PMC:4195273 / 36232-66664 JSONTXT

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implications of CART on energy metabolism from pharmacological interventions\nThe identification of the underlying mechanisms by which CART exerts effects on feeding and energy homeostasis have been challenging due to the lack of any knowledge of the corresponding CART receptor (s) and the absence of specific antagonists. Nevertheless, numerous studies incorporating both pharmacological and genetic manipulations of CART expression in murine models have been endeavored in the last decades to determine the sites of action and the effects on feeding behavior and metabolism of the peptide. Overexpression studies to discern the brain regions mediating CART-induced regulation of feeding have been the most common approach in rodents. Widely adopted as an appetite-regulating peptide of the CNS with hypothalamic expression levels modulated by nutritional status (Kristensen et al., 1998; Thim et al., 1998, 1999; Schwartz et al., 2000), CART was administrated i.c.v. to address the effects of overexpression during varying energy states (Kristensen et al., 1998; Lambert et al., 1998; Thim et al., 1998; Vrang et al., 1999b; Edwards et al., 2000; Kask et al., 2000; Larsen et al., 2000; Aja et al., 2001a,b; Bannon et al., 2001; Nakhate et al., 2011, 2013) (Table 1). The lateral ventricle (LV) of the forebrain or the 3rd ventricle (3V) have been the major injection targets. I.c.v. injection of recombinant CART peptide has been consistently demonstrated to inhibit food intake and body weight gain in a dose-dependent manner in both food-restricted and free-feeding conditions as well as both under standard chow or a nutritionally complete liquid diet, in either normal or diet-induced obese animals (Kristensen et al., 1998; Lambert et al., 1998; Thim et al., 1998; Vrang et al., 1999b; Edwards et al., 2000; Kask et al., 2000; Larsen et al., 2000; Abbott et al., 2001; Aja et al., 2001a,b; Bannon et al., 2001; Rohner-Jeanrenaud et al., 2002; Tachibana et al., 2003; Wortley et al., 2004; Qing and Chen, 2007; Nakhate et al., 2011, 2013). Furthermore, the catabolic capacity of CART appeared sufficient to prevent and attenuate the orexigenic effects of NPY, as i.c.v. and intra-PVN CART potently suppressed feeding in satiated rats subjected to NPY-induced hyperphagia (Kristensen et al., 1998; Lambert et al., 1998; Vrang et al., 1999b; Wang et al., 2000; Rohner-Jeanrenaud et al., 2002). Similarly, the anorectic potency of CART has also been demonstrated in a recent study focusing on the interaction between CART and the GABA type A receptor (GABA-A) active neurosteroid allopregnanolone (ALLO) and the inhibitor neurosteroid dehydroepiandrosterone sulfate (DHEAs) (Nakhate et al., 2013). It was shown in rodents that pre-treatment of i.c.v. CART effectively attenuated subcutaneous ALLO-induced hyperphagia and weight gain, as well as potentiating DHEAS-induced hypophagic and weight reducing effects (Nakhate et al., 2013).\nTable 1 Summary of the metabolic and behavioral effects of central CART administration via various intracerebroventricular and intranuclear delivery methods.\nYear Publication Targeting peptide/CART fragment Route of adminis-tration Species (genetic background) Diet Feeding behavior and body weight alterations Locomotor behavior\n1998 Kristensen et al., 1998 CART I (55–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding; ↓ Fast-induced feeding; ↓ NPY-induced feeding N/A\n1998 Lambert et al., 1998 CART (55–59) i.c.v. Rat Standard chow ~ Spontaneous feeding N/A\n1998 Lambert et al., 1998 CART (55–76) i.c.v. Rat Standard chow ↓ Spontaneous feeding; ↓ NPY-induced feeding N/A\n1998 Lambert et al., 1998 CART (62–76) i.c.v. Rat Standard chow ↓ Spontaneous feeding N/A\n1998 Thim et al., 1998 CART I (55–102); CART II (62–102) i.c.v. Mouse Standard chow ↓ Fast-induced feeding N/A\n1999 Vrang et al., 1999b CART I (42–89) i.c.v.g Rat Standard chow ↓ Spontaneous feeding (in food-restricted animals) and ↓ NPY-induced feeding N/A\n2000 Edwards et al., 2000 CART I (55–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding N/A\n2000 Kask et al., 2000 CART (62–76) i.c.v. Rat Standard chow ↓ Spontaneous feeding N/A\n2000 Volkoff and Peter, 2000 CART I (55–102); CART (62–76) i.c.v. Goldfish Standard chow ↓ Spontaneous feeding and ↓ NPY-induced feeding (in food-restricted animals) N/A\n2000 Larsen et al., 2000 CART I (42–89) i.c.v. (chronic infusion) Lean and obese Zucker (fa/fa) rats Standard chow ↓ Spontaneous feeding (in both free-feeding and food-restricted animals); dose-dependent ↓ in body weight Dose-dependent motor disturbances (combined gait and walking ataxia)\n2000 Okumura et al., 2000 CART I (55–102) i.c. Rat Standard chow ↓ Fast-induced feedingb N/A\n2000 Wang et al., 2000 CART I (55–102) intra-PVN Rat Standard chow ↓ NPY-induced feedingi N/A\n2001 Bannon et al., 2001 CART I (55–102); CART II (62–102) i.c.v. Mouse Standard chow ↓ Fast-induced feeding N/A\n2001 Abbott et al., 2001 CART I (55–102) Hypothalamic intranuclear injections (VMH, Arc, PVN, SON, DMH, LHA and AHA) Rat Standard chow ↑ Spontaneous feeding (only measured in satiated animals cannulated into the DMH or Arc); ↑ Fast-induced feeding N/A\n2001 Abbott et al., 2001 CART I (55–102) i.c.v. (3V) Rat Standard chow ↓ Spontaneous feeding; ↓ Fast-induced feeding (↓ feeding episodes) Behavioral abnormalities marked by reduced feeding episodes, flat-backed posture and movement-associated tremors (behavioral analysis performed for 24-h fasted animals only but not satiated animals)\n2001 Zheng et al., 2001 CART I (55–102) i.c.v. (LV and 4V) Rat Sucrose solution or standard chow ↓ Spontaneous feeding (↓ short-term sucrose intake and ↓ overnight chow intake)—effects more pronounced in 4V compared to LV administration Alterations in motor behavior (mild movement-associated tremors in part of 4V injected subjects)\n2001a Aja et al., 2001a CART I (55–102) i.c.v. Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet intake in licks and meal size in food restricted animals) Altered oral motor function and behavioral alterations (trance-like state, flat-backed and arched-backed postures, cage licking, movement-associated tremors)\n2001b Aja et al., 2001b CART I (55–102) i.c.v. (3V) Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet intake and observations of feeding in food restricted animals)—reductions significantly attentuated by aqueduct obstructiona Alterations in motor behavior (flat-backed and arched-backed postures and movement-associated tremors)—alterations significantly attentuated by aqueduct obstructiona\n2001b Aja et al., 2001b CART I (55–102) i.c.v. (4V) Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet intake and observations of feeding in food restricted animals)—reductions unaffected by aqueduct obstructiona Alterations in motor behavior (flat-backed and arched-backed postures and movement-associated tremors)—alterations unaffected by aqueduct obstructiona\n2002 Aja et al., 2002 CART I (55–102) i.c.v. (4V) Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet and water intake in food restricted animals); production of conditioned taste aversion N/A\n2002 Rohner-Jeanrenaud et al., 2002 CART I (55–102) i.c.v. (chronic infusion) Rat (normal and DIO) Standard chow or HFD ↓ Spontaneous feeding and ↓ NPY-induced feeding; ↓ body weight gain N/A\n2002 Zheng et al., 2002 CART I (55–102) i.c.v. (4V)h and intra-NTS Rat Sucrose solution or standard chow ↓ Spontaneous feeding (↓ short-term sucrose intake)—effects more pronounced in 4V compared to intra-NTS administration N/A\n2003 Smedh and Moran, 2003 CART I (55–102) i.c.v. (4V) Rat Sucrose solution ↓ Spontaneous feeding (↓ sucrose intake in food restricted animals); altered lick microstrcuture parametersc N/A\n2003 Kong et al., 2003 CART I (55–102) intra-Arcd Rat Standard chow ↑ Spontaneous feeding (in both free-feeding and food-restricted animals) and ↑ Fast-induced feeding; ↑ cumulative body weight gain; ↑ body weight loss following 24-hr fasting and food restrictioni N/A\n2004 Wortley et al., 2004 CART I (55–102) i.c.v. (3V) Rat Standard chow ↓ Spontaneous feeding N/A\n2005 Yang et al., 2005 CART I (55–102) intra-AcbSh Rat Standard chow ↓ Spontaneous feeding; ↓ Fast-induced feeding; ↓ GABA-A agonist muscimol-induced feeding N/A\n2007 Qing and Chen, 2007 rat CART cDNA i.c.v.e Rat (DIO) High fat/high sucrose diet ↓ Spontaneous feeding; ↓ Fast-induced feeding; ↓ body weight gain (↓ lean mass; fat mass unaffected) N/A\n2007 Jean et al., 2007 CART I (55–102) intra-AcbSh Mouse Standard chow ↓ Fast-induced feeding N/A\n2007 Jean et al., 2007 CART siRNA intra-AcbSh Mouse Standard chow ↑ Spontaneous feeding and ↓ stimulating 5-HT4R- or MDMA-induced anorexia in staved animals N/A\n2008 Smith et al., 2008 rAAV encoding full length rat CART cDNA (GenBank accession no. U10071) intra-PVNe Rat Standard chow or HFD ↑ Cumulative feeding and cumulative body weight gain; effects more accentuated on HFD N/A\n2009 Skibicka et al., 2009 CART I (55–102) i.c.v. (4V) or intra-NTS Rat Standard chow [4V injection]f ↓ Spontaneous feeding and body weight (in food-restricted animals); hypophagic response and weight loss attenuated by pre-treatment with hindbrain delivery of GLP-1R antagonist (exendin-9-39); intra-NTS injection produced no observable effect on feeding or body weight N/A\n2010 Hou et al., 2010 CART I (55–102) intra-Arc; intra-DMH Streptozotocin-diabetic rats Standard chow or HFD Chow diet: ↑ Spontaneous feeding (in satiated animals) (Arc) and ↑ Fast-induced feeding (DMH and Arc); HFD: ↑ Spontaneous feeding (Arc) N/A\n2011 Nakhate et al., 2011 CART I (54–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding and body weight; attenuated social isolation-induced hyperphagia and body weight gain N/A\n2013 Nakhate et al., 2013 CART I (54–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding; attenuated ALLO-induced hyperphagia and weight gain; potentiated DHEAS-induced anorexia and weight loss N/A\nCSF, cerebrospinal fluid; DIO, diet-induced obese; HFD, high fat diet; BAT, brown adipose tissue; i.c.v., intracerebroventricular; i.c., intracisternal; LV, lateral ventricle; 3V, third ventricle; 4V, fourth ventricle; Acb, nucleus accumbens; AcbSh, nucleus accumbens shell; AHA, anterior hypothalamic area; Arc, arcuate nucleus; DMH, dorsomedial nucleus; LHA, lateral hypothalamic area; NTS, nucleus of the solitary tract; PBN, parabrachial nucleus; PVN, paraventricular nucleus; SON, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus.\nEffects on body weight described where results presented, otherwise either unaffected or information unavailable.\nEffects on locomotor behavior described where results presented, otherwise either unaffected or information unavailable.\na Cerebral aqueduct occlusion to interrupt forebrain-hindbrain CSF flow.\nb Inhibition of gastric function (suppression of gastric acid secretion and gastric emptying); inhibition of gastric acid secretion remained in vagotomized animals; inhibition of gastric acid secretion blocked by pretreatment with central administration of CRF receptor antagonist α-helical CRF9-41.\nc Inhibition of gastric function (suppression of gastric emptying); inhibition of gastric emptying blocked by pretreatment with central administration of CRF receptor antagonist α-helical CRF9-41; CART-induced inhibition of gastric emptying proposed unlikely to contribute to CART-mediated inhibition of food intake.\nd Acute administration through repeated injections and chronic overexpression using stereotactically targeted gene transfer.\ne Chronic overexpression using recombinant adeno-associated virus vector containing rat CART cDNA.\nf ↑ Blood glucose levels; hyperglycemic response not altered by GLP-1R blockade in animals pre-treated with GLP-1R antagonist (exendin-9-39).\ng Induction of Fos expression in the PVN, DMH, SON and Arc (hypothalamus), central nucleus of amygdala (cerebrum), PBN and NTS (hindbrain).\nh Induction of Fos expression in NTS neurons.\ni ↑ UCP-1 expression thermogenic capacity in BAT. CART administration via the i.c.v. route was also able to eliminate the increase in feeding and deleterious weight gain caused by social isolation in rats (Nakhate et al., 2011), a consequence of the downregulation of the hypothalamic CART-containing system in various hypothalamic feeding-related areas caused by this condition (Nakhate et al., 2011). In the same study, whilst re-socialization of the isolation-reared rats restored the food intake, body weight, and hypothalamic CART-immunoreactivity back to controls levels, immunoneutralization of endogenous CART by i.c.v. CART antibody attenuated the restoration, confirming the important role of CART in feeding regulation under chronic psychological stress condition (Nakhate et al., 2011). This is consistent with other studies that used antibodies raised against different CART segments for blocking central CART signaling, where all of which were able to neutralize the anorectic property of CART and lead to a significant hyperphagic response (Kristensen et al., 1998; Lambert et al., 1998; Nakhate et al., 2010, 2011). In addition to eliciting an anorectic response, gastrointestinal effects including inhibition of gastric acid secretion and gastric emptying have also been reported as a result from i.c.v. CART (Okumura et al., 2000; Asakawa et al., 2001; Smedh and Moran, 2003, 2006; Tebbe et al., 2004). Chronic overproduction of CART mRNA via viral approaches or continuous infusion of recombinant CART peptide transferred through i.c.v. cannulas into genetically (fa/fa) (Larsen et al., 2000) or diet-induced (Rohner-Jeanrenaud et al., 2002; Qing and Chen, 2007) obese rats induces hypophagic effects during fed states and reduced hyperphagia following fasting were also observed. Such reduction in energy intake was accompanied by suppression of body weight gain mainly due to decrease in lean mass (Larsen et al., 2000; Qing and Chen, 2007), indicating the potential of CART in the long-term regulation of food consumption and body mass, under both normal condition and nutritionally induced obesity.\nIn conjunction with the characterization of physiological responses, neuronal activities stimulated by central CART has been investigated by structural studies for the purpose of identifying brain areas potentially crucial for CART-induced anorectic effects. Following i.c.v. CART administration, temporal expression patterns of the immediate early gene c-Fos (Dragunow and Faull, 1989; VanElzakker et al., 2008), which has been adopted to depict neuronal firing of actions potentials (Dragunow and Faull, 1989; VanElzakker et al., 2008), were found to concentrate in the hypothalamic and brainstem structures implicated in the central regulation of feeding (Vrang et al., 1999b; Zheng et al., 2002). In the hypothalamus in particular, high density of Fos expression was located in the PVN and the posterior DMH, while considerable Fos-IR cells were also identified in the Arc and SO. In the brainstem, Fos-positive cell nuclei were also concentrated in the PBN and, more importantly, in the NTS, which serves a key sensory relay nucleus with reciprocal connections with numerous forebrain and brainstem structures (Vrang et al., 1999b). Such CART-induced Fos activation in the NTS has been indicated independent from possible secondary effects triggered by chemo-activation at the area postrema (AP) directed to the NTS, as the chemosensitive neurons in the AP were devoid of Fos-IR cells (Vrang et al., 1999b). Moderately high Fos expression was also detected in cerebral nuclei associated with autonomic functions and energy balance (Smith and DeVito, 1984; Vrang et al., 1999b), including the central nucleus of the amygdala, where neuronal projections also reciprocally link with the PVN of the hypothalamus and the PBN and NTS of the hindbrain (Hopkins and Holstege, 1978; Holstege et al., 1985). The widespread Fos expression pattern elicited by forebrain i.c.v. CART has been demonstrated to encompass an anatomical continuum of neuronal activations across the cerebrum, hypothalamus and brainstem (Vrang et al., 1999b). The paralleled effects on appetite inhibition and metabolic regulation are believed to portray an integrated outcome of the interactions between central CART-interfered pathways residing primarily within the hypothalamic and brainstem neurons. For instance, as aforementioned, the administration of CART combined with other neuromodulatory such as CCK in mice generated synergistic effects on food intake and locomotion, while displaying concomitant enhancement in the number of Fos-positive neurons compared to injecting each peptide alone (Maletinska et al., 2008; Pirnik et al., 2010). The additive effect on Fos immunoreactivity was especially notable in the target areas common to both peptides, namely the hypothalamic PVN, DMH, VMH and Arc, as well as NTS at the brainstem (Maletinska et al., 2008; Pirnik et al., 2010), wherein the CCK-related satiety signals transmitted to the hindbrain were suggested to be further regulated by leptin action integrated in the Arc as well as neuronal signals from both PVN and LHA (Broberger, 2005; Morton et al., 2005; Maletinska et al., 2008).\nCART is widely expressed in the brain and particularly concentrated in the hypothalamus, suggestive of a diverse range of functions. While effective, delivery of ligands via the i.c.v. route is associated with the downside of the simultaneous stimulation of pathways in various parts of the brain, likely contrasting with effects attributable to the activation of specific neuronal populations. One such case are the CART neurons at the Arc, which respond to and are modulated by leptin signals, leading to the activation of selective neurons and associated downstream pathways (Kristensen et al., 1998; Schwartz et al., 2000). It is therefore unsurprising that the observed effects of the i.c.v. injection of substances like CART are not always replicated by targeted delivery of the same peptide into specific nuclei of the hypothalamus. Indeed, several studies have shown that targeting CART into individual hypothalamic nuclei results in revelation of the orexigenic effects of CART, leading to increased food intake and body weight (Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010). The strongest orexigenic effects were observed by injection of CART into the VMH, DMH and Arc, and a much lesser effect was observed when administered into the PVN, LHA, anterior hypothalamic area, and SO (Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010) (Table 1). Other effects following Arc and PVN CART delivery such as greater energy expenditure and thermogenic capacity, as indirectly measured by the expression and activity of UCP-1 in brown adipose tissue crucial in thermogenesis, has also been reported (Wang et al., 2000; Kong et al., 2003) (Table 1).\nDespite the dependence of endogenous CART expression on nutritional states discussed above, energy states of the animals or dietary options appeared to have little influence over the potency of CART administration-induced feeding stimulation (Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010). For instance, intra-arcuate delivery of CART resulted in elevated food intake in rodents under both fasted, food-restricted and satiated conditions, subjected to the dietary interventions of either regular chow or HFD (Abbott et al., 2001; Kong et al., 2003; Hou et al., 2010). Intriguingly, the orexigenic effects of CART were exhibited in both non-diabetic normal rats as well as streptozotocin-induced diabetic rats, where the intra-Arc CART-induced increase in feeding was reproduced under various energy states and dietary treatments (Hou et al., 2010). Similarly, in rats receiving chronic overexpression of recombinant CART virally delivered into the PVN, higher cumulative food intake and body weight gain was observed in both groups fed either normal chow or HFD compared to control groups, with more pronounced changes in the HFD group (Smith et al., 2008). Underlying such observations, the appetite-promoting effects of hypothalamic intranuclear CART administration may be attributed to a role of hypothalamic CART in stimulating the release of orexigenic neuropeptides locally (Smith et al., 2008; Hou et al., 2010). This is supported by experiments involving a static incubation system where an increase in the release of NPY- and AgRP-IR but not α-MSH-IR was detected in both Arc-containing hypothalamic explants incubated with CART peptide in vitro as well as in PVN-containing hypothalamic explants isolated from animals subjected to intra-PVN CART injection (Smith et al., 2008; Hou et al., 2010). Direct hypothalamic intranuclear CART injection at specific sites, therefore resulted in feeding behaviors opposite to the anorectic effects seen for i.c.v. CART.\nThe discrepancy between the anorexic effects of CART when injected i.c.v. vs. the predominately orexigenic effects of CART when delivered into specific hypothalamic nuclei suggests that CART expression/function in other brain areas may also be important to the regulation of food intake and energy homeostasis, also suggesting that CART may be involved in both anorexigenic and orexigenic circuits in the CNS (Parker and Bloom, 2012). Other potential areas for CART-mediated anorexic effects include the striatum, which is known to have upregulated CART expression following acute i.p. administration of psychostimulants (Douglass et al., 1995; Fagergren and Hurd, 1999; Hubert and Kuhar, 2008), reduced CART mRNA levels following fasting (Kristensen et al., 1998; Adams et al., 1999; Yang and Shieh, 2005), and has been shown to be involved in the mediation of reward and reinforcement (Koob and Bloom, 1988; Salamone, 1996; Upadhya et al., 2012) as well as the neuronal circuits controlling feeding behavior (Bakshi and Kelley, 1993; Gilbert and Cooper, 1995; Pothos et al., 1995; Stratford et al., 1997; Stratford and Kelley, 1999; Baldo et al., 2002; Yang and Shieh, 2005; Upadhya et al., 2012). Evidence for such a role was gained from experiments in a strain of CCK-1 receptor-deficient obese rats, where a significant reduction in CART immunoreactivity in the Arc was found potentially associated with a diminished anorectic effect of CART peptide compared to lean controls (Abraham et al., 2009). Furthermore, intra-accumbal CART peptide injection has been demonstrated to diminish both basal food consumption and food deprivation-induced feeding (Yang and Shieh, 2005; Jean et al., 2007), as well as potently attenuating the orexigenic effects of the GABA-A agonist muscimol (Yang and Shieh, 2005), albeit some inconsistency across different studies (Jaworski et al., 2008) (Table 1). The antagonistic effects of the GABA system and CART at the Acb were also demonstrated in the neurochemical phenotypes of hypothalamic neurons after the appetite-inducing microinjection of muscimol into the AcbSh, which increased Fos expression in orexin neurons at the perifornical area and NPY neurons at the Arc, while inhibiting that in Arc CART/POMC neurons (Zheng et al., 2003). In a recent study, subcutaneous injection of the GABA-A active neurosteroid ALLO significantly reduced CART immunoreactive cells and fibers in the AcbSh, as well as in other feeding-related hypothalamic nuclei such as the PVN, Arc and LHA (Nakhate et al., 2013). Direct CART administration into the Acb performed by an independent group generated no detectable influence on food reward assessed by food self-administration, yet triggered inhibitory effects on cocaine self-administration, an alternative measure of reward and reinforcement entailing dopaminergic functions (Jaworski et al., 2008).\nThe anorexia elicited by intra-accumbal CART was more sustainable in freely fed compared to starved animals, highlighting the significance of fuel status on CART function in feeding modulation (Yang and Shieh, 2005). Furthermore, complementary to the overexpression experiments, RNA-interference has been employed to investigate the effects of CART depletion in rodents (Jean et al., 2007; Job and Kuhar, 2012). Tissue-specific CART knockdown in the Acb via intra-accumbal administration of short interfering RNA (siRNA) or short hairpin (shRNA) against CART mRNA induced body weight gain and hyperphagia in fed mice (Jean et al., 2007; Job and Kuhar, 2012), as well as abolishing the anorectic effects of serotonin (5-hydroxytryptamine, 5-HT) 4 receptor (5-HT4R) stimulation as well as 3,4-N-methylenedioxymethamphetamine (MDMA, ecstasy) treatment in starved mice, further denoting the potential role of Acb CART in mediating the appetite suppressant properties in models of anorexia nervosa (Jean et al., 2007).\nDespite the body of evidence endorsing the plausibility of the Acb as a site for CART-directed anorexia, the appetite-regulating effects produced by intra-accumbal CART likely represent part of the reward and motivational responses derived from an interaction between CART and the dopaminergic system in the Acb. Multiple lines of evidence have suggested an inhibitory role of endogenous accumbal CART in addiction-relevant behaviors, which are speculated to act in concert with feeding modulation as well as the locomotive effects mediated by the dopaminergic circuits (Kim et al., 2003; Jaworski et al., 2008; Hubert et al., 2010; Job and Kuhar, 2012; Upadhya et al., 2012). For instance, substantial innervations have been described for CART-containing neurons in the ventral pallidum, a key nucleus harboring accumbal efferents, where CART-IR terminals were reported to compose symmetric synapses resembling inhibitory GABAergic synapses (Hubert et al., 2010). Whilst intra-accumbal administration of CART alone produced no effect on locomotor activity, co-injection with cocaine or amphetamine into the Acb inhibited the cocaine-like locomotor effects produced by Acb dopamine microinfusions, both intra-accumbal and intra-pallidal injections of CART peptide led to reduction in cocaine- and amphetamine-induced locomotor activity (Jaworski et al., 2003, 2008; Kim et al., 2003; Hubert et al., 2010). Correspondingly, CART depletion through intra-accumbal CART shRNA increased cocaine-mediated locomotion (Job and Kuhar, 2012), supporting an antagonistic property of Acb CART in the functions of cocaine and other psychostimulants.\nThe hindbrain, as a region described to convey post-prandial satiety effects to the hypothalamus, has been considered a potential candidate site for CART action (Marty et al., 2007; Subhedar et al., 2014). Supporting evidence include the moderate CART expression in terms of both transcript levels and immunoreactivity in caudal brain areas such as the locus coeruleus, NTS, PBN and the inferior olive (Douglass et al., 1995; Koylu et al., 1998), accorded with the increased Fos-IR identified in the NTS and PBN following i.c.v. CART into the LV (Vrang et al., 1999b). Comparable to i.c.v. injections into the LV or 3V, hindbrain delivery of CART peptide through the 4th ventricle (4V) led to reduction in food intake and body weight in both fed and food-deprived rodents, whilst the hypophagic effects showed no specificity to nutrients from either chow, sucrose or a nutritionally complete liquid diet (Aja et al., 2001b, 2002; Zheng et al., 2001, 2002; Skibicka et al., 2009) (Table 1). Importantly, the extent of feeding inhibition appeared more potent with CART administered into the 4V compared with LV injections (Zheng et al., 2001), raising the speculation that the hindbrain may house the key mediator for the hypophagic effects of i.c.v. CART (Aja et al., 2001b). Foundation for the idea involved the postulate that the anorectic effects triggered by forebrain i.c.v. CART indeed reflected the outcome of CART diffusion into hindbrain sites via the cerebrospinal fluid (CSF) (Aja et al., 2001b). Surmised from the CART-IR observed in cell bodies and central terminals of vagal afferent neurons projecting to the GI tract, a potential functional role of CART in meal termination and satiety may effectuate at the level of the brainstem (Broberger et al., 1999; Zheng et al., 2002). In rat, vagotomy caused considerable reduction in CART mRNA expression in several CART fibers in the vagus nerve and viscero-sensory nodose ganglion (Broberger et al., 1999). To verify such proposition, cerebral aqueduct occlusion was performed with an aqueductal plug to interrupt the forebrain-hindbrain CSF flow, and CART was injected into the 3V or 4V (Aja et al., 2001b) (Table 1). Interestingly, cerebral aqueduct blockage markedly attenuated the anorectic effects of 3V CART, whilst suppression of food intake remained unchanged when receiving 4V CART injection, signifying the independence of hindbrain CART in producing anorexia (Aja et al., 2001b). In contrast, hindbrain processing may be required or responsible for mediating a hypophagic response following forebrain or interbrain i.c.v. CART, further reinforcing the role of the brainstem in manifesting CART-driven anorectic effects, as concordant with the aforementioned higher potency in feeding inhibition with 4V as opposed to forebrain or interbrain i.c.v. CART (Zheng et al., 2001). Specifically, on the assumption that the repressed ingestive behaviors following LV or 3V CART may attribute to hindbrain CART action, the observations resulted from obtrusion of CSF flow could offer a possible explanation for the opposite feeding effects of orexigenic and anorexigenic natures induced by direct hypothalamic subnuclei (Wang et al., 2000; Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010) vs. hindbrain ventricular (Aja et al., 2001b, 2002; Zheng et al., 2001, 2002; Skibicka et al., 2009) CART administration respectively. Such phenomena promote reevaluation of the authenticity and proposed mechanisms involved in the hypophagia exhibited after forebrain ventricular CART detailed in other studies (Kristensen et al., 1998; Lambert et al., 1998; Vrang et al., 1999b; Larsen et al., 2000; Abbott et al., 2001; Aja et al., 2001a,b)."}

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implications of CART on energy metabolism from pharmacological interventions\nThe identification of the underlying mechanisms by which CART exerts effects on feeding and energy homeostasis have been challenging due to the lack of any knowledge of the corresponding CART receptor (s) and the absence of specific antagonists. Nevertheless, numerous studies incorporating both pharmacological and genetic manipulations of CART expression in murine models have been endeavored in the last decades to determine the sites of action and the effects on feeding behavior and metabolism of the peptide. Overexpression studies to discern the brain regions mediating CART-induced regulation of feeding have been the most common approach in rodents. Widely adopted as an appetite-regulating peptide of the CNS with hypothalamic expression levels modulated by nutritional status (Kristensen et al., 1998; Thim et al., 1998, 1999; Schwartz et al., 2000), CART was administrated i.c.v. to address the effects of overexpression during varying energy states (Kristensen et al., 1998; Lambert et al., 1998; Thim et al., 1998; Vrang et al., 1999b; Edwards et al., 2000; Kask et al., 2000; Larsen et al., 2000; Aja et al., 2001a,b; Bannon et al., 2001; Nakhate et al., 2011, 2013) (Table 1). The lateral ventricle (LV) of the forebrain or the 3rd ventricle (3V) have been the major injection targets. I.c.v. injection of recombinant CART peptide has been consistently demonstrated to inhibit food intake and body weight gain in a dose-dependent manner in both food-restricted and free-feeding conditions as well as both under standard chow or a nutritionally complete liquid diet, in either normal or diet-induced obese animals (Kristensen et al., 1998; Lambert et al., 1998; Thim et al., 1998; Vrang et al., 1999b; Edwards et al., 2000; Kask et al., 2000; Larsen et al., 2000; Abbott et al., 2001; Aja et al., 2001a,b; Bannon et al., 2001; Rohner-Jeanrenaud et al., 2002; Tachibana et al., 2003; Wortley et al., 2004; Qing and Chen, 2007; Nakhate et al., 2011, 2013). Furthermore, the catabolic capacity of CART appeared sufficient to prevent and attenuate the orexigenic effects of NPY, as i.c.v. and intra-PVN CART potently suppressed feeding in satiated rats subjected to NPY-induced hyperphagia (Kristensen et al., 1998; Lambert et al., 1998; Vrang et al., 1999b; Wang et al., 2000; Rohner-Jeanrenaud et al., 2002). Similarly, the anorectic potency of CART has also been demonstrated in a recent study focusing on the interaction between CART and the GABA type A receptor (GABA-A) active neurosteroid allopregnanolone (ALLO) and the inhibitor neurosteroid dehydroepiandrosterone sulfate (DHEAs) (Nakhate et al., 2013). It was shown in rodents that pre-treatment of i.c.v. CART effectively attenuated subcutaneous ALLO-induced hyperphagia and weight gain, as well as potentiating DHEAS-induced hypophagic and weight reducing effects (Nakhate et al., 2013).\nTable 1 Summary of the metabolic and behavioral effects of central CART administration via various intracerebroventricular and intranuclear delivery methods.\nYear Publication Targeting peptide/CART fragment Route of adminis-tration Species (genetic background) Diet Feeding behavior and body weight alterations Locomotor behavior\n1998 Kristensen et al., 1998 CART I (55–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding; ↓ Fast-induced feeding; ↓ NPY-induced feeding N/A\n1998 Lambert et al., 1998 CART (55–59) i.c.v. Rat Standard chow ~ Spontaneous feeding N/A\n1998 Lambert et al., 1998 CART (55–76) i.c.v. Rat Standard chow ↓ Spontaneous feeding; ↓ NPY-induced feeding N/A\n1998 Lambert et al., 1998 CART (62–76) i.c.v. Rat Standard chow ↓ Spontaneous feeding N/A\n1998 Thim et al., 1998 CART I (55–102); CART II (62–102) i.c.v. Mouse Standard chow ↓ Fast-induced feeding N/A\n1999 Vrang et al., 1999b CART I (42–89) i.c.v.g Rat Standard chow ↓ Spontaneous feeding (in food-restricted animals) and ↓ NPY-induced feeding N/A\n2000 Edwards et al., 2000 CART I (55–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding N/A\n2000 Kask et al., 2000 CART (62–76) i.c.v. Rat Standard chow ↓ Spontaneous feeding N/A\n2000 Volkoff and Peter, 2000 CART I (55–102); CART (62–76) i.c.v. Goldfish Standard chow ↓ Spontaneous feeding and ↓ NPY-induced feeding (in food-restricted animals) N/A\n2000 Larsen et al., 2000 CART I (42–89) i.c.v. (chronic infusion) Lean and obese Zucker (fa/fa) rats Standard chow ↓ Spontaneous feeding (in both free-feeding and food-restricted animals); dose-dependent ↓ in body weight Dose-dependent motor disturbances (combined gait and walking ataxia)\n2000 Okumura et al., 2000 CART I (55–102) i.c. Rat Standard chow ↓ Fast-induced feedingb N/A\n2000 Wang et al., 2000 CART I (55–102) intra-PVN Rat Standard chow ↓ NPY-induced feedingi N/A\n2001 Bannon et al., 2001 CART I (55–102); CART II (62–102) i.c.v. Mouse Standard chow ↓ Fast-induced feeding N/A\n2001 Abbott et al., 2001 CART I (55–102) Hypothalamic intranuclear injections (VMH, Arc, PVN, SON, DMH, LHA and AHA) Rat Standard chow ↑ Spontaneous feeding (only measured in satiated animals cannulated into the DMH or Arc); ↑ Fast-induced feeding N/A\n2001 Abbott et al., 2001 CART I (55–102) i.c.v. (3V) Rat Standard chow ↓ Spontaneous feeding; ↓ Fast-induced feeding (↓ feeding episodes) Behavioral abnormalities marked by reduced feeding episodes, flat-backed posture and movement-associated tremors (behavioral analysis performed for 24-h fasted animals only but not satiated animals)\n2001 Zheng et al., 2001 CART I (55–102) i.c.v. (LV and 4V) Rat Sucrose solution or standard chow ↓ Spontaneous feeding (↓ short-term sucrose intake and ↓ overnight chow intake)—effects more pronounced in 4V compared to LV administration Alterations in motor behavior (mild movement-associated tremors in part of 4V injected subjects)\n2001a Aja et al., 2001a CART I (55–102) i.c.v. Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet intake in licks and meal size in food restricted animals) Altered oral motor function and behavioral alterations (trance-like state, flat-backed and arched-backed postures, cage licking, movement-associated tremors)\n2001b Aja et al., 2001b CART I (55–102) i.c.v. (3V) Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet intake and observations of feeding in food restricted animals)—reductions significantly attentuated by aqueduct obstructiona Alterations in motor behavior (flat-backed and arched-backed postures and movement-associated tremors)—alterations significantly attentuated by aqueduct obstructiona\n2001b Aja et al., 2001b CART I (55–102) i.c.v. (4V) Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet intake and observations of feeding in food restricted animals)—reductions unaffected by aqueduct obstructiona Alterations in motor behavior (flat-backed and arched-backed postures and movement-associated tremors)—alterations unaffected by aqueduct obstructiona\n2002 Aja et al., 2002 CART I (55–102) i.c.v. (4V) Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet and water intake in food restricted animals); production of conditioned taste aversion N/A\n2002 Rohner-Jeanrenaud et al., 2002 CART I (55–102) i.c.v. (chronic infusion) Rat (normal and DIO) Standard chow or HFD ↓ Spontaneous feeding and ↓ NPY-induced feeding; ↓ body weight gain N/A\n2002 Zheng et al., 2002 CART I (55–102) i.c.v. (4V)h and intra-NTS Rat Sucrose solution or standard chow ↓ Spontaneous feeding (↓ short-term sucrose intake)—effects more pronounced in 4V compared to intra-NTS administration N/A\n2003 Smedh and Moran, 2003 CART I (55–102) i.c.v. (4V) Rat Sucrose solution ↓ Spontaneous feeding (↓ sucrose intake in food restricted animals); altered lick microstrcuture parametersc N/A\n2003 Kong et al., 2003 CART I (55–102) intra-Arcd Rat Standard chow ↑ Spontaneous feeding (in both free-feeding and food-restricted animals) and ↑ Fast-induced feeding; ↑ cumulative body weight gain; ↑ body weight loss following 24-hr fasting and food restrictioni N/A\n2004 Wortley et al., 2004 CART I (55–102) i.c.v. (3V) Rat Standard chow ↓ Spontaneous feeding N/A\n2005 Yang et al., 2005 CART I (55–102) intra-AcbSh Rat Standard chow ↓ Spontaneous feeding; ↓ Fast-induced feeding; ↓ GABA-A agonist muscimol-induced feeding N/A\n2007 Qing and Chen, 2007 rat CART cDNA i.c.v.e Rat (DIO) High fat/high sucrose diet ↓ Spontaneous feeding; ↓ Fast-induced feeding; ↓ body weight gain (↓ lean mass; fat mass unaffected) N/A\n2007 Jean et al., 2007 CART I (55–102) intra-AcbSh Mouse Standard chow ↓ Fast-induced feeding N/A\n2007 Jean et al., 2007 CART siRNA intra-AcbSh Mouse Standard chow ↑ Spontaneous feeding and ↓ stimulating 5-HT4R- or MDMA-induced anorexia in staved animals N/A\n2008 Smith et al., 2008 rAAV encoding full length rat CART cDNA (GenBank accession no. U10071) intra-PVNe Rat Standard chow or HFD ↑ Cumulative feeding and cumulative body weight gain; effects more accentuated on HFD N/A\n2009 Skibicka et al., 2009 CART I (55–102) i.c.v. (4V) or intra-NTS Rat Standard chow [4V injection]f ↓ Spontaneous feeding and body weight (in food-restricted animals); hypophagic response and weight loss attenuated by pre-treatment with hindbrain delivery of GLP-1R antagonist (exendin-9-39); intra-NTS injection produced no observable effect on feeding or body weight N/A\n2010 Hou et al., 2010 CART I (55–102) intra-Arc; intra-DMH Streptozotocin-diabetic rats Standard chow or HFD Chow diet: ↑ Spontaneous feeding (in satiated animals) (Arc) and ↑ Fast-induced feeding (DMH and Arc); HFD: ↑ Spontaneous feeding (Arc) N/A\n2011 Nakhate et al., 2011 CART I (54–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding and body weight; attenuated social isolation-induced hyperphagia and body weight gain N/A\n2013 Nakhate et al., 2013 CART I (54–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding; attenuated ALLO-induced hyperphagia and weight gain; potentiated DHEAS-induced anorexia and weight loss N/A\nCSF, cerebrospinal fluid; DIO, diet-induced obese; HFD, high fat diet; BAT, brown adipose tissue; i.c.v., intracerebroventricular; i.c., intracisternal; LV, lateral ventricle; 3V, third ventricle; 4V, fourth ventricle; Acb, nucleus accumbens; AcbSh, nucleus accumbens shell; AHA, anterior hypothalamic area; Arc, arcuate nucleus; DMH, dorsomedial nucleus; LHA, lateral hypothalamic area; NTS, nucleus of the solitary tract; PBN, parabrachial nucleus; PVN, paraventricular nucleus; SON, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus.\nEffects on body weight described where results presented, otherwise either unaffected or information unavailable.\nEffects on locomotor behavior described where results presented, otherwise either unaffected or information unavailable.\na Cerebral aqueduct occlusion to interrupt forebrain-hindbrain CSF flow.\nb Inhibition of gastric function (suppression of gastric acid secretion and gastric emptying); inhibition of gastric acid secretion remained in vagotomized animals; inhibition of gastric acid secretion blocked by pretreatment with central administration of CRF receptor antagonist α-helical CRF9-41.\nc Inhibition of gastric function (suppression of gastric emptying); inhibition of gastric emptying blocked by pretreatment with central administration of CRF receptor antagonist α-helical CRF9-41; CART-induced inhibition of gastric emptying proposed unlikely to contribute to CART-mediated inhibition of food intake.\nd Acute administration through repeated injections and chronic overexpression using stereotactically targeted gene transfer.\ne Chronic overexpression using recombinant adeno-associated virus vector containing rat CART cDNA.\nf ↑ Blood glucose levels; hyperglycemic response not altered by GLP-1R blockade in animals pre-treated with GLP-1R antagonist (exendin-9-39).\ng Induction of Fos expression in the PVN, DMH, SON and Arc (hypothalamus), central nucleus of amygdala (cerebrum), PBN and NTS (hindbrain).\nh Induction of Fos expression in NTS neurons.\ni ↑ UCP-1 expression thermogenic capacity in BAT. CART administration via the i.c.v. route was also able to eliminate the increase in feeding and deleterious weight gain caused by social isolation in rats (Nakhate et al., 2011), a consequence of the downregulation of the hypothalamic CART-containing system in various hypothalamic feeding-related areas caused by this condition (Nakhate et al., 2011). In the same study, whilst re-socialization of the isolation-reared rats restored the food intake, body weight, and hypothalamic CART-immunoreactivity back to controls levels, immunoneutralization of endogenous CART by i.c.v. CART antibody attenuated the restoration, confirming the important role of CART in feeding regulation under chronic psychological stress condition (Nakhate et al., 2011). This is consistent with other studies that used antibodies raised against different CART segments for blocking central CART signaling, where all of which were able to neutralize the anorectic property of CART and lead to a significant hyperphagic response (Kristensen et al., 1998; Lambert et al., 1998; Nakhate et al., 2010, 2011). In addition to eliciting an anorectic response, gastrointestinal effects including inhibition of gastric acid secretion and gastric emptying have also been reported as a result from i.c.v. CART (Okumura et al., 2000; Asakawa et al., 2001; Smedh and Moran, 2003, 2006; Tebbe et al., 2004). Chronic overproduction of CART mRNA via viral approaches or continuous infusion of recombinant CART peptide transferred through i.c.v. cannulas into genetically (fa/fa) (Larsen et al., 2000) or diet-induced (Rohner-Jeanrenaud et al., 2002; Qing and Chen, 2007) obese rats induces hypophagic effects during fed states and reduced hyperphagia following fasting were also observed. Such reduction in energy intake was accompanied by suppression of body weight gain mainly due to decrease in lean mass (Larsen et al., 2000; Qing and Chen, 2007), indicating the potential of CART in the long-term regulation of food consumption and body mass, under both normal condition and nutritionally induced obesity.\nIn conjunction with the characterization of physiological responses, neuronal activities stimulated by central CART has been investigated by structural studies for the purpose of identifying brain areas potentially crucial for CART-induced anorectic effects. Following i.c.v. CART administration, temporal expression patterns of the immediate early gene c-Fos (Dragunow and Faull, 1989; VanElzakker et al., 2008), which has been adopted to depict neuronal firing of actions potentials (Dragunow and Faull, 1989; VanElzakker et al., 2008), were found to concentrate in the hypothalamic and brainstem structures implicated in the central regulation of feeding (Vrang et al., 1999b; Zheng et al., 2002). In the hypothalamus in particular, high density of Fos expression was located in the PVN and the posterior DMH, while considerable Fos-IR cells were also identified in the Arc and SO. In the brainstem, Fos-positive cell nuclei were also concentrated in the PBN and, more importantly, in the NTS, which serves a key sensory relay nucleus with reciprocal connections with numerous forebrain and brainstem structures (Vrang et al., 1999b). Such CART-induced Fos activation in the NTS has been indicated independent from possible secondary effects triggered by chemo-activation at the area postrema (AP) directed to the NTS, as the chemosensitive neurons in the AP were devoid of Fos-IR cells (Vrang et al., 1999b). Moderately high Fos expression was also detected in cerebral nuclei associated with autonomic functions and energy balance (Smith and DeVito, 1984; Vrang et al., 1999b), including the central nucleus of the amygdala, where neuronal projections also reciprocally link with the PVN of the hypothalamus and the PBN and NTS of the hindbrain (Hopkins and Holstege, 1978; Holstege et al., 1985). The widespread Fos expression pattern elicited by forebrain i.c.v. CART has been demonstrated to encompass an anatomical continuum of neuronal activations across the cerebrum, hypothalamus and brainstem (Vrang et al., 1999b). The paralleled effects on appetite inhibition and metabolic regulation are believed to portray an integrated outcome of the interactions between central CART-interfered pathways residing primarily within the hypothalamic and brainstem neurons. For instance, as aforementioned, the administration of CART combined with other neuromodulatory such as CCK in mice generated synergistic effects on food intake and locomotion, while displaying concomitant enhancement in the number of Fos-positive neurons compared to injecting each peptide alone (Maletinska et al., 2008; Pirnik et al., 2010). The additive effect on Fos immunoreactivity was especially notable in the target areas common to both peptides, namely the hypothalamic PVN, DMH, VMH and Arc, as well as NTS at the brainstem (Maletinska et al., 2008; Pirnik et al., 2010), wherein the CCK-related satiety signals transmitted to the hindbrain were suggested to be further regulated by leptin action integrated in the Arc as well as neuronal signals from both PVN and LHA (Broberger, 2005; Morton et al., 2005; Maletinska et al., 2008).\nCART is widely expressed in the brain and particularly concentrated in the hypothalamus, suggestive of a diverse range of functions. While effective, delivery of ligands via the i.c.v. route is associated with the downside of the simultaneous stimulation of pathways in various parts of the brain, likely contrasting with effects attributable to the activation of specific neuronal populations. One such case are the CART neurons at the Arc, which respond to and are modulated by leptin signals, leading to the activation of selective neurons and associated downstream pathways (Kristensen et al., 1998; Schwartz et al., 2000). It is therefore unsurprising that the observed effects of the i.c.v. injection of substances like CART are not always replicated by targeted delivery of the same peptide into specific nuclei of the hypothalamus. Indeed, several studies have shown that targeting CART into individual hypothalamic nuclei results in revelation of the orexigenic effects of CART, leading to increased food intake and body weight (Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010). The strongest orexigenic effects were observed by injection of CART into the VMH, DMH and Arc, and a much lesser effect was observed when administered into the PVN, LHA, anterior hypothalamic area, and SO (Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010) (Table 1). Other effects following Arc and PVN CART delivery such as greater energy expenditure and thermogenic capacity, as indirectly measured by the expression and activity of UCP-1 in brown adipose tissue crucial in thermogenesis, has also been reported (Wang et al., 2000; Kong et al., 2003) (Table 1).\nDespite the dependence of endogenous CART expression on nutritional states discussed above, energy states of the animals or dietary options appeared to have little influence over the potency of CART administration-induced feeding stimulation (Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010). For instance, intra-arcuate delivery of CART resulted in elevated food intake in rodents under both fasted, food-restricted and satiated conditions, subjected to the dietary interventions of either regular chow or HFD (Abbott et al., 2001; Kong et al., 2003; Hou et al., 2010). Intriguingly, the orexigenic effects of CART were exhibited in both non-diabetic normal rats as well as streptozotocin-induced diabetic rats, where the intra-Arc CART-induced increase in feeding was reproduced under various energy states and dietary treatments (Hou et al., 2010). Similarly, in rats receiving chronic overexpression of recombinant CART virally delivered into the PVN, higher cumulative food intake and body weight gain was observed in both groups fed either normal chow or HFD compared to control groups, with more pronounced changes in the HFD group (Smith et al., 2008). Underlying such observations, the appetite-promoting effects of hypothalamic intranuclear CART administration may be attributed to a role of hypothalamic CART in stimulating the release of orexigenic neuropeptides locally (Smith et al., 2008; Hou et al., 2010). This is supported by experiments involving a static incubation system where an increase in the release of NPY- and AgRP-IR but not α-MSH-IR was detected in both Arc-containing hypothalamic explants incubated with CART peptide in vitro as well as in PVN-containing hypothalamic explants isolated from animals subjected to intra-PVN CART injection (Smith et al., 2008; Hou et al., 2010). Direct hypothalamic intranuclear CART injection at specific sites, therefore resulted in feeding behaviors opposite to the anorectic effects seen for i.c.v. CART.\nThe discrepancy between the anorexic effects of CART when injected i.c.v. vs. the predominately orexigenic effects of CART when delivered into specific hypothalamic nuclei suggests that CART expression/function in other brain areas may also be important to the regulation of food intake and energy homeostasis, also suggesting that CART may be involved in both anorexigenic and orexigenic circuits in the CNS (Parker and Bloom, 2012). Other potential areas for CART-mediated anorexic effects include the striatum, which is known to have upregulated CART expression following acute i.p. administration of psychostimulants (Douglass et al., 1995; Fagergren and Hurd, 1999; Hubert and Kuhar, 2008), reduced CART mRNA levels following fasting (Kristensen et al., 1998; Adams et al., 1999; Yang and Shieh, 2005), and has been shown to be involved in the mediation of reward and reinforcement (Koob and Bloom, 1988; Salamone, 1996; Upadhya et al., 2012) as well as the neuronal circuits controlling feeding behavior (Bakshi and Kelley, 1993; Gilbert and Cooper, 1995; Pothos et al., 1995; Stratford et al., 1997; Stratford and Kelley, 1999; Baldo et al., 2002; Yang and Shieh, 2005; Upadhya et al., 2012). Evidence for such a role was gained from experiments in a strain of CCK-1 receptor-deficient obese rats, where a significant reduction in CART immunoreactivity in the Arc was found potentially associated with a diminished anorectic effect of CART peptide compared to lean controls (Abraham et al., 2009). Furthermore, intra-accumbal CART peptide injection has been demonstrated to diminish both basal food consumption and food deprivation-induced feeding (Yang and Shieh, 2005; Jean et al., 2007), as well as potently attenuating the orexigenic effects of the GABA-A agonist muscimol (Yang and Shieh, 2005), albeit some inconsistency across different studies (Jaworski et al., 2008) (Table 1). The antagonistic effects of the GABA system and CART at the Acb were also demonstrated in the neurochemical phenotypes of hypothalamic neurons after the appetite-inducing microinjection of muscimol into the AcbSh, which increased Fos expression in orexin neurons at the perifornical area and NPY neurons at the Arc, while inhibiting that in Arc CART/POMC neurons (Zheng et al., 2003). In a recent study, subcutaneous injection of the GABA-A active neurosteroid ALLO significantly reduced CART immunoreactive cells and fibers in the AcbSh, as well as in other feeding-related hypothalamic nuclei such as the PVN, Arc and LHA (Nakhate et al., 2013). Direct CART administration into the Acb performed by an independent group generated no detectable influence on food reward assessed by food self-administration, yet triggered inhibitory effects on cocaine self-administration, an alternative measure of reward and reinforcement entailing dopaminergic functions (Jaworski et al., 2008).\nThe anorexia elicited by intra-accumbal CART was more sustainable in freely fed compared to starved animals, highlighting the significance of fuel status on CART function in feeding modulation (Yang and Shieh, 2005). Furthermore, complementary to the overexpression experiments, RNA-interference has been employed to investigate the effects of CART depletion in rodents (Jean et al., 2007; Job and Kuhar, 2012). Tissue-specific CART knockdown in the Acb via intra-accumbal administration of short interfering RNA (siRNA) or short hairpin (shRNA) against CART mRNA induced body weight gain and hyperphagia in fed mice (Jean et al., 2007; Job and Kuhar, 2012), as well as abolishing the anorectic effects of serotonin (5-hydroxytryptamine, 5-HT) 4 receptor (5-HT4R) stimulation as well as 3,4-N-methylenedioxymethamphetamine (MDMA, ecstasy) treatment in starved mice, further denoting the potential role of Acb CART in mediating the appetite suppressant properties in models of anorexia nervosa (Jean et al., 2007).\nDespite the body of evidence endorsing the plausibility of the Acb as a site for CART-directed anorexia, the appetite-regulating effects produced by intra-accumbal CART likely represent part of the reward and motivational responses derived from an interaction between CART and the dopaminergic system in the Acb. Multiple lines of evidence have suggested an inhibitory role of endogenous accumbal CART in addiction-relevant behaviors, which are speculated to act in concert with feeding modulation as well as the locomotive effects mediated by the dopaminergic circuits (Kim et al., 2003; Jaworski et al., 2008; Hubert et al., 2010; Job and Kuhar, 2012; Upadhya et al., 2012). For instance, substantial innervations have been described for CART-containing neurons in the ventral pallidum, a key nucleus harboring accumbal efferents, where CART-IR terminals were reported to compose symmetric synapses resembling inhibitory GABAergic synapses (Hubert et al., 2010). Whilst intra-accumbal administration of CART alone produced no effect on locomotor activity, co-injection with cocaine or amphetamine into the Acb inhibited the cocaine-like locomotor effects produced by Acb dopamine microinfusions, both intra-accumbal and intra-pallidal injections of CART peptide led to reduction in cocaine- and amphetamine-induced locomotor activity (Jaworski et al., 2003, 2008; Kim et al., 2003; Hubert et al., 2010). Correspondingly, CART depletion through intra-accumbal CART shRNA increased cocaine-mediated locomotion (Job and Kuhar, 2012), supporting an antagonistic property of Acb CART in the functions of cocaine and other psychostimulants.\nThe hindbrain, as a region described to convey post-prandial satiety effects to the hypothalamus, has been considered a potential candidate site for CART action (Marty et al., 2007; Subhedar et al., 2014). Supporting evidence include the moderate CART expression in terms of both transcript levels and immunoreactivity in caudal brain areas such as the locus coeruleus, NTS, PBN and the inferior olive (Douglass et al., 1995; Koylu et al., 1998), accorded with the increased Fos-IR identified in the NTS and PBN following i.c.v. CART into the LV (Vrang et al., 1999b). Comparable to i.c.v. injections into the LV or 3V, hindbrain delivery of CART peptide through the 4th ventricle (4V) led to reduction in food intake and body weight in both fed and food-deprived rodents, whilst the hypophagic effects showed no specificity to nutrients from either chow, sucrose or a nutritionally complete liquid diet (Aja et al., 2001b, 2002; Zheng et al., 2001, 2002; Skibicka et al., 2009) (Table 1). Importantly, the extent of feeding inhibition appeared more potent with CART administered into the 4V compared with LV injections (Zheng et al., 2001), raising the speculation that the hindbrain may house the key mediator for the hypophagic effects of i.c.v. CART (Aja et al., 2001b). Foundation for the idea involved the postulate that the anorectic effects triggered by forebrain i.c.v. CART indeed reflected the outcome of CART diffusion into hindbrain sites via the cerebrospinal fluid (CSF) (Aja et al., 2001b). Surmised from the CART-IR observed in cell bodies and central terminals of vagal afferent neurons projecting to the GI tract, a potential functional role of CART in meal termination and satiety may effectuate at the level of the brainstem (Broberger et al., 1999; Zheng et al., 2002). In rat, vagotomy caused considerable reduction in CART mRNA expression in several CART fibers in the vagus nerve and viscero-sensory nodose ganglion (Broberger et al., 1999). To verify such proposition, cerebral aqueduct occlusion was performed with an aqueductal plug to interrupt the forebrain-hindbrain CSF flow, and CART was injected into the 3V or 4V (Aja et al., 2001b) (Table 1). Interestingly, cerebral aqueduct blockage markedly attenuated the anorectic effects of 3V CART, whilst suppression of food intake remained unchanged when receiving 4V CART injection, signifying the independence of hindbrain CART in producing anorexia (Aja et al., 2001b). In contrast, hindbrain processing may be required or responsible for mediating a hypophagic response following forebrain or interbrain i.c.v. CART, further reinforcing the role of the brainstem in manifesting CART-driven anorectic effects, as concordant with the aforementioned higher potency in feeding inhibition with 4V as opposed to forebrain or interbrain i.c.v. CART (Zheng et al., 2001). Specifically, on the assumption that the repressed ingestive behaviors following LV or 3V CART may attribute to hindbrain CART action, the observations resulted from obtrusion of CSF flow could offer a possible explanation for the opposite feeding effects of orexigenic and anorexigenic natures induced by direct hypothalamic subnuclei (Wang et al., 2000; Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010) vs. hindbrain ventricular (Aja et al., 2001b, 2002; Zheng et al., 2001, 2002; Skibicka et al., 2009) CART administration respectively. Such phenomena promote reevaluation of the authenticity and proposed mechanisms involved in the hypophagia exhibited after forebrain ventricular CART detailed in other studies (Kristensen et al., 1998; Lambert et al., 1998; Vrang et al., 1999b; Larsen et al., 2000; Abbott et al., 2001; Aja et al., 2001a,b)."}

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implications of CART on energy metabolism from pharmacological interventions\nThe identification of the underlying mechanisms by which CART exerts effects on feeding and energy homeostasis have been challenging due to the lack of any knowledge of the corresponding CART receptor (s) and the absence of specific antagonists. Nevertheless, numerous studies incorporating both pharmacological and genetic manipulations of CART expression in murine models have been endeavored in the last decades to determine the sites of action and the effects on feeding behavior and metabolism of the peptide. Overexpression studies to discern the brain regions mediating CART-induced regulation of feeding have been the most common approach in rodents. Widely adopted as an appetite-regulating peptide of the CNS with hypothalamic expression levels modulated by nutritional status (Kristensen et al., 1998; Thim et al., 1998, 1999; Schwartz et al., 2000), CART was administrated i.c.v. to address the effects of overexpression during varying energy states (Kristensen et al., 1998; Lambert et al., 1998; Thim et al., 1998; Vrang et al., 1999b; Edwards et al., 2000; Kask et al., 2000; Larsen et al., 2000; Aja et al., 2001a,b; Bannon et al., 2001; Nakhate et al., 2011, 2013) (Table 1). The lateral ventricle (LV) of the forebrain or the 3rd ventricle (3V) have been the major injection targets. I.c.v. injection of recombinant CART peptide has been consistently demonstrated to inhibit food intake and body weight gain in a dose-dependent manner in both food-restricted and free-feeding conditions as well as both under standard chow or a nutritionally complete liquid diet, in either normal or diet-induced obese animals (Kristensen et al., 1998; Lambert et al., 1998; Thim et al., 1998; Vrang et al., 1999b; Edwards et al., 2000; Kask et al., 2000; Larsen et al., 2000; Abbott et al., 2001; Aja et al., 2001a,b; Bannon et al., 2001; Rohner-Jeanrenaud et al., 2002; Tachibana et al., 2003; Wortley et al., 2004; Qing and Chen, 2007; Nakhate et al., 2011, 2013). Furthermore, the catabolic capacity of CART appeared sufficient to prevent and attenuate the orexigenic effects of NPY, as i.c.v. and intra-PVN CART potently suppressed feeding in satiated rats subjected to NPY-induced hyperphagia (Kristensen et al., 1998; Lambert et al., 1998; Vrang et al., 1999b; Wang et al., 2000; Rohner-Jeanrenaud et al., 2002). Similarly, the anorectic potency of CART has also been demonstrated in a recent study focusing on the interaction between CART and the GABA type A receptor (GABA-A) active neurosteroid allopregnanolone (ALLO) and the inhibitor neurosteroid dehydroepiandrosterone sulfate (DHEAs) (Nakhate et al., 2013). It was shown in rodents that pre-treatment of i.c.v. CART effectively attenuated subcutaneous ALLO-induced hyperphagia and weight gain, as well as potentiating DHEAS-induced hypophagic and weight reducing effects (Nakhate et al., 2013).\nTable 1 Summary of the metabolic and behavioral effects of central CART administration via various intracerebroventricular and intranuclear delivery methods.\nYear Publication Targeting peptide/CART fragment Route of adminis-tration Species (genetic background) Diet Feeding behavior and body weight alterations Locomotor behavior\n1998 Kristensen et al., 1998 CART I (55–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding; ↓ Fast-induced feeding; ↓ NPY-induced feeding N/A\n1998 Lambert et al., 1998 CART (55–59) i.c.v. Rat Standard chow ~ Spontaneous feeding N/A\n1998 Lambert et al., 1998 CART (55–76) i.c.v. Rat Standard chow ↓ Spontaneous feeding; ↓ NPY-induced feeding N/A\n1998 Lambert et al., 1998 CART (62–76) i.c.v. Rat Standard chow ↓ Spontaneous feeding N/A\n1998 Thim et al., 1998 CART I (55–102); CART II (62–102) i.c.v. Mouse Standard chow ↓ Fast-induced feeding N/A\n1999 Vrang et al., 1999b CART I (42–89) i.c.v.g Rat Standard chow ↓ Spontaneous feeding (in food-restricted animals) and ↓ NPY-induced feeding N/A\n2000 Edwards et al., 2000 CART I (55–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding N/A\n2000 Kask et al., 2000 CART (62–76) i.c.v. Rat Standard chow ↓ Spontaneous feeding N/A\n2000 Volkoff and Peter, 2000 CART I (55–102); CART (62–76) i.c.v. Goldfish Standard chow ↓ Spontaneous feeding and ↓ NPY-induced feeding (in food-restricted animals) N/A\n2000 Larsen et al., 2000 CART I (42–89) i.c.v. (chronic infusion) Lean and obese Zucker (fa/fa) rats Standard chow ↓ Spontaneous feeding (in both free-feeding and food-restricted animals); dose-dependent ↓ in body weight Dose-dependent motor disturbances (combined gait and walking ataxia)\n2000 Okumura et al., 2000 CART I (55–102) i.c. Rat Standard chow ↓ Fast-induced feedingb N/A\n2000 Wang et al., 2000 CART I (55–102) intra-PVN Rat Standard chow ↓ NPY-induced feedingi N/A\n2001 Bannon et al., 2001 CART I (55–102); CART II (62–102) i.c.v. Mouse Standard chow ↓ Fast-induced feeding N/A\n2001 Abbott et al., 2001 CART I (55–102) Hypothalamic intranuclear injections (VMH, Arc, PVN, SON, DMH, LHA and AHA) Rat Standard chow ↑ Spontaneous feeding (only measured in satiated animals cannulated into the DMH or Arc); ↑ Fast-induced feeding N/A\n2001 Abbott et al., 2001 CART I (55–102) i.c.v. (3V) Rat Standard chow ↓ Spontaneous feeding; ↓ Fast-induced feeding (↓ feeding episodes) Behavioral abnormalities marked by reduced feeding episodes, flat-backed posture and movement-associated tremors (behavioral analysis performed for 24-h fasted animals only but not satiated animals)\n2001 Zheng et al., 2001 CART I (55–102) i.c.v. (LV and 4V) Rat Sucrose solution or standard chow ↓ Spontaneous feeding (↓ short-term sucrose intake and ↓ overnight chow intake)—effects more pronounced in 4V compared to LV administration Alterations in motor behavior (mild movement-associated tremors in part of 4V injected subjects)\n2001a Aja et al., 2001a CART I (55–102) i.c.v. Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet intake in licks and meal size in food restricted animals) Altered oral motor function and behavioral alterations (trance-like state, flat-backed and arched-backed postures, cage licking, movement-associated tremors)\n2001b Aja et al., 2001b CART I (55–102) i.c.v. (3V) Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet intake and observations of feeding in food restricted animals)—reductions significantly attentuated by aqueduct obstructiona Alterations in motor behavior (flat-backed and arched-backed postures and movement-associated tremors)—alterations significantly attentuated by aqueduct obstructiona\n2001b Aja et al., 2001b CART I (55–102) i.c.v. (4V) Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet intake and observations of feeding in food restricted animals)—reductions unaffected by aqueduct obstructiona Alterations in motor behavior (flat-backed and arched-backed postures and movement-associated tremors)—alterations unaffected by aqueduct obstructiona\n2002 Aja et al., 2002 CART I (55–102) i.c.v. (4V) Rat Ensure liquid diet ↓ Spontaneous feeding (↓ liquid diet and water intake in food restricted animals); production of conditioned taste aversion N/A\n2002 Rohner-Jeanrenaud et al., 2002 CART I (55–102) i.c.v. (chronic infusion) Rat (normal and DIO) Standard chow or HFD ↓ Spontaneous feeding and ↓ NPY-induced feeding; ↓ body weight gain N/A\n2002 Zheng et al., 2002 CART I (55–102) i.c.v. (4V)h and intra-NTS Rat Sucrose solution or standard chow ↓ Spontaneous feeding (↓ short-term sucrose intake)—effects more pronounced in 4V compared to intra-NTS administration N/A\n2003 Smedh and Moran, 2003 CART I (55–102) i.c.v. (4V) Rat Sucrose solution ↓ Spontaneous feeding (↓ sucrose intake in food restricted animals); altered lick microstrcuture parametersc N/A\n2003 Kong et al., 2003 CART I (55–102) intra-Arcd Rat Standard chow ↑ Spontaneous feeding (in both free-feeding and food-restricted animals) and ↑ Fast-induced feeding; ↑ cumulative body weight gain; ↑ body weight loss following 24-hr fasting and food restrictioni N/A\n2004 Wortley et al., 2004 CART I (55–102) i.c.v. (3V) Rat Standard chow ↓ Spontaneous feeding N/A\n2005 Yang et al., 2005 CART I (55–102) intra-AcbSh Rat Standard chow ↓ Spontaneous feeding; ↓ Fast-induced feeding; ↓ GABA-A agonist muscimol-induced feeding N/A\n2007 Qing and Chen, 2007 rat CART cDNA i.c.v.e Rat (DIO) High fat/high sucrose diet ↓ Spontaneous feeding; ↓ Fast-induced feeding; ↓ body weight gain (↓ lean mass; fat mass unaffected) N/A\n2007 Jean et al., 2007 CART I (55–102) intra-AcbSh Mouse Standard chow ↓ Fast-induced feeding N/A\n2007 Jean et al., 2007 CART siRNA intra-AcbSh Mouse Standard chow ↑ Spontaneous feeding and ↓ stimulating 5-HT4R- or MDMA-induced anorexia in staved animals N/A\n2008 Smith et al., 2008 rAAV encoding full length rat CART cDNA (GenBank accession no. U10071) intra-PVNe Rat Standard chow or HFD ↑ Cumulative feeding and cumulative body weight gain; effects more accentuated on HFD N/A\n2009 Skibicka et al., 2009 CART I (55–102) i.c.v. (4V) or intra-NTS Rat Standard chow [4V injection]f ↓ Spontaneous feeding and body weight (in food-restricted animals); hypophagic response and weight loss attenuated by pre-treatment with hindbrain delivery of GLP-1R antagonist (exendin-9-39); intra-NTS injection produced no observable effect on feeding or body weight N/A\n2010 Hou et al., 2010 CART I (55–102) intra-Arc; intra-DMH Streptozotocin-diabetic rats Standard chow or HFD Chow diet: ↑ Spontaneous feeding (in satiated animals) (Arc) and ↑ Fast-induced feeding (DMH and Arc); HFD: ↑ Spontaneous feeding (Arc) N/A\n2011 Nakhate et al., 2011 CART I (54–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding and body weight; attenuated social isolation-induced hyperphagia and body weight gain N/A\n2013 Nakhate et al., 2013 CART I (54–102) i.c.v. Rat Standard chow ↓ Spontaneous feeding; attenuated ALLO-induced hyperphagia and weight gain; potentiated DHEAS-induced anorexia and weight loss N/A\nCSF, cerebrospinal fluid; DIO, diet-induced obese; HFD, high fat diet; BAT, brown adipose tissue; i.c.v., intracerebroventricular; i.c., intracisternal; LV, lateral ventricle; 3V, third ventricle; 4V, fourth ventricle; Acb, nucleus accumbens; AcbSh, nucleus accumbens shell; AHA, anterior hypothalamic area; Arc, arcuate nucleus; DMH, dorsomedial nucleus; LHA, lateral hypothalamic area; NTS, nucleus of the solitary tract; PBN, parabrachial nucleus; PVN, paraventricular nucleus; SON, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus.\nEffects on body weight described where results presented, otherwise either unaffected or information unavailable.\nEffects on locomotor behavior described where results presented, otherwise either unaffected or information unavailable.\na Cerebral aqueduct occlusion to interrupt forebrain-hindbrain CSF flow.\nb Inhibition of gastric function (suppression of gastric acid secretion and gastric emptying); inhibition of gastric acid secretion remained in vagotomized animals; inhibition of gastric acid secretion blocked by pretreatment with central administration of CRF receptor antagonist α-helical CRF9-41.\nc Inhibition of gastric function (suppression of gastric emptying); inhibition of gastric emptying blocked by pretreatment with central administration of CRF receptor antagonist α-helical CRF9-41; CART-induced inhibition of gastric emptying proposed unlikely to contribute to CART-mediated inhibition of food intake.\nd Acute administration through repeated injections and chronic overexpression using stereotactically targeted gene transfer.\ne Chronic overexpression using recombinant adeno-associated virus vector containing rat CART cDNA.\nf ↑ Blood glucose levels; hyperglycemic response not altered by GLP-1R blockade in animals pre-treated with GLP-1R antagonist (exendin-9-39).\ng Induction of Fos expression in the PVN, DMH, SON and Arc (hypothalamus), central nucleus of amygdala (cerebrum), PBN and NTS (hindbrain).\nh Induction of Fos expression in NTS neurons.\ni ↑ UCP-1 expression thermogenic capacity in BAT. CART administration via the i.c.v. route was also able to eliminate the increase in feeding and deleterious weight gain caused by social isolation in rats (Nakhate et al., 2011), a consequence of the downregulation of the hypothalamic CART-containing system in various hypothalamic feeding-related areas caused by this condition (Nakhate et al., 2011). In the same study, whilst re-socialization of the isolation-reared rats restored the food intake, body weight, and hypothalamic CART-immunoreactivity back to controls levels, immunoneutralization of endogenous CART by i.c.v. CART antibody attenuated the restoration, confirming the important role of CART in feeding regulation under chronic psychological stress condition (Nakhate et al., 2011). This is consistent with other studies that used antibodies raised against different CART segments for blocking central CART signaling, where all of which were able to neutralize the anorectic property of CART and lead to a significant hyperphagic response (Kristensen et al., 1998; Lambert et al., 1998; Nakhate et al., 2010, 2011). In addition to eliciting an anorectic response, gastrointestinal effects including inhibition of gastric acid secretion and gastric emptying have also been reported as a result from i.c.v. CART (Okumura et al., 2000; Asakawa et al., 2001; Smedh and Moran, 2003, 2006; Tebbe et al., 2004). Chronic overproduction of CART mRNA via viral approaches or continuous infusion of recombinant CART peptide transferred through i.c.v. cannulas into genetically (fa/fa) (Larsen et al., 2000) or diet-induced (Rohner-Jeanrenaud et al., 2002; Qing and Chen, 2007) obese rats induces hypophagic effects during fed states and reduced hyperphagia following fasting were also observed. Such reduction in energy intake was accompanied by suppression of body weight gain mainly due to decrease in lean mass (Larsen et al., 2000; Qing and Chen, 2007), indicating the potential of CART in the long-term regulation of food consumption and body mass, under both normal condition and nutritionally induced obesity.\nIn conjunction with the characterization of physiological responses, neuronal activities stimulated by central CART has been investigated by structural studies for the purpose of identifying brain areas potentially crucial for CART-induced anorectic effects. Following i.c.v. CART administration, temporal expression patterns of the immediate early gene c-Fos (Dragunow and Faull, 1989; VanElzakker et al., 2008), which has been adopted to depict neuronal firing of actions potentials (Dragunow and Faull, 1989; VanElzakker et al., 2008), were found to concentrate in the hypothalamic and brainstem structures implicated in the central regulation of feeding (Vrang et al., 1999b; Zheng et al., 2002). In the hypothalamus in particular, high density of Fos expression was located in the PVN and the posterior DMH, while considerable Fos-IR cells were also identified in the Arc and SO. In the brainstem, Fos-positive cell nuclei were also concentrated in the PBN and, more importantly, in the NTS, which serves a key sensory relay nucleus with reciprocal connections with numerous forebrain and brainstem structures (Vrang et al., 1999b). Such CART-induced Fos activation in the NTS has been indicated independent from possible secondary effects triggered by chemo-activation at the area postrema (AP) directed to the NTS, as the chemosensitive neurons in the AP were devoid of Fos-IR cells (Vrang et al., 1999b). Moderately high Fos expression was also detected in cerebral nuclei associated with autonomic functions and energy balance (Smith and DeVito, 1984; Vrang et al., 1999b), including the central nucleus of the amygdala, where neuronal projections also reciprocally link with the PVN of the hypothalamus and the PBN and NTS of the hindbrain (Hopkins and Holstege, 1978; Holstege et al., 1985). The widespread Fos expression pattern elicited by forebrain i.c.v. CART has been demonstrated to encompass an anatomical continuum of neuronal activations across the cerebrum, hypothalamus and brainstem (Vrang et al., 1999b). The paralleled effects on appetite inhibition and metabolic regulation are believed to portray an integrated outcome of the interactions between central CART-interfered pathways residing primarily within the hypothalamic and brainstem neurons. For instance, as aforementioned, the administration of CART combined with other neuromodulatory such as CCK in mice generated synergistic effects on food intake and locomotion, while displaying concomitant enhancement in the number of Fos-positive neurons compared to injecting each peptide alone (Maletinska et al., 2008; Pirnik et al., 2010). The additive effect on Fos immunoreactivity was especially notable in the target areas common to both peptides, namely the hypothalamic PVN, DMH, VMH and Arc, as well as NTS at the brainstem (Maletinska et al., 2008; Pirnik et al., 2010), wherein the CCK-related satiety signals transmitted to the hindbrain were suggested to be further regulated by leptin action integrated in the Arc as well as neuronal signals from both PVN and LHA (Broberger, 2005; Morton et al., 2005; Maletinska et al., 2008).\nCART is widely expressed in the brain and particularly concentrated in the hypothalamus, suggestive of a diverse range of functions. While effective, delivery of ligands via the i.c.v. route is associated with the downside of the simultaneous stimulation of pathways in various parts of the brain, likely contrasting with effects attributable to the activation of specific neuronal populations. One such case are the CART neurons at the Arc, which respond to and are modulated by leptin signals, leading to the activation of selective neurons and associated downstream pathways (Kristensen et al., 1998; Schwartz et al., 2000). It is therefore unsurprising that the observed effects of the i.c.v. injection of substances like CART are not always replicated by targeted delivery of the same peptide into specific nuclei of the hypothalamus. Indeed, several studies have shown that targeting CART into individual hypothalamic nuclei results in revelation of the orexigenic effects of CART, leading to increased food intake and body weight (Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010). The strongest orexigenic effects were observed by injection of CART into the VMH, DMH and Arc, and a much lesser effect was observed when administered into the PVN, LHA, anterior hypothalamic area, and SO (Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010) (Table 1). Other effects following Arc and PVN CART delivery such as greater energy expenditure and thermogenic capacity, as indirectly measured by the expression and activity of UCP-1 in brown adipose tissue crucial in thermogenesis, has also been reported (Wang et al., 2000; Kong et al., 2003) (Table 1).\nDespite the dependence of endogenous CART expression on nutritional states discussed above, energy states of the animals or dietary options appeared to have little influence over the potency of CART administration-induced feeding stimulation (Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010). For instance, intra-arcuate delivery of CART resulted in elevated food intake in rodents under both fasted, food-restricted and satiated conditions, subjected to the dietary interventions of either regular chow or HFD (Abbott et al., 2001; Kong et al., 2003; Hou et al., 2010). Intriguingly, the orexigenic effects of CART were exhibited in both non-diabetic normal rats as well as streptozotocin-induced diabetic rats, where the intra-Arc CART-induced increase in feeding was reproduced under various energy states and dietary treatments (Hou et al., 2010). Similarly, in rats receiving chronic overexpression of recombinant CART virally delivered into the PVN, higher cumulative food intake and body weight gain was observed in both groups fed either normal chow or HFD compared to control groups, with more pronounced changes in the HFD group (Smith et al., 2008). Underlying such observations, the appetite-promoting effects of hypothalamic intranuclear CART administration may be attributed to a role of hypothalamic CART in stimulating the release of orexigenic neuropeptides locally (Smith et al., 2008; Hou et al., 2010). This is supported by experiments involving a static incubation system where an increase in the release of NPY- and AgRP-IR but not α-MSH-IR was detected in both Arc-containing hypothalamic explants incubated with CART peptide in vitro as well as in PVN-containing hypothalamic explants isolated from animals subjected to intra-PVN CART injection (Smith et al., 2008; Hou et al., 2010). Direct hypothalamic intranuclear CART injection at specific sites, therefore resulted in feeding behaviors opposite to the anorectic effects seen for i.c.v. CART.\nThe discrepancy between the anorexic effects of CART when injected i.c.v. vs. the predominately orexigenic effects of CART when delivered into specific hypothalamic nuclei suggests that CART expression/function in other brain areas may also be important to the regulation of food intake and energy homeostasis, also suggesting that CART may be involved in both anorexigenic and orexigenic circuits in the CNS (Parker and Bloom, 2012). Other potential areas for CART-mediated anorexic effects include the striatum, which is known to have upregulated CART expression following acute i.p. administration of psychostimulants (Douglass et al., 1995; Fagergren and Hurd, 1999; Hubert and Kuhar, 2008), reduced CART mRNA levels following fasting (Kristensen et al., 1998; Adams et al., 1999; Yang and Shieh, 2005), and has been shown to be involved in the mediation of reward and reinforcement (Koob and Bloom, 1988; Salamone, 1996; Upadhya et al., 2012) as well as the neuronal circuits controlling feeding behavior (Bakshi and Kelley, 1993; Gilbert and Cooper, 1995; Pothos et al., 1995; Stratford et al., 1997; Stratford and Kelley, 1999; Baldo et al., 2002; Yang and Shieh, 2005; Upadhya et al., 2012). Evidence for such a role was gained from experiments in a strain of CCK-1 receptor-deficient obese rats, where a significant reduction in CART immunoreactivity in the Arc was found potentially associated with a diminished anorectic effect of CART peptide compared to lean controls (Abraham et al., 2009). Furthermore, intra-accumbal CART peptide injection has been demonstrated to diminish both basal food consumption and food deprivation-induced feeding (Yang and Shieh, 2005; Jean et al., 2007), as well as potently attenuating the orexigenic effects of the GABA-A agonist muscimol (Yang and Shieh, 2005), albeit some inconsistency across different studies (Jaworski et al., 2008) (Table 1). The antagonistic effects of the GABA system and CART at the Acb were also demonstrated in the neurochemical phenotypes of hypothalamic neurons after the appetite-inducing microinjection of muscimol into the AcbSh, which increased Fos expression in orexin neurons at the perifornical area and NPY neurons at the Arc, while inhibiting that in Arc CART/POMC neurons (Zheng et al., 2003). In a recent study, subcutaneous injection of the GABA-A active neurosteroid ALLO significantly reduced CART immunoreactive cells and fibers in the AcbSh, as well as in other feeding-related hypothalamic nuclei such as the PVN, Arc and LHA (Nakhate et al., 2013). Direct CART administration into the Acb performed by an independent group generated no detectable influence on food reward assessed by food self-administration, yet triggered inhibitory effects on cocaine self-administration, an alternative measure of reward and reinforcement entailing dopaminergic functions (Jaworski et al., 2008).\nThe anorexia elicited by intra-accumbal CART was more sustainable in freely fed compared to starved animals, highlighting the significance of fuel status on CART function in feeding modulation (Yang and Shieh, 2005). Furthermore, complementary to the overexpression experiments, RNA-interference has been employed to investigate the effects of CART depletion in rodents (Jean et al., 2007; Job and Kuhar, 2012). Tissue-specific CART knockdown in the Acb via intra-accumbal administration of short interfering RNA (siRNA) or short hairpin (shRNA) against CART mRNA induced body weight gain and hyperphagia in fed mice (Jean et al., 2007; Job and Kuhar, 2012), as well as abolishing the anorectic effects of serotonin (5-hydroxytryptamine, 5-HT) 4 receptor (5-HT4R) stimulation as well as 3,4-N-methylenedioxymethamphetamine (MDMA, ecstasy) treatment in starved mice, further denoting the potential role of Acb CART in mediating the appetite suppressant properties in models of anorexia nervosa (Jean et al., 2007).\nDespite the body of evidence endorsing the plausibility of the Acb as a site for CART-directed anorexia, the appetite-regulating effects produced by intra-accumbal CART likely represent part of the reward and motivational responses derived from an interaction between CART and the dopaminergic system in the Acb. Multiple lines of evidence have suggested an inhibitory role of endogenous accumbal CART in addiction-relevant behaviors, which are speculated to act in concert with feeding modulation as well as the locomotive effects mediated by the dopaminergic circuits (Kim et al., 2003; Jaworski et al., 2008; Hubert et al., 2010; Job and Kuhar, 2012; Upadhya et al., 2012). For instance, substantial innervations have been described for CART-containing neurons in the ventral pallidum, a key nucleus harboring accumbal efferents, where CART-IR terminals were reported to compose symmetric synapses resembling inhibitory GABAergic synapses (Hubert et al., 2010). Whilst intra-accumbal administration of CART alone produced no effect on locomotor activity, co-injection with cocaine or amphetamine into the Acb inhibited the cocaine-like locomotor effects produced by Acb dopamine microinfusions, both intra-accumbal and intra-pallidal injections of CART peptide led to reduction in cocaine- and amphetamine-induced locomotor activity (Jaworski et al., 2003, 2008; Kim et al., 2003; Hubert et al., 2010). Correspondingly, CART depletion through intra-accumbal CART shRNA increased cocaine-mediated locomotion (Job and Kuhar, 2012), supporting an antagonistic property of Acb CART in the functions of cocaine and other psychostimulants.\nThe hindbrain, as a region described to convey post-prandial satiety effects to the hypothalamus, has been considered a potential candidate site for CART action (Marty et al., 2007; Subhedar et al., 2014). Supporting evidence include the moderate CART expression in terms of both transcript levels and immunoreactivity in caudal brain areas such as the locus coeruleus, NTS, PBN and the inferior olive (Douglass et al., 1995; Koylu et al., 1998), accorded with the increased Fos-IR identified in the NTS and PBN following i.c.v. CART into the LV (Vrang et al., 1999b). Comparable to i.c.v. injections into the LV or 3V, hindbrain delivery of CART peptide through the 4th ventricle (4V) led to reduction in food intake and body weight in both fed and food-deprived rodents, whilst the hypophagic effects showed no specificity to nutrients from either chow, sucrose or a nutritionally complete liquid diet (Aja et al., 2001b, 2002; Zheng et al., 2001, 2002; Skibicka et al., 2009) (Table 1). Importantly, the extent of feeding inhibition appeared more potent with CART administered into the 4V compared with LV injections (Zheng et al., 2001), raising the speculation that the hindbrain may house the key mediator for the hypophagic effects of i.c.v. CART (Aja et al., 2001b). Foundation for the idea involved the postulate that the anorectic effects triggered by forebrain i.c.v. CART indeed reflected the outcome of CART diffusion into hindbrain sites via the cerebrospinal fluid (CSF) (Aja et al., 2001b). Surmised from the CART-IR observed in cell bodies and central terminals of vagal afferent neurons projecting to the GI tract, a potential functional role of CART in meal termination and satiety may effectuate at the level of the brainstem (Broberger et al., 1999; Zheng et al., 2002). In rat, vagotomy caused considerable reduction in CART mRNA expression in several CART fibers in the vagus nerve and viscero-sensory nodose ganglion (Broberger et al., 1999). To verify such proposition, cerebral aqueduct occlusion was performed with an aqueductal plug to interrupt the forebrain-hindbrain CSF flow, and CART was injected into the 3V or 4V (Aja et al., 2001b) (Table 1). Interestingly, cerebral aqueduct blockage markedly attenuated the anorectic effects of 3V CART, whilst suppression of food intake remained unchanged when receiving 4V CART injection, signifying the independence of hindbrain CART in producing anorexia (Aja et al., 2001b). In contrast, hindbrain processing may be required or responsible for mediating a hypophagic response following forebrain or interbrain i.c.v. CART, further reinforcing the role of the brainstem in manifesting CART-driven anorectic effects, as concordant with the aforementioned higher potency in feeding inhibition with 4V as opposed to forebrain or interbrain i.c.v. CART (Zheng et al., 2001). Specifically, on the assumption that the repressed ingestive behaviors following LV or 3V CART may attribute to hindbrain CART action, the observations resulted from obtrusion of CSF flow could offer a possible explanation for the opposite feeding effects of orexigenic and anorexigenic natures induced by direct hypothalamic subnuclei (Wang et al., 2000; Abbott et al., 2001; Kong et al., 2003; Smith et al., 2008; Hou et al., 2010) vs. hindbrain ventricular (Aja et al., 2001b, 2002; Zheng et al., 2001, 2002; Skibicka et al., 2009) CART administration respectively. Such phenomena promote reevaluation of the authenticity and proposed mechanisms involved in the hypophagia exhibited after forebrain ventricular CART detailed in other studies (Kristensen et al., 1998; Lambert et al., 1998; Vrang et al., 1999b; Larsen et al., 2000; Abbott et al., 2001; Aja et al., 2001a,b)."}