DISCUSSION
The applicability of DBS is currently being evaluated in various psychiatric indications including TRD (Fisher et al, 2010; for a review see Luigjes et al, 2012). Yet, the investigation of its underlying mechanisms is limited in humans for understandable reasons, and preclinical data showing the behavioral efficacy of DBS as well as its neurobiological correlates are rare in psychopathologically relevant animal models. Motivated by these facts, we initially further validated the unique HAB mouse model of comorbid trait anxiety- and depression-like behavior (Kromer et al, 2005; Landgraf et al, 2007; Sah et al, 2012) by demonstrating that these mice are resistant to the antidepressant effects of chronic SSRI treatment, the first-line therapy for depression as well as many anxiety disorders (Koenig and Thase, 2009). In contrast, repeated NAcb-DBS rescued the enhanced depression- and anxiety-related behavior of HAB animals. This effect coincided with (i) distinct changes in stress-induced neuronal activation of depression-related brain areas, including cortical areas and the HPC, as well as (ii) increased adult hippocampal neurogenesis.

HAB Mice Displayed Features of TRD
HAB mice display enhanced depression-like behavior as well as altered stress-induced neuronal activity in networks related to depression and anxiety in comparison with their normal depression/anxiety NAB counterparts (Sah et al, 2012; Sartori et al, 2011b). In this study, we demonstrate for the first time that male HABs are insensitive to at least three SSRIs differing in half-life, plasma-binding, active metabolites, and Ki values of 5-HT uptake (Hiemke and Hartter, 2000). Interestingly, we observed that treatment resistance seemed to be restricted to drugs targeting solely the serotonergic system, as both the selective noradrenaline re-uptake inhibitor reboxetine and the tricyclic antidepressant desipramine significantly reduced the depression-like behavior of HAB mice. This finding was surprising further suggesting that male HAB mice represent a valid model to study treatment resistance against SSRIs in humans. Indeed, at least 33% of patients do not respond to first-line pharmacotherapies such as the SSRI citalopram (Koenig and Thase, 2009; Trivedi et al, 2006). Changed responsiveness to SSRI treatment in humans has been linked to SNPs affecting 5-HT synthesis and polymorphism in the 5-HTT promoter region as well as functional polymorphisms in the 5-HT1A and 5-HT1B receptor genes (for a review see Kroeze et al, 2012). However, while an aberrant 5-HT neurotransmission has been reported in male HAB rats (Keck et al, 2005), pathophysiological correlates underlying the selective insensitivity to SSRIs in HAB mice remain to be elucidated in further experiments.
To date, there are only a few reports on TRD in animal models of depression. For example, the Flinders sensitive rat line has recently been described as being insensitive to citalopram and nortryptilin when subjected to repeated maternal separation (Borsini, 2012; Carboni et al, 2010). Furthermore, congenital learned-helplessness rats and about a quarter of rats subjected to unpredictable mild stress do not respond to chronic treatment with desipramine or fluoxetine, or to electroconvulsive therapy, respectively (Sartorius et al, 2007; Wang et al, 2011). It is possible that in the past insensitivities to certain drug classes were less likely to be reported. However, more recently the restriction of predictive validity of animal models for depression has been (partially) eased as a consequence of the need to model TRD (Borsini, 2012; Samuels and Hen, 2011) and to test new antidepressant approaches not primarily acting via monoaminergic systems.

NAcb-DBS Elicited Antidepressant and Anxiolytic Effects in HAB Mice
In this study, we show for the first time that NAcb-DBS reduced depression-like behavior in an SSRI-insensitive animal model for enhanced depression-like behavior. While acute stimulation was not sufficient, repeated high-frequency stimulation caused a robust antidepressant effect in HAB mice. Similarly, in patients with TRD, significant reductions in depressive symptomatology reaching response or remission end points also seem to require more time (Bewernick et al, 2010; Lozano et al, 2008), despite some rapid effects on specific symptoms seen during or following early phases of DBS (Schlaepfer et al, 2008; Mayberg et al, 2005). These findings, thus, suggest that at least some of the behavioral changes induced by NAcb-DBS may require long-term changes such as modulation of neuroplasticity. Indeed, in rats, pERK expression (an early marker of cell plasticity) is enhanced in prefrontal and orbitofrontal regions as well as in the amygdala following DBS of the NAcb/ventral striatum (Rodriguez-Romaguera et al, 2012). In addition to its antidepressant effects, repeated NAcb-DBS reduced the enhanced trait anxiety of HAB mice to an extent comparable to that achieved after anxiolytic treatment (Gaburro et al, 2011; Kromer et al, 2005; Sartori et al, 2011a; for a review see Sartori et al, 2011b). Underlining the translational value of the present findings in HAB mice, similar effects were also observed in humans (Bewernick et al, 2012; Denys et al, 2010).
Various behaviors, including locomotion, seem to be rapidly altered by interference with the NAcb function. As both ablation (Parkinson et al, 1999) and electrical stimulation (van Kuyck et al, 2007) of the NAcb affect activity and exploratory behavior in animals and/or humans, we tested whether NAcb-DBS under our stimulation condition produces changes in general locomotor activity, which could potentially influence behavioral assessment of anxiolytic and antidepressant effects. This possibility could be excluded, as it was found that NAcb-sham and NAcb-DBS mice did not differ in terms of distance traveled in the open field, indicating no effect on locomotion using the present high-frequency stimulation settings.
To clarify whether the behavioral effects of NAcb-DBS were restricted to acting only on pathophysiologically deranged as opposed to physiological networks, we investigated the behavioral outcome of repeated NAcb-DBS in NABs. Neither single nor repeated NAcb-DBS affected the level of depression- and anxiety-like behavior in NAB mice, indicating that NAcb-DBS (at the chosen stimulation settings) preferentially acts on systems of altered/enhanced emotionality. In support of this idea, van der Plasse et al (2012) recently reported inefficacy of NAcb-DBS in terms of its ability to affect depression-like behavior displayed by normal rats. Mood-elevating effects of antidepressants are predominantly observed in depressed patients (Barr et al, 1997; Gelfin et al, 1998; Yeragani et al, 2003), suggesting that the neurobiological targets of antidepressants in pathophysiologically deranged systems differ from those in intact systems (Berton and Nestler, 2006). Therefore, while some aspects of therapeutic efficacy of DBS may be shared by normal animals (Hamani et al, 2010; Rodriguez-Romaguera et al, 2012) and pathophysiological models (Falowski et al, 2011; Friedman et al, 2012; Hamani et al, 2012), some critical changes underlying their antidepressant effect will remain undetected if animals reflecting physiological but not pathophysiological conditions are used.

Neurobiological Changes Induced by NAcb-DBS in HAB Mice

Alteration of challenge-induced neuronal activation patterns
The NAcb receives projections from the main monoaminergic nuclei including the raphe nuclei and the locus coeruleus and from regions associated with locomotion, emotion, and memory, including the globus pallidus, the amygdala, the cortex, and the HPC (Nauto and Domesick, 1984). In turn, NAcb projects to pallidal and nigral complexes, to cortical areas such as the medial prefrontal cortex, and to the thalamic and hypothalamic regions. Several of these brain areas have been implicated in the pathophysiology of depression and/or in the processing of antidepressant effects (Krishnan and Nestler, 2008). Furthermore, many of these areas are positioned along the superolateral arm of the medial forebrain bundle, a structure related to the reward circuitry, likely to be stimulated by most of, if not all, the different electrode placements (eg the anterior limb of the internal capsule, the subgenual cingulate gyrus, the NAcb) used in the treatment of TRD patients (Coenen et al, 2011). Regarding potential mechanisms of action identified so far, DBS preferentially modulates network fibers passing the electrode, while local effects of DBS on somatodendritc structures are minor (McIntyre and Grill, 1999; McIntyre et al, 2004; Nowak and Bullier, 1998a, 1998b). Specifically, direct inhibition of the electrode target area by muscimol injections or radiofrequency lesions does not seem to resemble effects of DBS on anxiety/depression networks (Hamani et al, 2010; Rodriguez-Romaguera et al, 2012). To identify distant parts of circuitries affected by NAcb-DBS at the present stimulation conditions, which may underlie the antidepressant effect observed in HAB mice, we used c-Fos mapping in specific brain areas (Singewald, 2007). The focus was laid upon those brain areas that have been previously shown to be associated with therapeutic modulation of enhanced depression-like behavior (Muigg et al, 2007; Sah et al, 2012; Winter et al, 2011).
One candidate area is the HPC, a highly stress-sensitive key brain structure dysregulated in depression (Floresco et al, 2001; Kingwell, 2010) in terms of reduced volume and dysfunctional activation under emotional challenge (Kempton et al, 2011; Lee et al, 2007; Milne et al, 2012; Tan et al, 2012; also see Disner et al (2011)). Interestingly, HAB rats (Muigg et al, 2007; Salomé et al, 2004), and more recently HAB mice (Muigg et al, 2009; Sah et al, 2012), display hypoactivation of the dentate gyrus (DG) by stress challenge. Here, we observed that NAcb-DBS enhanced the c-Fos induction in response to FST stress, suggesting that (i) neuronal DG activity is restored in HAB mice, and (ii) DG activity is strongly correlated with depression-like behavior. To our knowledge, so far changes in DG/HPC activity have not been reported in TRD patients undergoing DBS, while activity of the HPC is enhanced in addicted patients undergoing NAcb-DBS (Heldmann et al, 2012).
In addition, the c-Fos response was enhanced in the OFC and the lateral habenula, but attenuated in the prelimbic cortex following FST. In line with our results, pERK expression is enhanced in prefrontal regions, including the OFC, following ventral striatum/NAcb-DBS, suggesting functional connectivity between these spatially distinct structures (Rodriguez-Romaguera et al, 2012). In further support of our findings, McCracken and Grace (2007, 2009) propose an antidromic activation of NAcb-input fibers descending from the OFC by NAcb-DBS, resulting in the modulation of activity within the OFC and, thus, potentially affecting disturbed communication between prefrontal areas, limbic areas, and the OFC in an ultimately beneficial way.

NAcb-DBS enhanced the number of immature neurons in the DG of the HPC
Whereas stress as a triggering factor for depression attenuates adult neurogenesis in the HPC, antidepressant treatment enhances proliferation and survival in the hippocampal neurogenic niche and has been proposed to be at least partially required for antidepressant efficacy (for a review see Samuels and Hen, 2011). Given that HAB mice display reduced adult hippocampal neurogenesis in comparison with NABs (Sah et al, 2012), we also investigated whether the blunted neurogenesis of HABs would be affected by NAcb-DBS. Indeed, the number of DCX-positive cells was enhanced in the DG in NAcb-DBS-treated HAB mice in comparison with NAcb-sham controls. This finding points towards enhanced adult hippocampal neurogenesis, as DCX is exclusively expressed in neuronal precursors and not fully differentiated neurons reflecting the developmental stages of neuroblast 1 and 2 cells as well as immature neurons (for a review see Encinas et al, 2006). Similarly, DBS of the anterior thalamic nucleus slightly increases the number of neuroblast 1 cells in the HPC, while the symmetric division of amplifying neuronal progenitors is its main effect (Encinas et al, 2006, 2011; Toda et al, 2008). To gain insight into the effect of NAcb-DBS on different developmental stages of newly born cells, BrdU staining was used. Interestingly, only when BrdU was injected before, but not during NAcb-DBS, an enhanced number of BrdU-positive cells was found. These results suggest that the higher number of DCX-positive neurons reflects a cell cohort, which was actually born before the NAcb-DBS intervention and whose survival was then increased by NAcb-DBS. This is supported by a recent study showing that a single DBS session of the entorhinal cortex increases the survival rate of cells born up to 10 days before DBS in the DG (Stone et al, 2011). In this context, it is speculated that 1- to 3-week-old progenitors are especially sensitive to life events that promote survival (eg environmental enrichment) or deteriorate it (eg through stressful condition; Zhao et al, 2008). However, future work will need to verify whether these cells contribute to the reduction of depressive symptoms.
In summary, the data presented here demonstrate that NAcb-DBS selectively rescued the enhanced depression- and anxiety-related behaviors in an SSRI-resistant psychopathological mouse model of high trait depression/anxiety without affecting these behaviors in normal depression/anxiety NAB controls. The specific changes that occurred in challenge-induced neuronal activation suggest that the beneficial effects of NAcb-DBS are mediated via a distributed network that includes the HPC, and cortical and thalamic areas. Furthermore, enhanced adult neurogenesis following repeated NAcb-DBS indicates that long-term alterations may also be an important part of the mechanism(s) of NAcb-DBS, leading to a rescue of exaggerated anxiety- and depression-like symptoms.