AMPA and NMDA receptors in dentate gyrus mediate memory for sucrose in two port discrimination task.

Although the phenomenon of memory formation and recall associated with the use of psychotropic drugs has been extensively studied, mechanisms underlying memories for natural reward have not been clarified. Herein, we test the hypothesis that glutamatergic receptors in the dentate gyrus play a role in memories associated with sucrose. We used pellet self-administration protocol to generate memories in two-port nose-poke discrimination task using male Wistar rats. During non-rewarded probe trial, the conditioned animals readily discriminated the active port versus inactive port and showed massive increase in mRNA expression of AMPA receptor subunit genes (gria2, gria3) as well as c-Fos protein in the DG. Access to sweet pellet further enhanced c-Fos expression in the DG. However, animals pre-treated with AMPA receptor antagonist CNQX (intra-DG), on exposure to operant chamber (no pellet), showed decreased discrimination as well as c-Fos expression. We suggest that AMPA receptors in DG mediate recall and consolidation of memories associated with sucrose consumption. CNQX pre-treated animals, if presented with sweet pellet on nose poke, exhibited high discrimination index coupled with increased c-Fos expression. In these CNQX treated rats, the DI was again decreased following administration of NMDA receptor antagonist AP5. We suggest that, although AMPA receptors are blocked, the access to sweet pellet may induce surge of glutamate in the DG, which in turn may reinstate memories via activation of erstwhile silent synapses in NMDA dependant manner.

Response of nitrergic system in the brain of rat conditioned to intracranial self-stimulation.

The role of nitrergic system in modulating the action of psychostimulants on reward processing is well established. However, the relevant anatomical underpinnings and scope of the involved interactions with mesolimbic dopaminergic system have not been clarified. Using immunohistochemistry, we track the changes in neuronal nitric oxide synthase (nNOS) containing cell groups in the animals conditioned to intracranial self-stimulation (ICSS) via an electrode implanted in the lateral hypothalamus-medial forebrain bundle (LH-MFB) area. An increase in the nNOS immunoreactivity was noticed in the cells and fibers in the ventral tegmental area (VTA) and nucleus accumbens shell (AcbSh), the primary loci of the reward system. In addition, nNOS was up-regulated in the nucleus accumbens core (AcbC), vertical limb of diagonal band (VDB), locus coeruleus (LC), lateral hypothalamus (LH), superficial gray layer (SuG) of the superior colliculus, and periaqueductal gray (PAG). The brain tissue fragments drawn from these areas showed a change in nNOS mRNA expression, but in opposite direction. Intracerebroventricular (icv) administration of nNOS inhibitor, 7-nitroindazole (7-NI) showed decreased lever press activity in a dose-dependent manner in ICSS task. While an increase in the dopamine (DA) and 3, 4-dihydroxyphenylacetic acid (DOPAC) efflux was noted in the microdialysates collected from the AcbSh of ICSS rats, pre-administration of 7-NI (icv route) attenuated the response. The study identifies nitrergic centers that probably mediate sensory, cognitive, and motor components of the goal-directed behavior.

Mate calling alters expression of neuropeptide, cocaine- and amphetamine- regulated transcript (CART) in the brain of male frog Microhyla nilphamariensis.

Croaking is a unique component of reproductive behaviour in amphibians which plays a key role in intraspecies communication and mate evaluation. While gonadal hormones are known to induce croaking, central regulation of sound production is less studied. Croaking is a dramatic, transient activity that sets apart an animal from non-croaking individuals. Herein, we aim at examining the profile of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in actively croaking and non-croaking frog Microhyla nilphamariensis. In anurans, this peptide is widely expressed in the areas inclusive of acoustical nuclei as well as areas relevant to reproduction. CART immunoreactivity was far more in the preoptic area (POA), anteroventral tegmentum (AV), ventral hypothalamus (vHy), pineal (P) and pituitary gland of croaking frog compared to non-croaking animals. On similar lines, tissue fragments collected from the mid region of the brain inclusive of POA, vHy, AV, pineal and pituitary gland of croaking frog showed upregulation of CART mRNA. However, CART immunoreactivity in the neuronal perikarya of raphe (Ra) was completely abolished during croaking activity. The data suggest that CART signaling in the brain may be an important player in mediating croaking behaviour in the frog.

Convergent Energy State-Dependent Antagonistic Signaling by Cocaine- and Amphetamine-Regulated Transcript (CART) and Neuropeptide Y (NPY) Modulates the Plasticity of Forebrain Neurons to Regulate Feeding in Zebrafish.

Dynamic reconfiguration of circuit function subserves the flexibility of innate behaviors tuned to physiological states. Internal energy stores adaptively regulate feeding-associated behaviors and integrate opposing hunger and satiety signals at the level of neural circuits. Across vertebrate lineages, the neuropeptides cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) have potent anorexic and orexic functions, respectively, and show energy-state-dependent expression in interoceptive neurons. However, how the antagonistic activities of these peptides modulate circuit plasticity remains unclear. Using behavioral, neuroanatomical, and activity analysis in adult zebrafish of both sexes, along with pharmacological interventions, we show that CART and NPY activities converge on a population of neurons in the dorsomedial telencephalon (Dm). Although CART facilitates glutamatergic neurotransmission at the Dm, NPY dampens the response to glutamate. In energy-rich states, CART enhances NMDA receptor (NMDAR) function by protein kinase A/protein kinase C (PKA/PKC)-mediated phosphorylation of the NR1 subunit of the NMDAR complex. Conversely, starvation triggers NPY-mediated reduction in phosphorylated NR1 via calcineurin activation and inhibition of cAMP production leading to reduced responsiveness to glutamate. Our data identify convergent integration of CART and NPY inputs by the Dm neurons to generate nutritional state-dependent circuit plasticity that is correlated with the behavioral switch induced by the opposing actions of satiety and hunger signals.SIGNIFICANCE STATEMENT Internal energy needs reconfigure neuronal circuits to adaptively regulate feeding behavior. Energy-state-dependent neuropeptide release can signal energy status to feeding-associated circuits and modulate circuit function. CART and NPY are major anorexic and orexic factors, respectively, but the intracellular signaling pathways used by these peptides to alter circuit function remain uncharacterized. We show that CART and NPY-expressing neurons from energy-state interoceptive areas project to a novel telencephalic region, Dm, in adult zebrafish. CART increases the excitability of Dm neurons, whereas NPY opposes CART activity. Antagonistic signaling by CART and NPY converge onto NMDA-receptor function to modulate glutamatergic neurotransmission. Thus, opposing activities of anorexic CART and orexic NPY reconfigure circuit function to generate flexibility in feeding behavior.

Neuropeptide S facilitates extinction of fear via modulation of mesolimbic dopaminergic circuitry.

The inability to extinguish learned fear is a hallmark of trauma- and stress-related disorders. A form of inhibitory learning called fear extinction is an effective way to treat these disorders. However, the neurobiology of fear extinction has not been clarified. The involvement of a dopaminergic pathway from the ventral tegmental area (VTA) to the nucleus accumbens shell (AcbSh) in fear extinction has been suggested. Several neuropeptide systems, including neuropeptide S (NPS), modulate the activity of VTA dopaminergic neurons. Herein, we investigated the role of NPS in modulating the VTA-AcbSh circuit in fear extinction. While the NPS-containing neurons of the pericoerulear (periLC) area project to the VTA, the recipient cells are equipped with NPS receptors. Using a Pavlovian fear conditioning procedure, we tested the effect of NPS on fear extinction in male Wistar rats. Intra-VTA administration of NPS prior to fear extinction training facilitated the fear extinction learning and memory, however, NPS receptors antagonist had the opposite effect. Fear extinction training increased the dopamine efflux and cFOS immunoreactivity in the AcbSh area of NPS-treated rats compared with the vehicle-injected controls. We suggest that the NPS neurons of the periLC project to the VTA and might facilitate fear extinction by enhancing the activity of mesolimbic dopaminergic circuit.

In-house fabrication of bipolar electrode-cannula assembly for electrical stimulation and drug delivery at the same site in rat brain.

Strategies drawn at understanding the functional attributes of specific neural circuits often necessitate electrical stimulation and pharmacological manipulation at the same anatomical site. We describe a simple, inexpensive and reliable method to fabricate a bipolar electrode-cannula assembly for delivery of electric pulses and administration of neuroactive agents at the same site in the rat brain. The assembly consisting of a guide cannula, dummy cannula, internal cannula and bipolar electrode was fabricated using syringe needles, wires and simple electronic components. To test the usefulness of the device, it was implanted on the skull of a rat specifically targeting the posterior ventral tegmental area (pVTA). The rat was conditioned to press the lever in intracranial self-stimulation (ICSS) protocol in an operant chamber. The number of lever presses in a 30 min task was monitored. Intra-pVTA administration with bicuculline (GABAA receptor antagonist) increased the lever press activity, while muscimol (GABAA receptor agonist) had opposite effect. The results confirm that the group of neurons responding to the electrical stimulation probably receive GABAergic inputs. The device is light in weight, costs less than a dollar and can be fabricated from readily available components. It can serve a useful purpose in electrically stimulating any given target in the brain - before, during or after pharmacological manipulation at the same locus and may find application in neuropharmacological and neurobehavioral studies.

TET1-induced DNA demethylation in dentate gyrus is important for reward conditioning and reinforcement.

Neuroadaptations in neurocircuitry of reward memories govern the persistent and compulsive behaviors. The study of the role of hippocampus in processing of reward memory and its retrieval is critical to our understanding of addiction and relapse. The aim of this study is to probe the epigenetic mechanisms underlying reward memory in the frame of dentate gyrus (DG). To that end, the rats conditioned to the food baited arm of a Y-maze and subjected to memory probe trial. The hippocampus of conditioned rats displayed higher mRNA levels of Ten-eleven translocase 1 (Tet1) and brain-derived neurotrophic factor (Bdnf) after memory probe trial. The DNA hydroxymethylation and TET1 occupancy at the Bdnf promoters showed concomitant increase. Stereotactic administration of Tet1 siRNA in the DG before and after conditioning inhibited reward memory formation and recall, respectively. Administration of Tet1 siRNA impaired the reward memory recall that was reinstated following administration of exogenous BDNF peptide or after wash-off period of 8 days. Infusion of a MEK/ERK inhibitor, U0126 in the DG inhibited reward memory retrieval. The TET1-induced DNA demethylation at the Bdnf promoters raised BDNF levels in the hippocampus, thereby setting the stage for reward memory retrieval. The study underscores the causative role of TET1 in the DG for reward memory formation and recall.

Neuroplastic Changes in the Superior Colliculus and Hippocampus in Self-rewarding Paradigm: Importance of Visual Cues.

Coincident excitation via different sensory modalities encoding objects of positive salience is known to facilitate learning and memory. With a view to dissect the contribution of visual cues in inducing adaptive neural changes, we monitored the lever press activity of a rat conditioned to self-administer sweet food pellets in the presence/absence of light cues. Application of light cues facilitated learning and consolidation of long-term memory. The superior colliculus (SC) of rats trained on light cue showed increased neuronal activity, dendritic branching, and brain-derived neurotrophic factor (BDNF) protein and mRNA expression. Concomitantly, the hippocampus showed augmented neurogenesis as well as BDNF protein and mRNA expression. While intra-SC administration of U0126 (inhibitor of ERK 1/2 and long-term memory) impaired memory formation, lidocaine (local anaesthetic) hindered memory recall. The light cue-dependent sweet food pellet self-administration was coupled with increased efflux of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the nucleus accumbens shell (AcbSh). In conditioned rats, pharmacological inhibition of glutamatergic signalling in dentate gyrus (DG) reduced lever press activity, as well as DA and DOPAC secretion in the AcbSh. We suggest that the neuroplastic changes in the SC and hippocampus might represent memory engrams sculpted by visual cues encoding reward information.

Sensitivity of olfactory sensory neurons to food cues is tuned to nutritional states by Neuropeptide Y signaling.

Modulation of sensory perception by homeostatic feedback from physiological states is central to innate purposive behaviors. Olfaction is an important predictive modality for feeding-related behaviors and its modulation has been associated with hunger-satiety states. However, the mechanisms mapping internal states to chemosensory processing in order to modify behavior are poorly understood. In the zebrafish olfactory epithelium, a subset of olfactory sensory neurons (OSNs) and the terminal nerve projections express neuropeptide Y (NPY). Using a combination of neuronal activity and behavioral evaluation, we find that NPY signaling in the peripheral olfactory system of zebrafish is correlated with its nutritional state and is both necessary and sufficient for the olfactory perception of food-related odorants. NPY activity dynamically modulates the microvillar OSN activation thresholds and acts cooperatively with amino acid signaling resulting in a switch-like increase in OSN sensitivity in starved animals. We suggest that cooperative activation of phospholipase C by convergent signaling from NPY and amino acid receptors is central to this heightened sensitivity. This study provides ethologically relevant, physiological evidence for NPY signaling in the modulation of OSN sensitivity to food-associated amino acid cues. We demonstrate sensory gating directly at the level of OSNs and identify a novel mechanistic framework for tuning olfactory sensitivity to prevailing energy states. Cover Image for this issue: https://doi.org/10.1111/jnc.15091.

Neuropeptide CART modulates dopamine turnover in the nucleus accumbens: Insights into the anatomy of rewarding circuits.

Neuropeptide cocaine- and amphetamine-regulated transcript (CART) is known to influence the activity of the canonical mesolimbic dopaminergic pathway and modulate reward seeking behaviour. CART neurons of the lateral hypothalamus (LH) send afferents to the ventral tegmental area (VTA) and paraventricular thalamic nucleus (PVT) and these nuclei, in turn, send secondary projections to nucleus accumbens. We try to dissect the precise sites of CART's action in these circuits in promoting reward. Rats were implanted with bipolar electrode targeted at the lateral hypothalamus-medial forebrain bundle (LH-MFB) and trained to press the lever through intracranial self-stimulation (ICSS) protocol. CART (55-102) administered directly into posterior VTA (pVTA) or PVT of the conditioned rats significantly increased the number of lever presses, indicating reward-promoting activity of the peptide. Concomitant increase in dopamine (DA) and 3, 4-dihydroxyphenylacetic acid (DOPAC) efflux was noted in the microdialysate collected from the nucleus accumbens shell (AcbSh). On the other hand, immunoneutralization of endogenous CART with CART antibodies injected directly in the pVTA or PVT reduced the lever press activity as well as DA and DOPAC efflux in the AcbSh. Injection of CART (1-39) in pVTA or PVT was ineffective. We suggest that CART cells in the LH-MFB area send afferents to (a) pVTA and influence dopaminergic neurons projecting to AcbSh and (b) PVT, from where the secondary neurons may feed into the AcbSh. Excitation of the CARTergic pathway to the pVTA as well as the PVT seems to promote DA release in the AcbSh and contribute to the generation of reward.

LSD1-BDNF activity in lateral hypothalamus-medial forebrain bundle area is essential for reward seeking behavior.

Reward induces activity-dependant gene expression and synaptic plasticity-related changes. Lysine-specific histone demethylase 1 (LSD1), a key enzyme driving histone modifications, regulates transcription in neural circuits of memory and emotional behavior. Herein, we focus on the role of LSD1 in modulating the expression of brain derived neurotrophic factor (BDNF), the master regulator of synaptic plasticity, in the lateral hypothalamus-medial forebrain bundle (LH-MFB) circuit during positive reinforcement. Rats, trained for intracranial self-stimulation (ICSS) via an electrode-cannula assembly in the LH-MFB area, were assayed for lever press activity, epigenetic parameters and dendritic sprouting. LSD1 expression and markers of synaptic plasticity like BDNF and dendritic arborization in the LH, showed distinct increase in conditioned animals. H3K4me2 levels at Bdnf IV and Bdnf IX promoters were increased in ICSS-conditioned rats, but H3K9me2 was decreased. While intra LH-MFB treatment with pan Lsd1 siRNA inhibited lever press activity, analyses of LH tissue showed reduction in BDNF expression and levels of H3K4me2 and H3K9me2. However, co-administration of BDNF peptide restored lever press activity mitigated by Lsd1 siRNA. BDNF expression in LH, driven by LSD1 via histone demethylation, may play an important role in reshaping the reward pathway and hold the key to decode the molecular basis of addiction.

Transient systemic inflammation in adult male mice results in underweight progeny.

PROBLEM: While the testes represent an immune-privileged organ, there is evidence that systemic inflammation is accompanied by local inflammatory responses. We therefore examined whether transient systemic inflammation caused any inflammatory and functional consequences in murine testes. METHOD OF STUDY: Using a single systemic administration of Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) or peptidoglycan (PG) or polyinosinic-polycytidylic acid (polyIC)] in young adult male mice, we assessed testicular immune-inflammatory landscape and reproductive functionality. RESULTS: Our findings demonstrated a significant induction of testicular TNF-α, IL-1ß and IL-6 transcripts within 24 h of TLR agonist injection. By day 6, these cytokine levels returned to baseline. While there was no change in caudal sperm counts at early time points, eight weeks later, twofold decrease in sperm count and reduced testicular testosterone levels were evident. When these mice were subjected to mating studies, no differences in mating efficiencies or litter sizes were observed compared with controls. Nonetheless, the neonatal weights of progeny from LPS/PG/polyIC-treated sires were significantly lower than controls. Postnatal weight gain up to three weeks was also slower in the progeny of LPS/polyIC-treated sires. Placental weights at 17.5 days post-coitum were significantly lower in females mated to LPS- and polyIC-treated males. Given this likelihood of an epigenetic effect, we found lower testicular levels of histone methyltransferase enzyme, mixed-lineage leukaemia-1, in mice given LPS/PG/polyIC 8 weeks earlier. CONCLUSION: Exposure to transient systemic inflammation leads to transient local inflammation in the testes, with persistent sperm-mediated consequences for foetal development.

Denial of food to the hungry rat: A novel paradigm for induction and evaluation of anger-like emotion.

BACKGROUND: Anger is one of the primary emotions that profoundly impacts our daily life. Although the neural basis of anger needs to be explored on high priority, the field has not sufficiently advanced, perhaps due to the lack of a suitable animal model. NEW METHOD: We fabricated arenas in which the hungry rat can see and smell food but can not consume it. These animals seemed hyperactive and we monitored the (a) motor activity to access food, (b) biting behaviour, (c) blood pressure, heart rate and nor-epinephrine (NE) in plasma, (d) 5-HT and its metabolite in CSF, (e) effect of diazepam, 5-HT agonist, and antagonist on the behaviour, and (f) expression of immediate early gene in discrete areas of the brain. RESULTS: The fasted animal frantically tries to acquire food. It engages in intense biting of the separator plate; the behaviour was considered as an expression of anger-like emotion. These behaviours were attenuated following pre-treatment with diazepam, fluoxetine (both ip) or 5-HT1A receptor agonist (icv), but potentiated by 5-HT1A antagonist (icv). Concomitantly, an increase in the blood pressure, heart rate and NE in plasma, but a decrease in 5-HT and 5-HIAA in the CSF was noted. The animals showed activation of neuronal c-Fos in different brain areas compared to fasted or refed controls. COMPARISON WITH EXISTING METHODS: A novel animal paradigm for assessment of anger. CONCLUSIONS: The protocol enables us to generate and evaluate anger-like responses in rat and permits insights into the neurological basis of anger.

Nicotine-induced Brain Stimulation Reward is Modulated by Melanocortin-4 Receptors in Ovariectomized Rats.

Apart from reproduction, estrogen influences a multitude of processes. Increase in estrogen levels in women is known to promote reward probably mediated via the melanocortin and dopamine systems. Reduced estrogen in post-menopausal women attenuates reward, evoking the need for stimulation with greater rewarding salience. This is reflected in the well-recognized phenomena of difficulty in quitting and increased craving for nicotine in women following the onset of menopause. The present study aims at understanding the role of melanocortin receptors (MC-R) in nicotine-induced reward behavior following ovariectomy in rats. The MC4-R mRNA level was increased in ipsilateral nucleus accumbens (Acb) of the intact rats implanted with electrode in medial forebrain bundle and trained in intracranial self-stimulation (ICSS) paradigm. Additional groups of ICSS trained rats were ovariectomized (OVX) and subjected to reward evaluation. Trained OVX rats revealed a significant increase in threshold frequency and rightward shift in rate frequency curve, suggesting reward deficit behavior. However, pre-administration with nicotine, alpha-melanocyte stimulating hormone (α-MSH) or NDP-MSH (MC4-R agonist) to OVX animals restored the rewarding activity in ICSS protocol; HS014 (MC4-R antagonist) suppressed the lever press activity. Prior treatment with sub-effective doses of α-MSH or NDP-MSH potentiated the reward effect of nicotine, but was attenuated by HS014. Alpha-MSH-immunoreactivity was decreased in the Acb shell, arcuate and paraventricular nucleus of hypothalamus, and ventral bed nucleus of stria terminalis in the OVX rats, while nicotine treatment restored the same. We suggest a role for the endogenous MC system, perhaps acting via MC4-R, in the nicotine-induced reward in OVX rats.

Intracellular mechanisms and behavioral changes in mouse model of attention deficit hyperactivity disorder: Importance of age-specific NMDA receptor blockade.

Exposure of NMDA receptor antagonists during developmental stages leads to behavioral consequences like attention deficit hyperactivity disorder (ADHD). However, the underlying molecular mechanisms have remained poorly understood. Herein, we studied the phosphorylated Akt (pAkt) and caspase-3, the key regulators of neuronal cell survival/death, as the probable downstream targets of MK-801 often used to engender ADHD-like condition. Swiss albino mice at postnatal days (PND) 7, 14 or 21 were injected with a single dose of MK-801 and evaluated for hyperactivity (open field test) and memory deficit at adolescence (PND 30) and adult stages (PND 60). PND 7 or 14 treatment groups (but not PND 21) consistently showed hyperactivity at the adolescence stage. A significant increase in working and reference memory errors in radial arm maze was noted at the adolescence age. PND 7 group continued to display the symptoms even in adulthood. All the treatment groups showed a significant decrease in the percent alterations (Y-maze) and discrimination index (novel object recognition test) at adolescence age. A significant increase in caspase-3 expression was noted in the prefrontal cortex (PFC) and hippocampus, whereas increased pAkt was noticed only in the hippocampus, following a single injection of MK-801 at PND 7. Concurrently, PND 7 treatment group showed significantly decreased neuronal nuclei (NeuN) expression (a marker for mature neurons) in the dentate gyrus, cornu ammonis-3 and PFC, but not in cornu ammonis-1, at adolescence age. We suggest that the observed symptoms of ADHD at adolescence and adulthood stages may be linked to alteration in pAkt and caspase-3 followed MK-801 treatment at PND 7.

Cocaine- and amphetamine-regulated transcript peptide promotes reward seeking behavior in socially isolated rats.

Reward deficit, expressed as anhedonia, is one of the major symptoms associated with neuropsychiatric disorders, but the underlying maladaptations have not been understood. Herein, we test the hypothesis that the neuropeptide cocaine- and amphetamine-regulated transcript (CART) may participate in the process. The study is justified since the peptide is a major player in inducing satiety and also processing of reward. The rats were socially isolated to induce reward deficit and conditioned to self-stimulate via an electrode in lateral hypothalamus (LH)-medial forebrain bundle (MFB) region. Compared to group-housed control rats, the socially isolated animals showed decreased lever press activity and elevated ICSS threshold indicating anhedonia-like condition. However, the effects of social isolation were alleviated by CART administered via intracerebroventricular route. The changes in the expression of CART protein and mRNA were screened using immunofluorescence and qRT-PCR methods, respectively. Socially isolated rats showed reduction in the expression of CART in the LH, nucleus accumbens shell (AcbSh) and posterior ventral tegmental area (pVTA) and CART mRNA in the Acb and LH. Double immunostaining with antibodies against CART and synaptophysin revealed significant loss of colabeled elements in LH, AcbSh and pVTA. We suggest that down-regulation of endogenous CARTergic system in the LH-pVTA-AcbSh reward circuitry may be causal to motivational anhedonia like phenotype seen in neuropsychiatric conditions.

Involvement of neuropeptide CART in the central effects of insulin on feeding and body weight.

While insulin secreted from pancreas plays a pivotal role in the control of glucose homeostasis, it also interacts with hypothalamic sites and negatively influences the energy balance. The present study was undertaken to reveal the functional interaction between cocaine- and amphetamine-regulated transcript (CART), a well-known anorexic peptide, and insulin within the framework of hypothalamus in the regulation of feeding behavior and body weight. Insulin was administered daily by intracerebroventricular (icv) route, alone or in combination with CART (icv) for a period of seven days. Immediately thereafter, preweighed food was offered to the animals at the commencement of the dark phase. The food intake and body weight were measured daily just prior to next injection. Furthermore, brains of insulin-treated rats were processed for the immunohistochemical analysis of CART-containing elements in the hypothalamus. Treatment with insulin (6 mU, icv) for a period of 7 days caused a significant decrease in food intake and body weight as compared to control. Concomitant administration of CART (0.5 µg, icv) potentiated insulin-induced anorexia and weight loss. Insulin administration resulted in a significant increase in CART immunoreactivity in the hypothalamic arcuate, paraventricular, dorsomedial and ventromedial nuclei. We suggest that increased CART contents in the hypothalamus may be causally linked with anorexia and weight loss induced by insulin.

Maternal ethanol exposure reshapes CART system in the rat brain: Correlation with development of anxiety, depression and memory deficits.

Ethanol ingestion by a mother during pregnancy entails adverse consequences for her offspring. In this study, adult female rats were given access to ethanol from 8 days prior to mating to post-parturition weaning, and the effects on her offspring were evaluated. We investigated changes in the cocaine- and amphetamine-regulated transcript peptide (CART), a neuropeptide involved in the central effects of ethanol in the frame of reward and stress processing circuits. CART-immunoreactivity was augmented in the cells of Edinger-Westphal (EW) nucleus and lateral hypothalamus (LH) and fibers in the LH and ventral tegmental area (VTA) in 25-day-old pups. On the other hand, a significant decrease was seen in the expression of the peptide in paraventricular nucleus (PVN), arcuate nucleus (ARC), hippocampus (CA1 and CA2) and locus coeruleus (LC). The offspring at 85 days showed increased anxiety in elevated plus maze and immobility in forced swim test suggestive of depression. These rats also failed to discriminate between novel versus familiar object in object recognition test indicating memory deficits. Their brains showed decreased CART-immunoreactivity in nucleus accumbens shell, lateral bed nucleus of stria terminalis, PVN, ARC, LH, hippocampus and LC as compared to age-matched control offspring. However, CART-immunoreactive profile in EW and fibers in VTA of 85-day-old offspring was similar to that in the control. Thus, regional imbalance in the CART system of the offspring of alcoholic dams seems correlated with the affective and emotional abnormalities and memory deficits.

Neuropeptide CART prevents memory loss attributed to withdrawal of nicotine following chronic treatment in mice.

Although chronic nicotine administration does not affect memory, its withdrawal causes massive cognitive deficits. The underlying mechanisms, however, have not been understood. We test the role of cocaine- and amphetamine-regulated transcript peptide (CART), a neuropeptide known for its procognitive properties, in this process. The mice on chronic nicotine treatment/withdrawal were subjected to novel object recognition task. The capability of the animal to discriminate between the novel and familiar objects was tested and represented as discrimination index (DI); reduction in the index suggested amnesia. Nicotine for 49 days had no effect on DI, but 8-hour withdrawal caused a significant reduction, followed by full recovery at 24-hour withdrawal timepoint. Bilateral CART infusion in dorsal hippocampus rescued deficits in DI at 8-hours, whereas CART-antibody infusion into the dorsal hippocampus attenuated the recovery at 24-hours. Commensurate changes were observed in the CART as well as CART mRNA profiles in the hippocampus. CART mRNA expression and the peptide immunoreactivity did not change significantly following chronic nicotine treatment. However, there was a significant reduction at 8-hour withdrawal, followed by a drastic increase in CART immunoreactivity as well as CART mRNA at 24-hour withdrawal, compared with 8-hour withdrawal. Distinct α7-nicotinic receptor immunoreactivity was detected on the hippocampal CART neurons, suggesting cholinergic inputs. An increase in the synaptophysin immunoreactive elements around CART cells in the dentate gyrus, cornu ammonis 3 and subiculum at 24-hour post-withdrawal timepoint suggested neuronal plasticity. CART circuit dynamics in the hippocampus seems to modulate short-term memory associated with nicotine withdrawal.

A role of neuropeptide CART in hyperphagia and weight gain induced by olanzapine treatment in rats.

Although olanzapine is highly efficacious and most widely used second generation antipsychotic drug, the success of treatment has been hampered by its propensity to induce weight gain. While the underlying neuronal mechanisms are unclear, their elucidation may help to target alternative pathways regulating energy balance. The present study was undertaken to define the role of cocaine- and amphetamine-regulated transcript (CART), a well-known anorexic peptide, in olanzapine-induced hyperphagia and body weight gain in female rats. Olanzapine was administered daily by intraperitoneal route, alone or in combination with CART (intracerebroventricular) for a period of two weeks. Immediately after drug administrations, preweighed food was offered to the animals at the commencement of the dark phase. The food intake and body weight were measured daily just prior to next injection. Furthermore, the brains of olanzapine-treated rats were processed for the immunohistochemical analysis of CART-containing elements in the hypothalamus. Treatment with olanzapine (0.5 mg/kg) for the duration of 14 days produced a significant increase in food intake and body weight as compared to control. However, concomitant administration of CART (0.5 µg) attenuated the olanzapine-induced hyperphagia and weight gain. Olanzapine administration resulted in a significant reduction in CART immunoreactivity in the hypothalamic arcuate, paraventricular, dorsomedial and ventromedial nuclei. We suggest that decreased CART contents in the hypothalamus may be causally linked with the hyperphagia and weight gain induced by olanzapine.