCAM expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries.
Masaharu Kotani 1,6 , Shiki Okamoto 2 , Masato Imada 3 , Kouichi Itoh 4 , Atsushi Irie 5 , Hitoshi Sakuraba 6 , Hideo Kubo 7 1 Department of molecular biologu, Ohu Univ., Koriyama, Japan; 2 Dept. Deve. Physiol., Natl. Inst. Physiol. Sci., Okazaki, Japan; 3 Dept. Anatomy, Nihon Univ. Shl. Med., Tokyo, Japan; 4 Dept. Mol. Pharma., Univ. Tokushima Bunri, Sanuki, Japan; 5 Dept. Biochem. Cell Res., Tokyo Metro. Inst. Med. Sci., Tokyo, Japan; 6 Department of Clin. Genet, Tokyo Metro. Inst. Med. Sci., Tokyo, Japan; 7 Dept. Med. Biol, Tokyo Metro. Inst. Med. Sci., Tokyo, Japan
As RANDAM-2 shows the highest expression level with the proliferating stage of neural stem cells (NSCs), it is thought that the isolation of NSCs based on the expression level of RANDAM-2 is possible. In the present, we show that the isolated RANDAM-2 high+ cells enrich NSCs. The RANDAM-2 high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. In contrast, almost all of the RANDAM-2 low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. The results demonstrate that RANDAM-2 is a usefule marker for the isolation of NSCs by FACS.
Yasuharu Takamori 1 , Yasuhisa Tamura 1,3 , Yosky Kataoka 1,2 , Yilong Cui 1,3 , Hisao Yamada 1 1 Department of Anatomy and Cell Science, Kansai Medical University, Osaka, Japan; 2 Department of physiology, Osaka City University Graduate School of Medicine, Osaka, Japan; 3 Morecular Imaging Reserch Program, RIKEN FRS, Saitama, Japan Lamins are major structural proteins of nuclear envelope. Three lamin subtypes, A/C, B1 and B2 are mainly present in mammalian somatic cells. To investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. GFAP-positive primary progenitor cells possess lamin A/C (++), B1 (++), B2 (++), PSA-NCAM-positive subsequent progenitor cells possess lamin A/C (−), B1 (+++), B2 (+), and mature neurons possess lamin A/C (++), B1 (+), B2 (+++), in both neurogenic regions. These observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis.
Yusuke Tozuka 1 , Yuichi Tanaka 1 , Tatsuhiro Hisatsune 1 1 Department of Integrated Biosciences, University of Tokyo, Chiba, Japan Recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. It has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. In the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received GABAergic inputs. The GABAergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. Although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. Interestingly, these progenitor cells also received excitatory GABAergic inputs. This GABAergic inputs inhibited their cell proliferations. From these results, we here propose that adult progenitor cells are a direct target of GABAergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations.
PS3P-E033 New migration pattern in the postnatal neurogenesis of the dentate gyrus Takashi Namba 1,2 , Hideo Namiki 2 , Tatsunori Seki 1 1 Dept. of Anat, Juntendo Univ. Sch. of Med., Tokyo, Japan; 2 Integrative Biosci. and Biomed. Eng, Sch. of Sci. and Eng, Waseda Univ., Tokyo, Japan
In the hippocampus, granule cells continue to be generated from embryonic to adult stages. The early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. Previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (GCL). However, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the GCL. To observe them, we developed a time-lapse imaging system. Retrovirus-GFP was injected into the rat hippocampus at P5. Three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. The present data show that neuroblasts migrate from the hilus to the GCL, changing the direction of their movement. This is inconsistent with the previous report suggesting simple radial migration (Rickmann, et al., 1987) . The dividing pattern is currently under investigation.
Akiya Watakabe 1 , Noritaka Ichinohe 2 , Sonoko Ohsawa 1 , Tsutomu Hashikawa 3 , Kathleen S. Rockland 2 , Tetsuo Yamamori 1 1 Div. of Brain Biol, NIBB, Okazaki, Japan; 2 Lab. for Cortical Organization and Systematics, BSI, RIKEN, Wako, Japan, 3 Lab. for Neural Architecture, BSI, RIKEN, Wako, Japan By using gene expression profiles, we have tried to classify layer 6 neurons in several areas of monkey neocortex. We previously reported that Nurr1, CTGF and Sema3E mRNAs are specifically expressed in subsets of layer 6 neurons. We further show here that cholecystokinin (CCK) mRNA is expressed in a subset of excitatory neurons in layer 6. By double ISH, layer 6 neurons in monkeys are roughly divisible into CCK(+) and Sema3E(+) subgroups. Each subgroup was further subdivided by other markers. Tracer experiments showed that CCK and Sema3E mRNA expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. From this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. The implication of our findings will be discussed in terms of constancy of laminar structure across areas and species.
Research funds: KAKENHI 17024055 PS3P-E035 RBP-J regulates the cortical laminar formation Kenji Tanigaki 1 , Kazue Muraki 1 , Norio Yamamoto 2 , Tasuku Honjo 2 1 Shiga Medical Center, Research Institute, Shiga, Japan; 2 Department of Medical Chemistry, Kyoto University, Kyoto, Japan Precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. To examine involvement of Notch-RBP-J signaling in the cortex laminar formation, we deleted RBP-J from neural progenitors in anatomically restricted areas by in vivo electroporation of Cre-expressing plasmids. Such studies revealed that RBP-J deficiency caused transformation of glutamatergic pyramidal neurons in layer II/III to layer IV neurons with concomitant loss of astrocytes. The loss of RBP-J accelerated neuronal differentiation and changed their laminar fates. In addition, time-lapse studies indicated the migration defect of RBP-J-deficient neurons. The results showed that Notch-RBP-J signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors.
PS3P-E036 Search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles Ayano Kawaguchi 1 , Tomoko Ikawa 1 , Yuya Kasukawa 2 , Hironori Ueda 2,3 , Kazuki Kurimoto 4 , Michinori Saitou 4 , Fumio Matsuzaki 1,5 1 Lab. for asymmetric cell division, CDB, RIKEN, Kobe, Japan; 2 Functional Genomics Subunit, CDB, RIKEN, Kobe, Japan; 3 Lab. for systems biology, CDB, RIKEN, Kobe, Japan; 4 Lab. for mammalian germ cell biology, CDB, RIKEN, Kobe, Japan; 5 CREST, JST, Japan
In the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. To overcome this problem, we randomly picked individual VZ/SVZ cells of mouse embryos, and constructed cDNAs from each of them by global PCR amplification method. We could classify these "single cell derived cDNAs" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. 30 samples that showed typical marker gene expression pattern of the groups were applied for GeneChip analysis. The obtained data were confirmed by quantitative PCR and in situ hybridization. By this strategy, we identified nine genes that were specifically expressed in the SVZ progenitor cells.
Research funds: KAKENHI (15700264)
Ryosuke Tatsuno 1 , Tomoaki Sai 2,3 , Masahiro Otsu 2 , Kuniko Akama 1 , Takashi Nakayama 4 , Tosifusa Toda 5 1 Grad. Sch. of Sci. and Tech., Chiba Univ., Chiba, Japan; 2 Lab. Regener Neurosci., Tokyo Metropol. Univ. Fac. Health Sci., Tokyo, Japan; 3 Dept. Orthop. Surg., Jikei Univ. Sch. Med., Tokyo. Japan; 4 Dept. Biochem., Yokohama City Univ. Sch. Med., Yokohama, Japan; 5 Proteomics Collab. Res., Tokyo Metropol. Inst. of Gerontol., Tokyo, Japan Embryonic stem (ES) cells possess pluripotency and self-renewal. However, the proteomic analysis of neural stem cells and neurons differentiated in vitro from ES cells has not so proceeded yet. We investigated the expression levels of proteins during in vitro differentiation of mouse ES cells into neurons via neural stem cells by Neural Stem Sphere (NSS) method, using 2-D gel electrophoresis and MALDI-TOF MS. We identified vimentin, creatine kinase, ATP synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in ES cells and neurons. These results suggest that the neural stem cells have characteristic protein expression profile.
PS3P-E038 Identification of SE90, a novel gene expressed in the nural progenitor cells Shin-ichi Sakakibara, Kazuhiko Nakadate, Shiichi Ueda Department of Histology and Neurobiology, Dokkyo University School of Medicine, Tochigi, Japan Identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. We compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. A subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. Among two thousand of the differentially expressed subtracted clones, we identified 150 genes that significantly upregulated in neural stem cell culture. These included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. In situ hybridization and the developmental northern analysis demonstrated that these mRNAs were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. We further analyzed the expression pattern of the novel gene SE90 in developing and matured CNS. Teiichi Furuichi 1 , Akira Sto 1,2 , Yukiko Sekine 1 , Noriyuki Morita 1 , Tetsushi Sadakata 1 , Satoshi Shoji 1 , Jin-hong Huang 1 , Toshio Kojima 2 1 Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Japan; 2 Comparative Systems Biology Team, RIKEN Genome Sciences Center, Yokohama 230-0045, Japan
Mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. We have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, RT-PCR, GeneChip, cDNA microarray, and in situ hybridization. We have informatively systematized all the profiles in an online neuroinformatics database CDT-DB (http://www.cdtdb.brain.riken.jp) with various search functions. We have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. Further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development.
Miwako Ozaki 1 , Makoto Mizuno 2 , Kazuhisa Sakai 4 , Yoshimoto Kiyohara 4 , Kazuhiko Yamaguchi 3 , Tsutomu Hashikawa 4 , Hiroyuki Nawa 2 1 Institute of Biomedical Engineering, Waseda University, Tokyo, Japan; 2 Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan; 3 Laboratory for Memory and Learning, BSI, RIKEN, Saitama, Japan; 4 Laboratory for Neural Architecture, BSI, RIKEN, Saitama, Japan Neuregulin (NRG), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. In order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared Ig-NRG1 knock out mice (NRG1 type I and IV were disrupted). The mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. In addition, the number of vesicles in presynaptic neurons decreased in their synapses. The study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors.
PS3P-E041 PSD-95 protein expression in rat oromaxillofacial motoneurons during postnatal development Kohji Ishihama 1,2 , Satoshi Wakisaka 1 , Shiho Honma 1 , Akira Ito 1,2 , Kei Azuma 1,2 , Mikihiko Kogo 1 1 Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, Osaka, Japan; 2 First Department of Oral and Maxillofacial Surgery, Osaka University Graduate School of Dentistry, Osaka, Japan Postsynaptic density (PSD), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. PSD-95 implicates in formation and maturation of excitatory synapses. PSD-95 regulates the localization of the NMDA receptor by means of binding with NR2. Rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that NMDA receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. Here we examined the temporal distributions of PSD-95 protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. There was early emergence of PSD-95 expression in the interneurons located at medial of the trigeminal motor nucleus.
Masami Miura, Masao Masuda, Toshihiko Aosaki Neural Circuits Dynamics Research Group, Tokyo Metropolitan Institute of Gerontology, Japan
The striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. Recent studies suggest that striatal interneurons play an important role in processing of cortical input. We investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. We found that fast-spiking (FS) interneurons sent GABAergic inhibitory input to cholinergic interneurons, which were GABA A receptor-mediated and suppressed by GABA B receptor agonist SKF97541. In turn, cholinergic interneurons sent cholinergic excitatory input to FS interneurons. Because the excitatory postsypnatic potentials (PSPs) were blocked by hexamethonium and dihydro--erythroidine, the PSPs were nicotinic acetylcholine receptor-mediated. These results suggest that GABAergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits.
PS3P-E043 Ocular following responses (OFRs) to a brief background motin are modulated in relation to preparation for upcoming pursuit Hiromitsu Tabata, Kenichiro Miura, Kenji Kawano Dept. Integ Brain Sci., Grad. Schl of Med., Kyoto Univ., Kyoto, Japan
Recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (SPEMs) rather than preparing for saccades or stationary fixation. Here, we report that the increase in ocular responses based on the anticipation of SPEMs was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. The result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. In other words, the anticipation of upcoming SPEMs could affect the generation of OFRs. Furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed SPEMs toward one direction, implying that the prediction of the upcoming SPEM direction might cause the directional asymmetry of the visuomotor transmission gain.
PS3P-E044 Comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging Toshinobu Shimoi 1 , Kenji Mizutani 2 , Hiroto Ogawa 3 , Kohji Hotta 1 , Kotaro Oka 1 1 Ctr. for Biosci. and Info, Keio Univ., Yokohama, Japan; 2 Neuro, Karolinska Inst, Stockholm, Sweden; 3 Bio, Saitama Med. Sch., Saitama, Japan
In this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (CPG) in the earthworm by calcium imaging as multiple recording. The candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. We obtained the responses of up to 50 cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion (25% or less for all neurons on the ventral surface). We analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. As a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. These results were great progress to identify motor neurons related with locomotory CPG in the earthworm.
PS3P-E045 Three dimensional (3D) pursuit eye movement signals in cerebellar dorsal vermis Takuya Nitta, Teppei Akao, Sergei Kurkin, Kikuro Fukushima Department of Physiology, Hokkaido University School of Medicine, Sapporo, Japan For pursuit of a target moving in 3D space, signals for frontal and vergence-pursuit must be synthesized. Studies in our laboratory have demonstrated that 3D pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. However, the majority of floccular Purkinje (P-) cells discharged after onset of vergence-pursuit. Cerebellar dorsal vermis is another cerebellar area for frontal pursuit. To examine whether 3D pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit P-cells in 3 monkeys. Of a total of 77 P-cells that were examined during both frontal and vergence-pursuit, 46% discharged for both, 40% only for vergence, and 14% only for frontal pursuit. These results indicate that most of vermal pursuit P-cells discharged for vergence and that about half of them had 3D pursuit signals. Majority (70%) of these P-cells discharged before onset of vergence eye movements with the typical lead time of 50 ms, suggesting their involvement in the initiation of vergence-pursuit.
Research funds: KAKENHI (17022001) PS3P-E046 Information processing in FEF-rNRTP pathway for smooth pursuit Seiji Ono, Michael J. Mustari Division of Sensory-Motor Systems, Yerkes National Primate Research Center, Emory University, Atlanta GA, USA The frontal eye field (FEF) cortex is known to play a role in smooth pursuit (SP). This role is supported by FEF projections to the rostral nucleus reticularis tegmenti pontis (rNRTP) which projects heavily to the vermis. Using multiple linear-regression modeling, we have shown that SP neurons in rNRTP were biased towards eye acceleration. However, the functional characteristics of SP related FEF neurons that project to rNRTP have never been described. Therefore, we used micro-electrical stimulation to deliver single pulses in rNRTP to antidromically activate FEF neurons. The majority of SP related FEF neurons that we identified as projecting to rNRTP were most sensitive to eye acceleration and much less sensitive to eye velocity. The neurons in FEF-rNRTP pathway carry signals that could play a primary role in SP initiation. Our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum.
Research funds: NIH Grants EY13308, RR00165
Aya Takemura 1 , Yumi Murata 1,3 , Kenji Kawano 1,2 1 Neurosci. Res. Insti, AIST, Tsukuba, Japan; 2 Dept. Integ Brain Sci., Grad. Sch. Med., Kyoto Univ., Japan; 3 Grad. Sch. Compreh Hum Sci., Univ. Tsukuba, Japan
Previous studies in monkeys suggest that the medial superior temporal (MST) area is involved in visual motion processing. To understand the role of the MST in optokinetic nystagmus (OKN) and afternystagmus (OKAN), we examined the effects of bilateral chemical lesions in the MST in two monkeys. When each monkey was injected with ibotenic acid (15 mg/ml, 36-37 l total), the initial rapid rise in OKN was reduced. Consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). By contrast, the steady state OKN was not affected and the OKAN persisted. The initial amplitude and falling time constant of the OKAN increased. The results suggest that the MST is part of the direct pathway for the initial rapid rise in the OKN, but is not involved in the velocity storage mechanism for the steady state OKN and OKAN. Smooth pursuit is performed by coordination of eye and head movements. We have reported that the majority of FEF pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. To examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at 20 • /s with random intervals. The majority of pursuit neurons discharged before the onset of head movements with the mean lead time of 58 ms. Discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. These results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. Rather, our results suggest that the main origin reflects pursuit commands.
PS3P-E049 The local feedback loop of the saccadic system: An analysis of the eye movements induced by PDB stimulation Rikako Kato Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki, Japan Saccadic amplitude are controlled by a comparator that calculates dynamic motor error. Some models place the comparator in the superior colliculus while others assign this role to the reticular formation. To decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. We stimulated collicular axons descending in the PDB. Our data demonstrate that electrical stimulation of the PDB evokes saccades and they always terminate before the end of the stimulus train. The characteristics of evoked saccades are comparable to those spontaneously generated by the cat. Our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus.
Katsuo Fujiwara 1 , Kenji Kunita 2 , Kaoru Maeda 1 , Takeo Kiyota 1 1 Department of Human Movement and Health, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan; 2 Institute for Health and Sport Sciences, Osaka City University, Osaka, Japan
We investigated changes in visual evoked potential (VEP) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. The subjects were 17 undergraduate students. A shutter goggle was used as a VEP stimulator which was opened periodically for 30 ms with 800-ms intervals. The oscillation trial (0.5-Hz frequency and 2.5-cm amplitude) (65-75 s) was repeated 5 times. Postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. The mean speed decreased as trial was repeated, and reached a plateau before the 5th trial. A significant correlation was shown between 5th-1st trial differences in mean speed and VEP amplitude (r = 0.79). This indicates that the role of visual information is different among subjects with various adaptation processes of postural control.
PS3P-E051 Primary motor cortex contributes to generating manual following response Toshitaka Kimura 1 , Naoki Saijo 1 , Hiroaki Gomi 1,2 1 NTT CS Labs, Kanagawa, Japan; 2 ERATO Shimojo Implicit Brain Function Proj, JST, Saitama, Japan A large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (MFR). The MFR exhibits similar features to the ocular following response (OFR) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. This suggests that computational mechanism is shared for both responses. However, the neural basis of the MFR motor command generation remains unclear, while OFR is known to be generated subcortically. Here we show, by using transcranial magnetic (TMS) and electrical (TES) stimulation over the primary motor cortex (M1), that (1) an EMG response evoked by TMS was facilitated during MFR, while that by TES was not, and (2) intracortical inhibition within M1 assessed by paired-pulses TMS was reduced during MFR. These results suggest that MFR is generated through activity of interneuronal networks within M1. Such cortical mechanisms for MFR generation are distinct from the subcortical processes for OFR generation.
Naoki Saijo 1 , Hiroaki Gomi 1,2 1 NTT CS Labs., Kanagawa, Japan; 2 ERATO Shimojo Implicit Brain Function Proj, JST, Saitama, Japan When a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. However, the computational mechanism to generate quick adjustment is still unclear. Here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. We observed the hand responses to the target shifts in 8 radial directions applied during reaching. The data show that the direction of the initial phase (150-200 ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase (250-300 ms) was little biased. Additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. These results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency.
PS3P-E053 Spatial relationship between gaze and reaching-target modulates manual following response Naotoshi Abekawa 1 , Hiroaki Gomi 1,2 1 NTT CS Labs., Kanagawa, Japan; 2 ERATO Shimojo Implicit Brain Function Proj, JST, Saitama, Japan
To explore the functional mechanism of the manual following response (MFR) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. On a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. In the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. The checker pattern suddenly moved either rightward or leftward brief after reaching start. In the second condition, subjects did the same task with watching the lower marker. In both conditions, the MFR amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the MFR amplitude. This suggests that the spatial relationship between gaze and target is important in modulating MFR.
Misako Komatsu, Eizo Miyashita Dept. Compu. Intelligence & Systems Sci., Tokyo Tech., Yokohama, Japan When a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. Moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. The result was consistent even when the position of the fixation point was changed. The above tendency was considered to occur in eye-or gaze-centered coordinates. It is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? To answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. The subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. All the results were consistent with the previous ones regardless of the position of monitor or the fixation point. These results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position.
PS3P-F055 Influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot Tetsuro Muraoka 1 , Takashi Obu 2 , Kazuyuki Kanosue 3 1 ASMeW, Waseda University, Saitama, Japan; 2 Graduate School of Human Sciences, Waseda University, Saitama, Japan; 3 Faculty of Sports Sciences, Waseda University, Saitama, Japan
The aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. The subjects (n = 6) were supine, and their hand was prone. They performed cyclical flexion-extension coordinations of the hand and foot in the iso-(ISO) or opposite-(OPPO) directions, and those with an elastic load against wrist flexion (EL-ISO and EL-OPPO) at 1.5, 1.75, and 2.0 Hz. Over 99% success rate was observed in all tasks except OPPO (78-97%). The in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except OPPO. It was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot.
Research funds: the Special Coordination Funds for Promoting Science and Technology, MEXT, Japan PS3P-F056 Simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot Takashi Obu 1 , Tetsuro Muraoka 3 , Kazuyuki Kanosue 1,2,3 1 Faculty of Human Sciences, Waseda University, Saitama, Japan; 2 Faculty of Sport Sciences, Waseda University, Saitama, Japan; 3 ASMeW, Waseda University, Saitama, Japan
In human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). The purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. Eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at 1, 1.33, and 1.66 Hz. They were asked to perform 4 tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. All tasks were performed successfully. Muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. It may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement.
PS3P-F057 Activities of erector spinae muscles during jaw clenching in man Kayoko Yasunaga 1,2 , Tadachika Yabushita 1 , Kazuo Toda 2 , Kunimichi Soma 1 1 Orthodontic Science, Tokyo Med. & Dent. Univ., Tokyo, Japan; 2 Div. Integrative Sensory Physiology, Nagasaki Univ., Nagasaki, Japan
Recent studies focused the functional relationships between the masticatory and the posture system. The hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. Therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. Bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. As a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. Our results suggested that there were some relationship between spinal muscle activities and jaw clenching.
The effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-Naomi Wada 1 , Sachiko Motoyama 1 , Futoshi Mori 1 , Shigemi Mori 2 1 Department of Veterinary Physiology, Yamaguchi University, Yamaguchi, Japan; 2 National Institute for Physiological Science, Okazaki, Japan
The one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. Many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. The purpose of our experiments is to show the influences of bipedal habits on central nervous systems. We have established the bipedal walking model using rats (RBM) by amputation of forelimbs and training of upright posture and bipedal walking. After training of upright posture and bipedal walking for 12-20 weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. In the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. The results of out experiments showed that bipedal habits inhibit the spinal reflex pathways.
PS3P-F059 Neuronal activity in primary motor cortex during quadrupedal locomotion of the Japanese monkey Katsumi Nakajima 1 , Futoshi Mori 2 , Akira Murata 1 , Masahiko Inase 1 1 Dept. of Physiol., Kinki Univ. Schl. of Med., Osakasayama, Japan; 2 Dept. of Vet. Physiol., Facult. of Agr., Yamaguchi Univ., Yamaguchi, Japan
To elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in M1 of the monkey walking quadrupedally on the treadmill. Tungsten microelectrodes were inserted into M1 hindlimb region using a custom-made micromanipulator. We found that all neurons recorded in M1 modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. The neuron exhibiting phasic modulation peaked once or twice per step. The peak activity occurred at widely different times during the step cycle in different recorded neurons. As the treadmill speed increased, most of recorded neurons increased their discharge frequency. All these results suggest that M1 output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates.
Research funds: KAKENHI (16500271) PS3P-F060 Activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys Sayuki Takara 1,2 , Nobuhiko Hatanaka 1,2 , Masahiko Takada 3 , Atsushi Nambu 1,2 1 School of Life Science, The Graduate University for Advanced Studies, Japan; 2 Division of System Neurophysiology, National Institute for Physiological Sciences, Japan; 3 Tokyo Metropolitan Institute for Neuroscience, Japan
The putaminal (Put) neurons receive motor cortical inputs and change their activity in relation to movements. To investigate how these inputs contribute to Put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified Put neurons during the performance of a memory-guided reaching task. Based on orthodromic spikes evoked by cortical stimulation, individual Put neurons were defined in terms of whether they receive input from the primary motor cortex (MI), the supplementary motor area (SMA), or both. The results showed that MI-recipient neuron activity was responsive to the movement, while SMA-recipient neuron activity was responsive to the cue stimuli and/or the delay period. The activity of neurons receiving convergent inputs was related to both the movement and the delay period. We previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, RM, and raphe pallidus, RP) induced suppression or facilitation on respiratory neural activities in cats and rats. In this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (VRGs) in the medulla and upper cervical inspiratory neurons (UCINs). Animals were anesthetized, immobilized and artificially ventilated. Stimulation of cerebral peduncle (CP) induced short latency facilitation and succeeding suppression on activities of UCINs. Stimulation of RM or CP evoked inhibitory postsynaptic potentials in the caudal VRGs. These results suggest that RM and cerebral cortex directly inhibit main respiratory output neurons in VRG.
Ken Muramatsu 1 , Sei-Ichi Sasaki 2 , Yuichiro Cho 1 , Kenji Sato 1 1 Anatomy and Physiological Science, Tokyo Medical and Dental University, Tokyo, Japan; 2 Department of Physiology, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan Distribution of average diameters of external anal sphincter (EAS) motoneurons and peripheral motor fibers were examined in cats. To identify EAS motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. EAS motoneurons were found in the Onuf's nucleus of S1 and S2 spinal levels. To examine size of peripheral motor fibers, ganglionectomy was performed onL7 -S3 spinal segments which contain afferent fibers of EAS muscles. After 3 weeks survival period, anal branches of the pudendal nerve was examined. Histograms of the Distribution of average diameters of cell body and motor fiber shows unimodal distri bution. Also, distribution of muscle spindles of EAS muscle were examined by serially sectioning the distal colon and staining with Mayer's haemotoxylin and eosin. No muscle spindles were found. These results suggest that EAS muscle is controlled without gamma loop.
Mariko Miura, Yoshiki Iwamoto, Kaoru Yoshida Neurophysiol., Univ. Tsukuba, Tsukuba, Japan Saccade accuracy is ensured by an adaptation mechanism. The speed and magnitude of adaptation vary greatly across experiments even for the same subject. One factor that might cause this variability is adaptation history. The present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. Gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. Adaptation experiments were repeated for 4 consecutive days. We compared adaptation in Day1 and that in Day4. The gain decrease for the first 700 saccades in Day 4 (0.140 ± 0.036) was larger than that in Day 1 (0.080 ± 0.021) (p = 0.026, n = 5, paired-t test). The rate of adaptation in Day 4 (1.739 ± 0.393 × 10 −4 /sac) was higher than that in Day 1 (1.120 ± 0.240 × 10 −4 /sac) (p = 0.011). The overall gain change (1800 saccades) in Day 4 (0.219 ± 0.030) was larger than that in Day 1 (0.112 ± 0.022) (p = 0.002). Thus, both the speed and magnitude of adaptation were increased by preceding adaptation. The present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation.
Research funds: KAKENHI (16300129) PS3P-F064 Asymmetry of the anticipatory convergence eye movement Haruo Toda, Takehiko Bando Div. Integr. Physiol., Grad. Sch. Med. Sci., Niigata Univ., Niigata, Japan Typically, convergence eye movement is known as symmetric adduction of the both eyes. But asymmetrical convergence also found in the natural condition. These asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. The lateral suprasylvian (LS) areas are extrastriate cortices which receive visual information from V1. The LS has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. Cats (n = 7) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of LED), preceding target movement by 4 s. After training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in 60% of trials. In three cats, we used training with obliquely approaching target. After training, asymmetrical anticipatory eye movements were observed. Based on these findings, related LS neuronal activities and results from lesion study, we will discuss the role of LS in asymmetry of anticipatory and visually-evoked convergence eye movement.
Yusuke Uchida 1 , Xiaofeng Lu 1,2 , Shogo Ohmae 1 , Toshimitsu Takahashi 1,2 , Shigeru Kitazawa 1,2 1 Dept. of Neurophysiol., Juntendo Univ. Grad. Sch. of Med., Tokyo, Japan; 2 CREST, JST, Tokyo, Japan
We examined reward related neural activity in the supplementary eye field (SEF). For this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of 16 targets that were arranged in a radial pattern. A target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. Reward was delivered during or after target-hold period. We found that many SEF cells became active during the period of reward delivery (R-cell). More than half of R-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (RD-cell). Interestingly, most of RD-cells displayed activity with the clear directional tuning. These results demonstrate reward dependent activity specific to spatial direction in the SEF, and further suggest that SEF cells provide reinforcement mechanism.
Research funds: KAKENHI 17022033 PS3P-F066 Frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity Hiromasa Kitazawa 1 , Soichi Nagao 1,2 1 Lab. for Motor Learning Control, RIKEN BSI, Saitama, Japan; 2 SORST, JST, Saitama, Japan
Smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. Smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. Role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (FPA) is not known. To reveal possible involvement of FPA in the adaptation of smooth pursuit, we identifying FPA by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. We found that inactivation of FPA not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated FPA, but also appreciably depressed its adaptation, suggesting that FPA is involved in the adaptation of smooth pursuit.
Shinji Matsutani Department of Functional Morphology, Kitasato University School of Nursing, Kanagawa, Japan Distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. The tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. As already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. Distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. In most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. It was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. While most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. The centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons.
PS3P-F068 Projection targets of the Drosophila taste receptor neurons in the primary gustatory center of the brain Takaaki Miyazaki 1,2 , Kei Ito 1,2,3 1 Dept. of Comput. Biol., Grad. Sch. of Frontier Sci., Univ. of Tokyo, Japan; 2 Center for Bioinform., IMCB, Univ. of Tokyo, Japan; 3 BIRD, JST, Japan
In order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. Drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. Gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (SOG) of the brain. To understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the GAL4 enhancer-trap strains that visualize specific neural fibers in the SOG and the labial nerve. Screening 4,000 strains, we identified about 180 candidate lines. The projection targets of the labeled neurons were classified into seven areas. The terminals of the already identified sensory neurons appear to fall into specific subsets of these areas.
Research funds: BIRD, JST PS3P-F069 Immunoreactivity and voltage-gated channels of mouse taste bud cells Kennji Kimura 1 , Yoshitaka Ohtubo 1 , Takashi Kumazawa 2 , Kiyonori Yoshii 1 1 Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu, Japan; 2 Department of Applied Chemistry, Saitama Institute of Technology, Fukaya, Japan Mammalian taste buds comprise four heterogeneous cell types, type I to IV, and their collaboration seems to generate taste sensation. We investigated the electrophysiological properties of these cell types except type IV with taste buds preserved in mouse lingual epithelia. Type I cells elicited smaller TTX-sensitive, TEA-sensitive, and TEAinsensitive currents in magnitude than other cell types. Type II cells elicited a smaller TEA-sensitive current and a larger TEA-insensitive current than type III cells. These results suggest that type II and III cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types.
Research funds: KAKENHI (16300094) and the 21st COE Program (center #19) granted by MEXT of Japan PS3P-F070 Inositol Monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of C. elegans Yoshinori Tanizawa 1 , Atsushi Kuhara 1 , Hitoshi Inada 1 , Eiji Kodama 1 , Takafumi Mizuno 1 , Ikue Mori 1,2 1 Lab. of Mol. Neurobiol., Nagoya Univ., Japan; 2 Institute for Advanced Research, Nagoya Univ., Japan Inositol monophosphatase (IMPase) is suggested to be relevant to bipolar disorder. Although Lithium is believed to exert therapeutic effect by inhibiting IMPase in patients, the mechanism underlying Lithium therapy is largely unknown. Here we show that the loss of IMPase causes defects in behavior and localization of synapses in C. elegans. Mutations in ttx-7 gene encoding IMPase exhibit defective thermotaxis behavior, which is attributable to the loss of IMPase activity in the most essential integrative interneuron RIA in the nervous system. The ttx-7 mutations also cause mislocalization of synaptic proteins in RIA. Both behavioral and synaptic defects in ttx-7 mutants were rescued by expression of IMPase at adult stage and Inositol application, and were mimicked by Lithium application in wild type animals. These results suggest that IMPase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly.
Research funds: KAKENHI PS3P-F071 Postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (OB) of rats H Ohmomo, F Shutoh, A. Ina, S. Yoshida, H. Nogami, S. Hisano Lab. Neuroendocr., Graduate Sch., Univ. Tsukuba, Tsukuba, Japan Olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. However, little is known about development of these OB glutamatergic neurons in early postnatal life. Vesicular glutamate transporters (VGLUT) have been used as the best histological markers to identify glutamatergic neurons. We here studied expressions of two VGLUT isoforms (VGLUT1 and -2) during rat OB development from postnatal day 1 (P1) to P10 by in situ hybridization and immunohistochemistry. At P1 VGLUT1 immunoreactivity (IR) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. VGLUT1 mRNA signals were detectable in the mitral cell layer from P1 to P10. In contrast, VGLUT2 IR was prominent in the glomerulus at all days examined, and only at P1 and P3 in mitral cells. Despite mitral VGLUT2 IR disappeared at P10, the mRNA signals were still detectable. These results suggest that glutametergic neurons in the rat OB continue to develop even after birth.
PS3P-F072 V1R genes multiplied in amphibian and expressed in the main olfactory system Atsuko Date-Ito 1,2 , Masumi Ichikawa 3 , Yuji Mori 2 , Kimiko Hagino-Yamagishi 1 1 Tokyo Metrop. Inst. Med. Sci., Tokyo, Japan; 2 The Univ. of Tokyo, Tokyo, Japan, 3 Tokyo Metrop. Inst. Neurosci., Tokyo, Japan
In rodent, V1R gene family is expressed specifically in the vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. However, teleost fishes lacking for the VNO have a single V1R gene, which is expressed in the olfactory epithelium (OE). To examine when the V1Rs function as pheromone receptors in the course of evolution, we analyzed the amphibian Xenopus tropicalis genome, and identified 23 V1R sequences. These V1Rs were not expressed in the VNO, but most of them were expressed in the OE of the middle cavity, which is considered for reception of water-soluble odorants. From these results, we speculate that the amphibian V1Rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. Our results raise the possibility that pheromonal information is transmitted via the main olfactory system.
PS3P-F073 Analyses of ligand binding sites and SNPs on sweet taste receptor system in human Noriatsu Shigemura, A.A. Islam, Yuki Nakamura, Shinya Shirosaki, Yuzo Ninomiya Sect. Oral Neurosci., Grad. Sch. Dent Science, Kyushu Univ., Japan
Recent studies have shown that T1r2/T1r3 heterodimer plays a role as a sweet taste receptor. But, mice lacking T1r3 showed diminished but not abolished behavioral and nerve responses to sugars, suggesting T1r3-independent sweetener binding site also exist in mice. In this study, to predict binding sites on T1r2/T1r3 and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. We also used Gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. The ten sweet compounds were classified into five groups [(1) sucrose, glcose, fructose, (2) saccharin, aspartame, acesulfame-K, glycine, (3) d-phenylalanine, (4) d-tryptophan, (5) l-proline]. In sequencing analysis, four and two SNPs with amino acid substitution were revealed in T1r2 and T1r3, respectively. These results suggest that there may be at least five binding sites in human sweet receptor system. The individual differences in sweet sensitivities may be due to these SNPs.
Keiko Yasumatsu 1 , Sachiko Saito 2 , Yuko Murata 3 , Ding Ming 4 , Tatsu Kobayakawa 5 , Robert F. Margolskee 4 , Yuzo Ninomiya 1 1 Sect. Oral Neurosci., Grad. Sch. Dent. Sci., Kyushu Univ., Fukuoka, Japan; 2 Saito Sachiko Taste and Smell Research Institution, Ibaraki, Japan; 3 National Res. Institute of Fisheries Sci., Kanagawa, Japan; 4 Dept. of Physiol. & Biophys., Mount Sinai Sch. Med., New York, USA; 5 National Institute of Advanced Industrial Science and Technology, Ibaraka, Japan
The effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. The results showed that DHA and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. We also found fatty-acid inhibition on bitter responses in an in vitro G-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in Ggustducin KO mice. These results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of T2R receptors which coupled with Ggustducin.
PS3P-F075 Newborn infant body odor attenuates their mother's postpartum moods Shota Nishitani 1 , Mayumi Kokuryo 1 , Tsunetake Miyamura 2 , Kazuyuki Shinohara 1 1 Div. Neurobiol. & Behav., Nagasaki University, Japan; 2 Obstet. & Gynecol. of Miyamura Hospital, Japan
Mothers are attracted to the body odor of newborn infants, but little is known about its reason. In the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. The body odors of newborn infants were collected from their undershirts. Postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. We used the POMS to assess the effects of infant body odors on postpartum moods. This study was approved by the ethics committee of Nagasaki University. The infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. These results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers.
Research funds: Japan Science and Technology Agency (JST), Research Institute of Science and Technology for Society (RISTEX) PS3P-F076 Human prefrontal activity in taste encoding: An fNIRS study Masako Okamoto 1 , Mari Matsunami 2 , Haruka Dan 1 , Tomoko Kohata 2 , Kaoru Kohyama 1 , Ippeita Dan 1 1 National Food Research Institute, Tsukuba, Japan; 2 Nippon Suisan Kaisha, Ltd., Japan Taste remains one of the least-explored human senses. Using multichannel functional near-infrared spectroscopy (fNIRS), we examined the lateral prefrontal cortex (LPFC) of healthy volunteers (N = 10) while they tasted and encoded the quaternary taste mixtures. The contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-LPFC and the right posterior portion of the LPFC. The activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of LPFC in intentional encoding, at least at a macro structural level. This study also demonstrates that, by using fNIRS, LPFC functions on taste can be examined with experimental paradigms comparable to those used for other senses. Recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. We sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (I-␣) and location of dipoles estimated from the potentials. Electroencephalographic dipole tracing identified the location of dipoles from the I-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. In this study, we compared the respiratory pattern during odor stimulations, I-␣, dipole localizations without habituation with those with habituation of odors. Onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. Habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active.
Akio Tsuboi, Takaaki Miyazaki, Takeshi Imai Dept. of Biophys. & Biochem., Univ. of Tokyo, Tokyo, Japan Vertebrate odorant receptor (OR) genes are divided phylogenetically into two distinct classes, the fish-like class I and the terrestrialspecific class II. In the present study, we systematically analyzed mouse class I OR genes (42 subfamilies) to elucidate the expression profiles in the olfactory epithelium (OE) and the projection sites of their olfactory sensory neurons (OSNs) in the olfactory bulb (OB). In situ hybridization (ISH) revealed that most class I OR genes (36 subfamilies) were expressed in the dorso-medial zone (zone 1) of the OE. Furthermore, there appeared to be no significant differences in the distributions of OSNs expressing class I genes within zone 1. These results indicate that there is a clear boundary between zone 1 and non-zone 1 areas in the OE. Some class I ORs are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. Our ISH analysis has revealed that OSNs expressing the class I ORs in zone 1 tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the OB.
Research funds: KAKENHI (16500196) PS3P-G079 Taste response characteristics of putative interneurons in the rat gustatory cortex Tatsuko Yokota, Kunihiro Eguchi, Katsunari Hiraba Department of Physiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan Previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. Taste-sensitive neurons in the rat gustatory cortex were classified according to (1) best-taste profiles and (2) spike-widths which were found to form a bimodal distribution (narrow and broad). Narrow-spike neurons had a significantly larger response to NaCl than broad-spike neurons, but no differences were found to other tastants. The proportion of narrow-spike neurons in the N-best neurons was higher than that in the H or NH-best neurons. These results indicate that putative interneurons may play an important role in the coding of salt taste information.
Research funds: KAKENHI (11671860) of Japan to T.Y.
Yuki Sato, Nobuhiko Miyasaka, Yoshihiro Yoshihara Laboratory for Neurobiology of Synapse, RIKEN BSI, Wako, Japan
In the fish olfactory system, individual olfactory sensory neurons (OSNs) are thought to express only one or at most a few different odorant receptors (ORs) from the large OR family consisting of ∼100 members. Here, we investigated the mechanisms underlying OR gene choice by using transgenic zebrafish that carried a modified BAC containing a zebrafish OR gene cluster. Replacement of the OR coding regions in the BAC transgene with reporter genes allowed the reporters to be expressed in a small population of OSNs in the transgenic fish. In situ hybridization analysis using OR-specific probes revealed that OR genes expressed in reporter-positive cells were mostly restricted within the same OR subfamilies to which the replaced ORs belonged. Additionally, the reporter-expressing OSNs projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. These findings suggest the hierarchical regulation of OR gene choice, whereby an individual OSN may express one OR gene from a limited subpopulation that is chosen from the entire repertoire in advance.
Research funds: KAKENHI (16300105) PS3P-G081 Identification of perisomatic-targeting granule cells in the mouse olfactory bulb Hiromi Naritsuka 1 , Kazuhisa Sakai 2 , Tsutomu Hashikawa 2 , Kensaku Mori 1 , Masahiro Yamaguchi 1 1 Dep. Physiol. Grad. Sch. Med., Univ. of Tokyo, Tokyo, Japan; 2 Laboratory for Neural Architecture, BSI, RIKEN, Saitama, Japan
In the olfactory bulb (OB), odor information is processed by the local circuit that includes inhibitory interneurons. Granule cells (GCs) are major interneurons in the OB, but their diversity is not well understood. In the OB of adult transgenic mice expressing GFP under the control of nestin gene regulatory regions, we observed GCs with strong GFP expression (referred to as type S cells). Their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical GCs make synapses with dendrites of mitral and tufted cells. Type S cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. Electron microscopic analysis revealed the existence of reciprocal synapses between type S cell protrusions and mitral cell somata. Characteristic morphology of perisomatic-targeting GCs indicates that they have functions distinct from typical GCs in the OB.
Keiko Moriya-Ito, Kentaroh Endoh, Yuuki Ishimatsu, Masumi Ichikawa Department of Neuroscience Basic Technology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan A coculture system of accessory olfactory bulb (AOB) neurons and vomeronasal neurons was established for studying the functional roles of AOB neurons in pheromonal signal processing. In this study, the effect of vomeronasal neurons on the development of AOB neurons was examined in a coculture system. The densities of dendritic spines were lower in the coculture than in single culture. The ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. The volume of spine head was larger in the coculture than in single culture. By electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. The synapses between AOB neurons and vomeronasal neurons were recognized in the coculture. These observations suggest that synapse formation of AOB neurons is modified by synaptic contact with vomeronasal neurons.
PS3P-G083 NaCl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells Ryusuke Yoshida, Tadahiro Ohkuri, Keiko Yasumatsu, Noriatsu Shigemura, Yuzo Ninomiya Sect. of Oral Neurosci., Grad. Sch. of Dental Sci., Kyushu Univ., Fukuoka, Japan
Previous electrophysiological studies showed that the chorda tympani nerve contains two types of NaCl-responsive fibers, amiloride sensitive (N-type) and insensitive (E-or H-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. In this study, we examined NaCl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. Some taste cells respond to apical restricted NaCl stimulation with increase in firing frequency and their responses were concentration dependent. Amiloride mixed with apical NaCl solution inhibited NaCl responses in some taste cells [amiloride sensitive (AS) cells] but not in others [amiloride insensitive (AI) cells]. AI cells responded to other electrolytes such as KCl and HCl. These results suggest the existence of at least two types of NaCl sensitive cells, AS and AI cells. N-or E-type fiber may selectively innervate AS or AI cells respectively.
Research funds: KAKENHI (15209061), KAKENHI (17791325) PS3P-G084 Integration of olfactory and oral sensory input in the rat insular cortex Hideki Kashiwadani, Kensaku Mori Department of Physiology, University of Tokyo, Tokyo, Japan Axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). However cellular mechanisms underlying the integration of multimodality are poorly understood yet. In this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. We found that more than 25% of recorded neurons in the insular cortex responded to odors. About half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. When odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. The integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation.
Research funds: KAKENHI (17023012) PS3P-G085 Odor combination selectivity of the rat piriform cortex neurons Ikue Yoshida, Kensaku Mori Dept. Physiol. Grad. Sch. Med., Univ. of Tokyo, Tokyo, Japan Olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. However, the manner of integration at the level of individual cortical neurons is not well understood yet. Using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (aPC) to 8 classes of odorous compounds, each class being present in odors from many different vegetables and fruits. Individual neurons typically responded to more than 2 classes of odorants. Each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. Single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. These results suggest that individual neurons in the aPC can be characterized by the odor combination selectivity and that the aPC neurons may integrate signals from different odorant classes.
Research funds: KAKENHI (13GS0007) PS3P-G086 Odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium Takahiro Ishikawa 1 , Takaaki Sato 2 , Akira Shimizu 1 , Ken-ichiro Tsutsui 1 , Toshio Iijima 1 1 Div. of Systems Neuroscience, Grad. Sch. of Life Sciences, Univ. of Tohoku, Sendai, Japan; 2 Res. Inst. for Cell Engineering, AIST, Amagasaki, Japan
To examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (APC), we analyzed odorinduced local field potential (LFP) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. In APC, odor-induced LFPs consisted of a phasic initial component followed by a fast oscillatory activity in the beta range (20 Hz). By comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. The beta oscillation might be generated by the repetition of these network activities. These electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations.
PS3P-G087 Chemotaxis of C. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant Lin Lin, Hiroyuki Oikawa, Miyako Sasaki, Tokumitsu Wakabayashi, Ryuzo Shingai Department of Welfare Engineering, Iwate University, Morioka, Japan
To investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of C. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. Lysine and chloride ions are water soluble chemoattractants. When 3 M lysine was spotted on ammonium chloride background, 0.003-0.01 M and 0.05 M background did not influence lysine chemotaxis, while 0.03 M background augmented and 0.07-0.1 M background suppressed the chemotaxis. In contrast, when 0.1 M ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. Interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa.
PS3P-G088 Glutamate receptors are regulated by the Ras-MAPK pathway in neural circuit-dependent odor adaptation in C. elegans Takaaki Hirotsu 1,2,3 , Takeshi Ishihara 1 , Eisuke Nishida 3 , Yuichi Iino 2 1 Dept. Biol., Fac. Sci., Kyushu Univ., Japan; 2 Mol. Genet. Res. Lab., Univ. of Tokyo, Japan; 3 Grad. Sch. Biostudies., Kyoto Univ., Japan C. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for 5 min. This plasticity, called early adaptation, requires AIY interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. The Ras-MAPK pathway is activated by odorant exposure in AIY and plays essential roles for early adaptation. The function of GLR-1, a non-NMDA type glutamate receptor, in AIY is also important for early adaptation. GLR-1 appears to localize at postsynaptic sites in AIY. This localization was changed by odorant exposure in early adaptation. Mutation of the Ras-MAPK pathway impaired localization of GLR-1. in vitro kinase analyses revealed the possibility that MAPK directly phosphorylates GLR-1. These results suggest that the Ras-MAPK pathway controls odor adaptation by directly regulating GLR-1 localization in AIY neurons.
Kohei Ueno 1 , Yoshiaki Kidokoro 2 1 Dept. Behav. Sci., Grad. Sch. Med., Gunma Univ., Maebashi, Japan; 2 Inst. Mol. Cel. Reg., Gunma Univ., Maebashi, Japan Sodium chloride (NaCl) is the major substance that induces NaCl taste. In rodents, some strains prefer NaCl solutions (∼1%), but others do not or even avoid them. Although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. In the 26th NS annual meeting, we have shown that NaCl preference in several wild-type strains of Drosophila melanogaster is variable and P-element insertion in a single gene suppressed NaCl preference. Here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (SNPs) in the gene. Moreover, we found that one of the SNPs was correlated with NaCl preference among wild-type strains. We generated transgenic flies and rescued the low preference phenotype of P-element insertion strain using the GAL4/UAS system. Finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. Taken together, we suggest that the gene is a novel NaCl receptor gene.
PS3P-G090 Spatial and temporal organization of odor representation by moth antennal lobe output neurons Shigehiro Namiki 1,2 1 Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan; 2 Department of Mechano-Informatics, Graduate School of Information Science and Technology, University of Tokyo, Tokyo, Japan
The antennal lobe (AL) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. Both systems have common structures called glomeruli, functional units of olfactory processing. Odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. But the details of olfactory coding mechanisms are still unclear. We confirmed that projection neurons (PNs, AL output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. By pooling data from many PNs that innervate identified glomeruli I reconstructed odor representations. I found that olfactory information is encoded by distributed spatiotemporal activity of a PN population and that there are no clear correlation between the similarity of slow temporal patterns of PNs and spatial distances of innervating glomeruli.
Research funds: BRAIN PS3P-G091 Medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties Makoto Yokosuka 1 , Yoshinori Sahara 2 , Shinichiro Horie 2 , Masumi Ichikawa 3 , Shun Nakamura 2 1 St. Marianna Univ. Schl. Med., Kawasaki, Japan; 2 Ntl. Inst. Neurosci., NCNP, Tokyo, Japan; 3 Tokyo Metropol. Inst. Neurosci., Tokyo, Japan
We characterize the electrophysiological and morphological properties of the medial nucleus amygdala (MEA) neurons using whole-cell recordings in mice slice preparations. Most MEA neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. Biocytin labeling showed that MEA neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. Norepinephrine increased the frequency of spontaneous IPSCs in some neurons, while serotonin increased spontaneous EPSCs in others. Morphologically and physiologically heterogeneous MEA neurons seem likely to produce multiplex outputs of many instinct behaviors.
Hideyuki Matsumoto, Kensaku Mori Department of Physiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Olfactory sensation sometimes lasts even after odorant stimulation has ceased. Neuronal mechanisms for the olfactory afterimage are not well understood yet. Single unit recordings from mitral/tufted cells in the mouse olfactory bulb (OB) showed that some neurons continued to discharge for more than 10 s even after the cessation of odorant stimulation. The induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. Spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. These results suggest that neuronal mechanism in the OB may be responsible for the induction of the post-stimulus sustained discharges. The respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. This implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells.
PS3P-G093 Synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb Yusuke Tsuno, Hideki Kashiwadani, Kensaku Mori Department of Physiology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan
Olfactory cortex (OC) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. Since the olfactory bulb (OB) receives the modulatory inputs heavily, neuronal activity in the OB might change in a state-dependent manner. In the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. Transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. In addition, we observed synchronous slow oscillations (about 1 Hz) in the granule cell layer of the OB, layer III of the OC, and the occipital cortex. Thus the OB shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex.
Research funds: KAKENHI (17023012) PS3P-G094 REM sleep deprivation decreases Na-K ATPase phosphorylation Gitanjali Das, Birendra N. Mallick School of Life Sciences, Jawaharlal Nehru University, New Delhi, India It has been hypothesized that "One of the functions of REM sleep is to maintain brain excitability" REM sleep deprivation increases noradrenaline in the brain that increases the Na-K ATPase activity causing increased brain excitability. However, the molecular mechanism of such increased Na-K ATPase activity was unknown; although it was known that dephosphorylated state is the active form of Na-K ATPase. Rats were REM sleep deprived by flower-pot method; large platform and recovery from lost REM sleep were carried out as controls. At the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for Western Blotting against phosphoserine and phosphothreonine antibodies as well as for Na-K ATPase activity. After REM sleep deprivation the activity increased, while the level of phosphorylated form of Na-K ATPase decreased in the same sample. This confirms our hypothesis that REM sleep deprivation induced increased activity is due to dephosphorylation of Na-K ATPase.
Research funds: ICMR (Govt. Of India) and UPOE (Govt Of India)
Takeshi Fujii 1,2 , Ken Yoshikawa 2 , Yuki Takatori 1 , Koichiro Kawashima 2 1 Dept. of Pharmacol., Fac. of Pharmaceut Sci., Doshisha Women's Coll., Japan; 2 Dept. of Pharmacol., Kyoritsu Univ. of Pharmacy, Japan Stimulation of muscarinic (mAChR) and nicotinic (nAChR) receptors with respective agonists induces Ca 2+ signals in T cells. In the present study, using RNA interference approach, we investigated roles of mAChR and nAChR subtypes in Ca 2+ signals in CCRF-CEM (CEM) cells, a human T cell line, as a model of T cells. CEM cells express M 1 , M 3 , M 4 and M 5 mAChR subtypes, and ␣3, ␣5, ␣6, ␣7, ␣9, ␣10 and 4 nAChR subunits. Transfection of anti-M 3 , anti-M 5 and anti-␣7 small interfering RNA (siRNA) significantly down-regulated respective mRNA expression, while no changes were observed in gene expression of other mAChR subtypes or nAChR subunits. Ca 2+ signals evoked by oxotremorine-M, a non-selective mAChR agonist, were reduced by anti-M 3 or anti-M 5 siRNA. Ca 2+ signals evoked by nicotine were reduced by anti-␣7 siRNA. These findings indicate that M 3 , M 5 mAChR and ␣7 nAChR subtypes play major roles in Ca 2+ signals to acetylcholine in T cells, and suggest that these receptors are involved in regulation of immune function.
Research funds: KAKENHI (16590060) PS3P-G101 Is "seronegative" MG explained by autoantibodies to MuSK?
Kazuhiro Shigemoto 1 , Sachiho Kubo 2 , Seiji Matsuda 3 , Naoki Maruyama 2 1 Dept. of Preventive Medicine, Ehime Univ. Schl. of Med., Ehime, Japan; 2 Dept. of Mol. Path., Tokyo Metro Inst. for Gerontology, Tokyo, Japan; 3 Dept. of Integrated Basic Medical Science, Ehime Univ. Schl. of Med., Ehime, Japan Muscle-specific kinase (MuSK) is critical for the synaptic clustering of nicotinic acetylcholine receptors (AChR). MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering at the postsynaptic membrane. Although autoantibodies against the ectodomain of MuSK have been found in a proportion of patients with generalized myasthenia gravis (MG), it is unclear whether MuSK autoantibodies are the causative agent of generalized MG. In the present study, rabbits immunized with MuSK ectodomain protein manifested MG-like muscle weakness with a reduction of AChR clustering at the NMJ. The autoantibodies activated MuSK and blocked AChR clustering induced by agrin or by mediators that do not activate MuSK. Thus, MuSK autoantibodies rigorously inhibit AChR clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of MG. (Shigemoto et al., J. Clinical Investigation, 2006) Research funds: KAKENHI (16590831) PS3P-G102 Dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration Shin-ichi Kyuhou, Hisae Gemba Department of Physiology, Kansai Medical University, Japan
In Purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day 50. Electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day 45. Analysis of spontaneous cortical field potentials by fast Fourier transform, revealed that high frequency oscillation (HFO) of around 25 Hz appeared prominently in the auditory cortex. Local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the HFO in the auditory cortex, indicating that the thalamus is involved in the generation of the HFO. The real time polymerase chain reaction analysis demonstrated the upregulation of the mRNA of NMDA receptors in the auditory cortex. These results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice.
Research funds: Grant C1 from Kansai Medical University PS3P-H103 Unusually folded SOD1 species sequester specific motor molecules and inhibit the axonal transport of their cargos Minako Tateno 1 , Yumiko Simazaki 1 , Fuminori Saitoh 1 , Ryosuke Takahashi 2 , Toshiyuki Araki 1 1 National Institute of Neuroscience (NCNP), Tokyo, Japan; 2 Dept. of Neurology, Kyoto University, Kyoto, Japan
Misfolding of mutant SOD1 protein is thought to be responsible for the selective loss of motoneurons in SOD1-related familial amyotrophic lateral sclerosis (ALS), although the molecular mechanisms underlying the toxicity of such unusually folded SOD1 species are not yet clarified. Since we have detected accumulation of unusual SOD1 species in motoneuronal axons from G93A SOD1-Tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual SOD1 species. Immunoprecipitation analyses revealed specific interaction of unusual SOD1 species with certain kinds of motor molecules. Moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant SOD1-Tg compared with WT SOD1-Tg mice. These data strongly suggest that the toxic property of unusual SOD1 proteins is partially ascribable to the transport inhibition of specific cargos.
Research funds: Grant-in-Aid for Scientific Research C (17590907) PS3P-H104 Relationship between the amount of the cathepsin D expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model Masahiro Shibata, Masato Koike, Yasuo Uchiyama Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Japan Mice deficient in cathepsin D (CD), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (NCL). Here we report that the phenotype of mice in which CD is partially expressed is decided depending on the amount of the protein expression of CD. The proteolytic activity and protein expression of CD in the mutant mice were approximately 30% of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day 24. The mice started to show NCL symptoms on P25, and their life span was prolonged for one to three days, compared to that of the CD-null mice. The protein expression of CD in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. These results indicate that a threshold of the CD expression required for the manifestation of NCL symptoms in the mice may be present in the range from 30% to 50% of that in the wild-type mice.
Research funds: KAKENHI (10343253) PS3P-H105 Neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels Mamoru Satoh, Atsuyoshi Shimada, Noriko Kawamura, Yoichi Chiba, Yuko Saitoh, Hiromi Keino, Masanori Hosokawa Dept. Pathol., Inst. Develop. Res., Aichi Human Service Center, Aichi, Japan
We previously reported that expanded polyglutamine (polyQ) tracts induced cellular toxicity of Neuro2a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. However, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyQ. In this report, we compared the toxicity of polyQ among cells expressing polyQ tracts with a variety of intracellular distribution but at similar expression levels. Damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. Cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyQ tracts were relatively spared. These data suggest that the severity of cell damages depends on the type of intracellular distribution of polyQ tracts in cells expressing polyQ tracts at similar level.
Ayumi Takamura 1 , Katsumi Higaki 1 , Junichiro Matsuda 2 , Yoshiyuki Suzuki 3 , Eiji Nanba 1 1 Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Tottori, Japan; 2 National Institute of Biomedical Innovation, Osaka, Japan; 3 Clinical Research Center, International University of Health and Welfare, Tochigi, Japan G M1 -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. Due to a deficiency of lysosomal -galactosidase, excessive lysosomal accumulation of GM1 is observed in patients and animal model brains. However pathogenesisi of this disease is still unclear. Since GM1 is known to be a major sialoglycolipid constituent of plasma membrane (PM) in neuron, we examined the analysis of brain of mouse model. Cerebellar granule cells from this mouse showed GM1 accumulation of lysosome and PM and the membrane fluidity was also reduced. GM1-bound phosphorylated TrkA was markedly decreased in cultured neuron and brain tissues. Subsequent PLC␥, known as a downstream signal of TrkA, was also impaired. These results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in G M1 -gangliosidosis.
Katsuya Inoue 1,2 , Katsuaki Endo 1 , Takamitsu Fujikawa 1 , Seijyun Fukuda 2 , Tatsuo Nakamura 2 1 Department of Physical Therapy, University of Aino, Osaka, Japan; 2 Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan Regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. The experiment was designed to reveal the process after spinal cord injury by asphyxia. To establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. Twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. After 40 min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. We also traced time course of electrical and functional changes after 40 min occulusion.
PS3P-H108 Development of a rodent behavioral model to study the direct interactions of reward and learning Adam Weitemier, Niall P. Murphy RIKEN Brain Science Institute, Japan Cognitive and reward processes often occur simultaneously, and perhaps interdependently. Learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. Conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. Recent studies have demonstrated shared neurobiology between memory and reward. A direct behavioral interaction between reward and memory has never been studied. Cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. We are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. We will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall.
Tetsuya Ando 1 , Yuya Kawanaka 1 , Minoru Saito 2 , Hiroaki Mochizuki 1 , Ken Honjo 1 , Hirofumi Toda 1,3 , Toshifumi Tomoda 3 , Akira Sawa 4 , Katsuo Furukubo-Tokunaga 1 1 Grad. School of Life & Envir. Sci., Univ. Tsukuba, Japan; 2 Molecular Physiol., Tokyo Metropolitan Inst. Neurosci. Tokyo, Japan; 3 Beckman Res. Inst., City of Hope. California, USA; 4 Dept. of Psych. & Neurosci. Johns Hopkins Univ. School of Medicine. Baltimore, USA
The disrupted-in-Schizophrenia-1 (DISC1) gene, originally identified at the breakpoint of a chromosome (1;11) (q42.1; q14.3) translocation in a Scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. However, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. To address DISC1 functions in vivo, we expressed DISC1 in Drosophila and examined developmental and behavioral phenotypes. Overexpression of DISC1 resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. We anticipate that the Drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia.
PS3P-H110 Effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy Yuji Miyatake 1 , Ayumi Kamo 1 , Kenji Minato 1 , Hitoshi Haruna 1 , Hiritsugu Fukuda 2 , Yuji Murata 2 , Takayoshi Hosono 1 1 Department of Bomedical Engineering, Osaka Electro-Communication University, Japan; 2 Graduate School of Medicine, Osaka University, Japan
We investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (HIE) in HIE-model rats using Olton T-maze and anatomy. The common carotid artery of 22 of 25 7-day-old rats was ligated and cut under anesthesia. After the operation the rats were put in a box containing 8% oxygen at 40 • C for 15 min. After the insult, 11 of the 22 rats were put in a box at 35 • C for 12 h (hypothermia, H-group). The other 11 rats were returned to their mother without hypothermia (normothermia, N-group). Sham operations were performed on three rats (S-group). Eight weeks after the operation, their learning and memory ability was assessed by Olton T-Maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the N-group was significantly smaller than in the H-group and S-group.
Withdrawn PS3P-H112 Tau hyperphosphorylation in Ts1Cje, a partial trisomy 16 mouse model for Down syndrome Ebrahim Abdul 1 , A. Shimohata 1 , W. Yu 1 , M. Yamaguchi 1 , M. Murayama 3 , D. Chui 3 , T. Akagi 2 , T. Takeuchi 1 , K. Amano 1 , H.S. Karthik 1 , T. Hashikawa 2 , H. Sago 4 , C.J. Epstein 5 , A. Takashima 3 , K. Yamakawa 1 1 Research Scientist; 2 Lab. for Neural Arch.; 3 Lab. for Alzheimers Disease; 4 Div. of Fetal Med. NCCHD; 5 UCSF, USA Although Down syndrome (DS) or Trisomy 21 is the most common genetic cause of mental retardation, its neuropathology remains unclear. Ts1Cje, a DS mouse model partially trisomic for chromosome 16, shows learning and behavioral abnormalities mimicking DS mental retardation. The trisomic segment, corresponding to parts of human chromosome 21q22, has about 97 genes. Importantly, SOD1 and APP, which may contribute to the DS phenotype, are excluded from the Ts1Cje trisomic segment. Here we report that Ts1Cje brains show hyperphosphorylation of tau in the absence of NFT formation, as well as increased Gsk3 and Jnk/Sapk activities without alterations in APP metabolism. Our results suggest that genes on the trisomic Ts1Cje segment other than APP and SOD1 can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of DS mental retardation.
Research funds: KAKENHI Number: 17790737 PS3P-H113 Increased oxidative stress and mitochondrial dysfunction in Ts1Cje, a Down syndrome mouse model Atsushi Shimohata 1 , Ebrahim A. S. 1 , M. Yamaguchi 1 , W. Yu 1 , H. Sago 2 , C.J. Epstein 3 , K. Yamakawa 1 1 Lab. for Neurogenetics, RIKEN-BSI, Japan; 2 Div. of Fetal Med. NCCHD, Japan; 3 Dept. Pediatrics, UCSF, USA Down's syndrome (DS), caused by chromosome 21(HSA21) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. Ts1Cje is one of a number of segmentally trisomic DS mouse models, and is triplicated for a region of mouse chromosome 16 extending from Sod1 to Znf295, containing 97 genes syntenic with HSA21. Since these mice show learning and behavioral abnormalities mimicking DS mental retardation, Ts1Cjespecific trisomic segment genes may be involved in the DS phenotype. In the present study, we observed increased levels of Reactive Oxygen Species (ROS), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in Ts1Cje brains. Collectively, our results implicate dosage imbalanced genes other than Sod1 and App in both ROS generation and mitochondrial dysfunction, which in turn possibly contribute to the Ts1Cje DS mental retardation-like phenotype.
PS3P-H114 Polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex Manabu Makinodan 1,2 , Kouko Tatsumi 2 , Takayuki Manabe 2 , Takahira Yamauchi 1,2 , Eri Makinodan 2 , Juro Shimoda 1 , Toshifumi Kishimoto 1 , Akio Wanaka 2 1 Department of Psychiatry, Nara Medical University, Kashihara, Japan; 2 Department of 2nd Anatomy, Nara Medical University, Kashihara, Japan Polyinosinic-polycytidylic acid (poly I:C) elicits maternal immune response similar to anti-viral ones. Recent studies demonstrated that poly I:C injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. In the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly I:C-injected mice, but not in the control group born from PBS-injected mice. They showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. On the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. The hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods.
Joanna Doumanis, Ritsuko Kazama, Adrian Moore, Nobuyuki Nukina RIKEN Brain Science Institute, Japan
The fruitfly Drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. To model the polyglutamine expansion disease, Huntington Disease (HD), we have established stable, inducible cell lines expressing N-terminal truncated huntingtin fused to EGFP with an expanded (62Q) polyglutamine repeat in a Drosophila larval central nervous system-derived cell line. Induction of expression results in the formation of protein aggregates, characteristic of HD. Utilising RNAi, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. 7200 genes, encompassing around 50% of the Drosophila genome, were screened, resulting in the identification of 370 candidates that either suppress or enhance aggregation. Most candidates identified have mammalian orthologues, validating the use of Drosophila to screen for genes relevant to human disease. We established in vivo models of HD by expressing polyQ-EGFP in the Drosophila nervous system and are further characterising selected candidates in our model. The rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. The present study investigated effects of harmaline on olivocerebellar systems of mice and rats. Systemic administration of harmaline produced generalized tremors in both types of rodents. Immunohistochemical studies revealed significant degeneration of Purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. However, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ION). Numbers of neurons in the mouse ION did not decrease, suggesting the possibility that microgliosis in ION might not be a simple neurotoxic effect. Presumably, differences in sensitivity of Purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. Recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors.
PS3P-H117 Analysis of ␣-synuclein expression in young mouse model of multiple system atrophy Kimiko Nakayama, Yasuyo Suzuki, Ikuru Yazawa Laboratory of Research Resources, National Institute for Longevity Sciences, Aichi, Japan
Multiple System Atrophy (MSA) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. Glial cytoplasmic inclusions (GCIs) are diagnostics of MSA. GCIs are shown to be abnormal accumulation of filamentous ␣-synuclein. Yazawa et al. (2005) generated a transgenic (Tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the 2 , 3 ,-cyclic nucleotide 3 -phosphodiesterase (CNP) promoter. TG mouse study demonstrated that formation of GCI-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. To elucidate the mechanisms of neurodegeneration in Tg mice, we prepared primary cultures of neurons and glial cells from Tg mice. The cells are examined the effects of ␣-synuclein accumulation.
PS3P-H118 Dysregulation of sodium channel 4 subunit by expanded polyglutamine in huntington disease transgenic mice Fumitaka Oyama, Haruko Miyazaki, Kazumasa Okamura, Yoko Machida, Kurosawa Masaru, Takashi Sakurai, Nobuyuki Nukina Laboratory for Structural Neuropathology, RIKEN BSI, Wako-shi, Japan Sodium channel 4 (4) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. We have identified 4 as an EST that was significantly downregulated in the striatum of HD model mice and found that reduction in 4 started at a presymptomatic stage of the HD model mice. In contrast, spinal cord neurons, which generate only negligible levels of expanded polyQ aggregates, maintained normal levels of 4 expression even at the symptomatic stage. Expanded polyQ with NLS expression suppressed the promoter activity of 4 gene in PC12 cells. Forskolin, an activator of the cAMP/PKA pathway, did not affect b4 promoter activity, indicating that 4 is not cAMP-responsive gene. These findings strongly suggest that sodium channel 4 subunit is a novel molecule, which is an upstream non-cAMP-responsive gene in HD pathogenesis.
PS3P-H119 Repeat length-and age-dependent changes in behavioral phenotypes of DRPLA transgenic mice harboring a single copy of a full-length human DRPLA gene Kazushi Suzuki 1 , Yuji Takahashi 1 , Jun Goto 1 , Mutsuo Oyake 2 , Toshiya Sato 3 , Shoji Tsuji 1 1 Department of Neurology, The University of Tokyo, Tokyo, Japan; 2 Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan; 3 Center for Bioresource-Based Research, Brain Research Institute, Niigata University, Niigata, Japan
We carried out detailed analyses of the behavioral phenotypes of DRPLA transgenic mice carrying an expanded CAG repeat of 76 (Q76), 96 (Q96), 113 (Q113), or 129 (Q129). In the accelerating rotarod (9W), the latencies of Q76, Q96, Q113 and Q129 were 113%, 81%, 34% and 24%, respectively. In the open field, moving distances of Q96, Q113, and Q129 were decreased to 98%, 50%, and 20%, respectively, while that of Q76 was increased to 134%. Home cage activity was decreased depending on the repeat length. The Q113 mice, however, showed increased ratios of the activity during the light time to that during the total day at 8 weeks (115%) and 24 weeks (208%), suggesting that DRPLA mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns.
PS3P-H120 The mice lacking Schnurri-2 show multiple behavioral abnormalities related to psychiatric disorders Keizo Takao 1 , Nobuyuki Yamasaki 1 , Keiko Toyama 1 , Tsuyoshi Takagi 2 , Shunsuke Ishii 2 , Tsuyoshi Miyakawa 1 1 HMRO, Kyoto University Graduate School of Medicine, Kyoto, Japan; 2 RIKEN, Tsukuba, Japan Schnurri-2 (Shn-2) is a zinc finger transcription factor, a mouse homologue of human HIV-EP2, that binds to nuclear factor kappa B-binding site in the HIV long terminal repeat. Shn-2 is known to play important roles in the mammalian immune systems. However, the role of Shn-2 in the central nervous system (CNS) is still unknown. To investigate the functional significance of Shn-2 in mammalian brain, we analyzed the Shn-2 knockout (KO) mice using a comprehensive behavioral test battery. Shn-2 KO mice were dramatically hyperactive under novel environment and in their home cage. They also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. Anxiety-like behavior and depression-like behavior were also significantly reduced in Shn-2 mice. Our results demonstrate a critical role of Shn-2 in CNS and suggest that Shn-2 KO mice may serve as an animal model of psychiatric disorders.
Research funds: KAKENHI (16680015, 16653065, 17017021, 17025023) , JST BIRD PS3P-H121 Comprehensive brain-behavior phenotyping of CaMKII␣ heterozygous knockout mice Nobuyuki Yamasaki, Koichi Tanda, Keiko Toyama, Yasuyuki Fukui, Keizo Takao, Tsuyoshi Miyakawa HMRO, Kyoto University Graduate School of Medicine, Kyoto, Japan Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. Several behavioral abnormalities of CaMKII␣ mutant mice were reported, but systematic assessments of behaviors of CaMKII␣ mutant mice have not been conducted. To analyze the behavioral effects of CaMKII␣ deficiency, we subjected CaMKII␣ heterozygous knockout mice to a comprehensive behavioral test battery. The mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. To identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. The potential involvement of CaMKII␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed.
Research funds: KAKENHI (16680015, 16653065, 17017021, 17025023) , JST BIRD PS3P-H122 Effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: A factor analytic study Hiroshi Ougino, Nobuyuki Yamasaki, Koichi Tanda, Keiko Toyama, Keizo Takao, Tsuyoshi Miyakawa HMRO, Kyoto University Graduate School of Medicine, Kyoto, Japan
We have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. For the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. In this study, we investigated the effects of background strains (C57BL/6J, C57BL/6N, C57BL/6C, 129SvEv, BALB/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than 1200 mice (, including wild type and mutant mice from 25 strains of genetically engineered mice), which had been tested in our laboratory. Also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. The potential implications of our findings for the improvement of the behavioral test battery will be discussed. Calcium-and calmodulin-dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor, cAMP responseelement binding protein (CREB). CaMKIV has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. However, functions of CaMKIV in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. To gain more insight into behavioral significance of CaMKIV, we subjected CaMKIV−/− mice to a battery of behavioral tests. CaMKIV−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. These results indicated selective and specific involvement of CaMKIV in regulating emotional behavior.
Research funds: KAKENHI (16680015, 16653065, 17017021, 17025023) , JST BIRD PS3P-H124 Comprehensive behaivoral analysis of ryanodine receptor type3 knockout mouse Suzuko Ohsako 1 , Koichi Tanda 1,2 , Nobuyuki Yamasaki 1 , Keiko Toyama 1 , Hiroshi Takeshima 3 , Tsuyoshi Miyakawa 1 1 Kyoto University Graduate School of Medicine, Kyoto, Japan; 2 Dep. of Pediatrics, Kyoto Prefectural Univ. of Medicine, Kyoto, Japan; 3 Dep. of Biochem. and Mol Biol., Tohoku Univ. Graduate School of Medicine, Miyagi, Japan Ca 2+ signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. Ryanodine receptors (RyRs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. RyR3 is highly expressed in the hippocampus, caudate putamen, and thalamus. To investigate the behavioral effects of RyR3 deficiency, we subjected RyR3 knockoout mice to a battery of behavioral tests. RyR3 knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. These results suggest a role of RyR3 in regulating general locomotor activity and social behavior.
Research funds: KAKENHI (16680015, 16653065, 17017021, 17025023) , JST BIRD PS3P-H125 Comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse Keiko Toyama 1 , Koichi Tanda 1,2 , Nobuyuki Yamasaki 1 , Tsuyoshi Miyakawa 1 1 HMRO, Kyoto University Graduate School of Medicine, Kyoto, Japan; 2 Dept. of Pediatrics, Kyoto Prefectural Univ. of Medicine, Kyoto, Japan Nitric Oxide (NO) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. NO is synthesized from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS). In neurons, NO is produced by neuronal nitric oxide synthase (nNOS), representing one of three NOS isoforms expressed in most tissues. To elucidate function of nNOS/NO in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nNOS knockout mice to a battery of behavioral tests. nNOS knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. Furthermore, they displayed increased social contacts in novel environment and homecage. These results indicate that nNOS/NO is involved in regulation of their behaviors.
Research funds: KAKENHI (16680015, 16653065, 17017021, 17025023) , JST BIRD PS3P-H126 Primate model of attention-deficit/hyperactivity-disorders (ADHD) Shintaro Funahashi 1 , Keiko Shimizu 2 1 Grad. Sch. Human and Environmental Std, Kyoto Univ., Kyoto, Japan; 2 Primate Res. Inst., Kyoto Univ., Inuyama, Japan ADHD is one of the prevalent childhood psychiatric disorders. Children with ADHD show hyperactive behavior and attention problems, suggesting prefrontal (PFC) contribution to ADHD. ADHD is also known as dopamine (DA) related dysfunctions, because methylphenidate is the most effective drug for the treatment of ADHD. PFC is the cortical area where the strongest DA innervation is observed. Injection of DA-related drugs to PFC produces behavioral deficits in cognitive tasks. These suggest that DA-related dysfunction in PFC could be a candidate of biological causes of ADHD. To prove this notion, we injected 6-OHDA into bilateral PFC to destroy DA innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. 6-OHDA injected monkeys showed significant increase of spontaneous activity in test cages. Oral administration of methylphenidate reduced spontaneous activity in 6-OHDA injected monkeys. These results suggest that monkeys injected 6-OHDA into PFC are good candidates of the primate model of ADHD.
Research funds: KAKENHI (17021022) PS3P-I127 Training-induced recovery of precision grip after primary motor cortex damage in the adult monkey Yumi Murata 1,2,3 , Noriyuki Higo 1,3 , Takao Oishi 1,3,4 , Akiko Yamashita 5 , Keiji Matsuda 1 , Motoharu Hayashi 4 1 Neurosci. Res. Inst, AIST, Tsukuba, Japan; 2 Grad. Sch. Compreh. Hum. Sci., Univ. of Tsukuba, Tsukuba, Japan; 3 CREST, JST, Kawaguchi, Japan; 4 Dept. Cell Mol. Biol., Primate Res. Inst., Kyoto Univ., Inuyama, Japan; 5 Div. Applied Sys. Neurosci., Nihon Univ. Sch. Med., Tokyo, Japan
In the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (MI), in order to investigate the effects of postlesion training on motor recovery. We derived a hand representation map in MI, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. After one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. Untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. This suggests that recovery of precision grip requires postlesion training.
Research funds: a Grant-in-Aid for Scientific Research on Priority Areas from MEXT (17021055) Mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. In order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen ENU (N-Ethyl-N-nitrosourea). We screened about 2,500 G1 animals (DBA/2J × ENU-treated C57BL/6J) for home-cage activity, open-field activity, and passive avoidance response, and obtained 13 lines of dominant behavioral mutants. By linkage analysis, the causative genes were mapped in 5 of 13 mutant lines. Hyperactivity was predominant phenotype, and 7 of 13 mutants showed hyperactivity in home-cage and/or open-field. We will report the recent results of initial characterization and the progress of fine mapping in these ENUinduced mutants.
PS3P-I129 Ubiquitin signal in neurons of cathepsin Ddeficient mouse brains with special reference to the autophagic process Masato Koike, Masahiro Shibata, Yasuo Uchiyama Dept. of Cell Biol. and Neurosci., Osaka Univ. Grad. Sch. of Med., Suita, Japan
We have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from CD−/− and CB−/−CL−/− mice, murine models for neuronal ceroid lipofuscinoses (NCLs) (Koike et al., 2005) . Until recently, it remains unknown what signaling is essential for autophagosome formation. Interestingly, in the conditional Atg7-knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (Komatsu et al., 2005) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. We therefore examined the immunohisto/cytochemical localization of ubiquitin and LC3, and found that in our NCL model mice, positive signals for ubiquitin and LC3 were co-localized on the membranes of granular structures in the neuronal perikarya. These results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in NCLs.
Research funds: Grant-in-Aid for Young Scientists (B)(17790141) and Creative Scientific Research (16GS0315)
PS3P-I130 Activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via AMPA/kainate glutamate receptors Tadahiro Katayama 1 , Eiichi Jodo 1 , Yoshiaki Suzuki 2 , Ken-yo Hoshino 2 , Yukihiko Kayama 1 1 Dept. of Physiology, Fukushima Medical University, Fukushima, Japan; 2 Dept. of Neuropsychiatry, Fukushima Medical University, Fukushima, Japan It has been shown that tonic activation of the medial prefrontal cortex (mPFC) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (PCP). However, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mPFC. Here, we examined effects of local application of those antagonists on increased firing activity of mPFC neurons by systemic PCP in anesthetized rats. After tonic activation of mPFC neurons by PCP had been established, CNQX, SCH23390, mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. CNQX reduced PCP-induced augmentation of firing activity to the baseline level, while others gave little changes. These results suggest that PCPinduced activation of mPFC neurons be mediated primarily via AMPA/kainate receptors.
PS3P-I131 Increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase IV (CaMKIV)-knockout mice Jiro Kasahara 1 , Hiroyuki Sakagami 2 , Hisatake Kondoh 2 , Kohji Fukunaga 1 1 Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan; 2 Department of Histology, Graduate School of Medicine, Tohoku University, Sendai, Japan Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is expressed abundantly in the nuclei of neurons and thought to regulate Ca-dependent gene expressions mediated by the transcriptional factors such as CREB. Recently, we found that chronic treatments of the rats with antidepressants increased CaMKIV activity and CREB phosphorylation in the prefrontal cortex, suggesting the importance of CaMKIV in the effects of antidepressants. This result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in CaMKIV-knockout mice by some experimental paradigms. From the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of CaMKIV for the regulation of depressiveness and the effects of antidepressants.
PS3P-I132 Severity of audiogenic seizures is influenced by multiple factors in Vlgr1-mutated mice Hideshi Yagi 1,2 , Makoto Sato 1,2 1 Division of Cell Biology and Neuroscience, Department of Morphological and Functional Sciences, Faculty of Medical Sciences, University of Fukui, Fukui, Japan; 2 Research and Education Program for Life Science, University of Fukui, Fukui, Japan Epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. We made Vlgr1-mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. To evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made C57BL/6 backcrossed Vlgr1-mutated mice and 129/SvS4 backcrossed Vlgr1-mutated mice. These two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. Furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. These observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our Vlgr1-mutated mice are good model of reflex epilepsies that are evoked by multifactors.
PS3P-I133 Reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine Tomoko Kadota 1 , Ken Kadota 1,2 1 Department of Bioenvironmental Medicine, University of Chiba, Chiba, Japan; 2 Chiba Institute of Psychiatry, Chiba, Japan
Our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (MAP) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (MFC). The present study further examined morpholological changes of rats that were administered a daily dose of 5 mg/kg of MAP i.p. for 12 days (d 1 d 12) and then withdrawn from the drug for 7 28 days (WD 7 WD 28). The regimen reduced the densities of parvalbumin positive cells (PAC); these were probably GABAergic cells and distributed in the strata covering layers II, III and V in the anterior cingulate cortex (Cg 1) and MFC. The decrease in the density of PAC was first observed in Cg 1 and then in MFC. The reduction began on d 6 and advanced to higher levels on d 12 and subsequently WD 7. These findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in Cg 1 and MFC, particularly in layers II and III.
PS3P-I134 Up-regulation of  2 -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy Teruyoshi Kondo, Yoshihiro Ishibashi, Kei-Ichiro Nakamura Department of Anatomy, Division of Microscopic and Developmental Anatomy, Kurume University School of Medicine, Kurume, Japan Stimulation of  2 -adrenergic receptor ( 2 -AR) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. Present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of  2 -AR-immunoreactivity (IR) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons.  2 -ARimmunoreactive cells coexpressed GFAP-IR and were positive for nestin which is characteristic of reactive astrocytes. A population of  2 -AR-immunoreactive cells were labeled with Ki-67, a marker of cell proliferation, indicating some of them went into cell mitotic state. Interestingly, a major population of  2 -AR-immunoreactive cells also exhibited FGF-2-IR. These findings suggest that  2 -AR may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as FGF-2.
PS3P-I135 Effects of lateral fluid percussion injury (FPI) on the optical signals in dentate gyrus of the rat brain slice preparations Shin Yamashita 1 , Norihiro Muraoka 2 , Hiroshi Hasuo 1 , Takashi Akasu 1 , Minoru Shigemori 2 1 Dept. of Physiology, Kurume Univ. Sch. of Med., Kurume, Japan; 2 Dept. of Neurosurgery, Kurume Univ. Sch. of Med., Kurume, Japan
We investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (DG) using optical recording techniques with voltage-sensitive dye (RH482). Horizontal hippocampal slices were obtained from the control and the FPI rats (one week after the single moderate impact). Electrical stimulation of perforant path (PP) produced the optical signal spread in the molecular layer of DG. Temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). Increment of stimulus intensity (10-50 V) increased the amplitude of both fast and slow peaks. The intensity for producing the maximal response was 30-40 V. The amplitude of slow peak in FPI group was about 50% larger than that in control group, while the amplitudes of fast peak were not different in the two groups. These data suggest that the excitatory PP synapse onto granule cells of DG is facilitated after FPI.
PS3P-I136 Comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and Ca 2+ imaging Chikafusa Bessho, Yasuharu Mitsushima, Ryo Matsumoto Department of Physics, Kyoto Sangyo University, Kyoto, Japan Recently K. Shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. We examined neural activities in mouse brain (hippocampal) slices by the modified method and Ca 2+ imaging. The slices (300 m) were prepared from the block in an ice cold ACSF medium using microslicer and incubated for 1 h in the oxygenated medium at room temperature. A slice was placed on a recording chamber perfused with the medium at a flow rate of 1 ml/min. Green autofluorescence (>520 nm) of the slices illuminated by blue light (460-490 nm) was observed by an inverted microscope. Images of the autofluorescence were recorded using a calcium imaging system. Ca 2+ imaging was also performed in the slices. Slices were incubated in ACSF medium containing 10 M of fluo3/4 AM for 1 h at 37 • C. The Ca 2+ image was recorded with an excitation wavelength of 460-490 nmand an emission wave length of >520 nm. The autofluorescence and Ca 2+ responses wereobserved in slices perfused with L-glutamate (10 mM).
Takuya Hayashi 1 , Hiroshi Sato 1 , Shinichi Abe 2 , Takashi Hanakawa 2 , Hiroshi Watabe 1 , Hidenao Fukuyama 2 , Babak Aldekani 3 , Hidehiro Iida 1 1 Department of Investigative Radiology, National Cardiovascular Center Research Institute, Osaka, Japan; 2 Human Brain Research Center, Kyoto University, Kyoto, Japan; 3 Nathan Kline Institute for Psychiatric Research, NY, USA We show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (DWI). In macaque, the DWIbased striatal connectivity of Brodmann's area 9 corresponded to that revealed by the tracer (MnCl 2 ) tractography. The DWI-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. In addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. These results suggest that human striatum has a dominant and specific role in processing prefrontal information.
Research funds: H17-kokoro-025 PS3P-I138 Optical analysis of synaptic transmission by a fluorescent glutamate probe Shigeyuki Namiki, Hirokazu Sakamoto, Sho Iinuma, Kenzo Hirose Department of Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan Glutamate is an essential excitatory neurotransmitter in the central nervous systems. For optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called EOS. By imaging with EOS, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. We also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. Furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. Neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. High concentrations of sucrose increased the frequency of the release. These features are reminiscent of those of miniature EPSC in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. In conclusion, our probe directly visualizes the presynaptic release.
Shingo Miyata 1,2 , Yasutake Mori 1,2 , Tsuya Taneda 1,2 , Hiroaki Okuda 1,2 , Masaya Tohyama 1,2 1 Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan; 2 21st COE program, Tokyo, Japan Local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mRNAs. A great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. However, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. Therefore, it is problematic to visualize the dynamic state of translation by the present methods. To solve the problem, we applied FRET (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eIFs), thereby showing that BDNF and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons.
PS3P-I140 A model selection of GLM applied to fMRI data using AIC Jobu Watanabe 1 , Fumikazu Miwakeichi 2 , Andreas Galka 3,4 , Ryuta Kawashima 4 , Tohru Ozaki 3 , Sunao Uchida 1,5 1 Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Japan; 2 Department of Medical System Engineering, Faculty of Engineering, Chiba University, Chiba, Japan; 3 Institute for Statistical Mathematics, Tokyo, Japan; 4 Institute of Experimental and Applied Physics, University of Kiel, Keil, Germany; 5 New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan; 6 Faculty of Sport Sciences, Waseda University, Tokorozawa, Japan
In the General Linear Model (GLM) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. The Akaike Information Criterion was applied as a basis of comparison and selection among several GLMs that analyze block-designed functional magnetic resonance imaging (fMRI) data. The GLMs with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. We demonstrate that a combination of these explanatory variables can effectively improve the model and that AIC is a useful tool for model selection in fMRI studies.
Ryuzo Shingai, Katsunori Hoshi, Tokumitsu Wakabayashi Department of Welfare Engineering, IwateUniversity, Morioka, Japan
To investigate the relationship between the behavior and function of the nervous system of Caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. We devised an automated analysis system of C. elegans locomotion. The system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. The system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for 20-30 min and then a gradual increase in the duration of forward movement. Accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion.
PS3P-J148 Flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex Daiki Kamatani, Ryuichi Hishida, Masaharu Kudoh, Katsuei Shibuki Dept. Neurophysiol., Brain Res. Inst., Niigata Univ., Niigata 951-8585, Japan
We have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. However, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. In the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. The cortical activities in layer IV were observed as fluorescence responses after thalamic stimulation. The magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. These cortical responses were suppressed by antagonists of glutamate receptors such as CNQX and APV, and almost completely abolished in the presence of CNQX plus APV. In preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice.
PS3P-J149 Effects of implicit emotional processes on encoding-related activations of episodic memory: An eventrelated fMRI study Yayoi Shigemune 1,2 , Takashi Tsukiura 1 , Hiroko Mochizuki-Kawai 1 , Chisato Suzuki 1,2 , Toshio Iijima 2 1 Neurosci. Res. Inst., AIST, Tsukuba, Japan; 2 Div. Systems Neurosci., Tohoku Univ. Grad. Sch. Life Sci., Sendai, Japan
In this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fMRI. Nineteen healthy right-handed male participated in this study. We prepared emotional pictures with three kinds of emotional valence (Negative: Nega, Neutral: Neu and Positive: Posi) and line drawings for encoding. In the fMRI scanning, subjects memorized line drawings, which were presented after the emotional pictures. After the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. We found significant activations of the right anterior cingulate gyrus specifically in the Nega condition, the right lingual gyrus in the Neu condition and the right amygdala and pulvinar in the Posi condition. These results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence.
PS3P-J150 Different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fMRI Masahiko Haruno 1 , Kenji Kansaku 2 , Yu Aramaki 2 , Mitsuo Kawato 1 1 ATR CNS, Kyoto, Japan; 2 Institute of Physiology, Okozoki, Japan Efficient decision making requires multiple reward predictions in switching different contexts and learning. We conducted a human fMRI experiment (n = 14) to examine stimulus-action-dependent and stimulus-dependent reward predictions. In condition A, each of two fractal figures specifies a monetary reward associated with a button push (left or right). If the button is pressed correctly, 50 or 10 yen is provided with a probability of 0.8, but only with 0.2 if pressed wrongly. The key difference in condition B is that a fractal determines the reward but not the action. Subjects had learned the two conditions fully before scanning. At the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (P < 0.01) in A, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in B. The striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions.
Research funds: NICT PS3P-J151 Brain networks for communicative speech production: Feeling inference and speech content production Yuko Sassa 1,2 , Motoaki Sugiura 3 , Hyeonjeong Jeong 1,4 , Keisuke Wakusawa 1,5 , Kaoru Horie 6 , Shigeru Sato 6 , Ryuta Kawashima 1 1 NICHe, Tohoku University, Sendai, Japan; 2 RISTEX, JST, Tokyo, Japan; 3 Miyagi University of Education, Sendai, Japan; 4 GSICS, Tohoku University, Sendai, Japan; 5 Department of Pediatrics, Tohoku University, Sendai, Japan; 6 The LBC Research Center, Tohoku University, Japan
Communicative speech production often accompany inference of the targetperson's feeling. In this fMRI study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. During presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (Speech), inferred feeling (Feeling), or described the action (Des). Greater activation in the contrasts Speech-Feeling was observed in themedial prefrontal cortex, and that in the contrast Feeling-Des wasobserved in the right superior temporal sulcus extending to the temporalpole. The results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively.
Research funds: the 21st COE Program PS3P-J152 The construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy Takafumi Miyoshi 1 , Yasuhisa Fujibayashi 2 , Yoshiharu Yonekura 2 , Tatsuya Asai 2 1 Department of Human and Intelligence Systems, University of Fukui, Fukui, Japan; 2 Biomedical Imaging Research Center, University of Fukui, Fukui, Japan People with severe motor disabilities can increase the quality of life if they can communicate with the external world. A brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. Brain activity was measured non-invasively by multi-channel near infrared spectroscopy (NIRS) during various motor tasks from healthy subjects. These spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. We propose that NIRS signals may be used to construct a brain-computer interface.
PS3P-J153 Imaging of brain activity by near infrared spectroscopy in response to various sounds Tatsuya Asai 1 , Kuniyoshi Shinya 1 , Tetsuo Araki 2 , Masahiro Kusakabe 2 , Yasuhisa Fujibayashi 3 , Yoshiharu Yonekura 3 1 Department of Nuclear Power and Energy Safety Engineering, University of Fukui, Fukui, Japan; 2 Department of Human and Intelligence Systems, University of Fukui, Fukui, Japan; 3 Biomedical Imaging Research Center, University of Fukui, Fukui, Japan Brain activity can be monitored non-invasively by near infrared spectroscopy (NIRS). In the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel NIRS from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. We have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. These results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans.
Kazuo Kitamura 1,2 , Winfried Denk 3 , Michael Hausser 2 1 Department of Cellular Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan; 2 University College London, London, UK; 3 Max-Plank Institute, Heidelberg, Germany
We describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. The method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. The same electrode can then be placed on the neuron under visual control to allow gigaseal formation. We demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. We also show that the method can be used for in vivo juxtacellular labelling in identified cell types. This approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations.
Research funds: Wellcome Trust, Gatsby Foundation, JSPS and Uehara Foundation PS3P-K161 Analysis on viability of GABAergic neurons in cerebral cortical slices of adult mice Yasuyo Tanaka 1 , Yasuhiro Tanaka 1 , Takeshi Kaneko 1,2 1 Dept. of Morphological Brain Science, Kyoto Univ., Kyoto, Japan; 2 CREST, JST, Kawaguchi, Japan Whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. We analyzed the effect of slice cutting and incubation conditions on viability of cortical GABAergic neurons, using GAD67-GFP knock-in mice. We considered GFP positive cells as having survived. We observed more GFP-positive cells in the slices when NaCl in cutting solution was replaced with N-methyl-D-glucamine (NMDG) chloride, choline chloride or sucrose. However, the viability was lower after 3 h incubation in NMDG-based solution than in NaCl-based solution. Cutting at 0 • C did not reduce the number of GFP-positive cells, but decreased GFP fluorescence in single neurons as compared with cutting at 20 • C. The viability after 3 h incubation was better kept at 20 • C than at 0 or 37 • C. We thus recommend that slices be cut at 20 • C in Na-free solution, and incubated at 20 • C in NaCl-based solution. We thank Dr Yanagawa for his generous gift of knock-in mice.
Research funds: KAKENHI (16200025,17022020,17650100) PS3P-K162 Contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state Takumi Satoh 1 , Yoshichika Yoshioka 2 1 Faculty of Engineering, Iwate University, Morioka, Japan; 2 Iwate Medical University, Takizawa, Japan We evaluated GSH signals by the MEGA-PRESS (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (MRS). GSH gave a single positive signal (2.95 ppm) by MEGA-PRESS. In contrast, GSSG gave a multiplet of reversed signals (3.03, 3.23, and 3.34 ppm). A phantom solution mimicking the normal condition (GSH:GSSG = 100:1) gave a single positive peak. GSH was prominent and GSSG signals were minimal. Thus, the signals originated from GSH, not from GSSG. In the phantom solution (creatine: GSH: aspartate: GABA = 7:3:1:1), the creatine signal overshadowed the other signals. Through MEGA-PRESS, a single peak of GSH stood out over other signals. In vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from GSH. The estimated concentration of GSH in the human brain was 1.9 mM. In conclusion, MEGA-PRESS allowed us to assess GSH levels in vivo non-invasionally.
Hiroshi Kadota, Hirofumi Sekiguchi, Yasoichi Nakajima, Yutaka Kohno, Makoto Miyazaki Department of Sensory and Communicative Disorders, Research Institute, National Rehabilitation Center for Persons with Disabilities, Tokorozawa, Japan
We investigated the brain regions related to the inhibition of habitual responses by using functional MRI. We used the rock-paper-scissors game as an example of a familiar habitual behavior. It is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. In this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." A comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. With regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (Brodmann area 9) and the right superior and middle frontal gyri (Brodmann area 10) in the lose group. These findings suggest that these regions play a role in the inhibition of habitual responses.
PS3P-K164 Cortical commissural connection in macaque and human callosum using diffusion MRI Rishu Piao 1 , Takuya Hayashi 1 , Hiroshi Sato 1 , Shinichi Abe 1,2 , Takashi Hanakawa 2 , Hidenao Fukuyama 2 , Hidehiro Iida 1 1 National Cardiovascular Center, Osaka, Japan; 2 Human Brain Research Center, Kyoto University, Kyoto, Japan
We investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (DWI). We used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in 9 subcortical areas. In macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. However, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. The human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. This study revealed that the commissural connectivity of the two species has a common intra-callosal topography.
PS3P-K165 Optimal resolution of EEG/MEG source imaging by spatial filtering Wan Xiaohong 1,2 1 NICHe, Department of Qutantum Science and Energy Engineering, Tohoku University, Sendai, Japan, 2 NICHe, Tohoku University, Sendai, Japan Nowadays, electro-and magnetoencephalography (EEG/MEG) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. Although we are well aware that it is impossible to accurately estimate the 3-D neural cortical activity using the 2-D EEG/MEG surface potential topography, the upper limit of these techniques is not well described. During the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. However, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. In this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. This motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. Essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution.
PS3A-C192 Sensitivity of serotonin synthesis to synthesis inhibitor GTP cyclohydrolase I in senescence-accelerated mouse-prone inbred strain (SAMP8) Nobuyuki Karasawa 1 , Kazuko Watanabe 2 , Keiki Yamada 3 1 Seijoh Universitry, Tokai, Japan, 2 Dept. Physio., Sch. Med., Gifu Univ., Gifu, Japan, 3 Dept. Anat., Sch. Health Sci., Fujita Health Univ., Toyoake, Japan
To study the relationship between aging and levels of monoaminergic neurons, 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (SAMP8) mice. Time course of immunoreactive intensity for serotonergic (5-HT) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. Results showed that 5-HT neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and 5-HT neurons.
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