Dadasaheb M. Kokare

Interaction between neuropeptide Y and alpha-melanocyte stimulating hormone in amygdala regulates anxiety in rats.

Neuropeptide Y (NPY) and alpha-melanocyte stimulating hormone (alpha-MSH) have been implicated in pathophysiology of feeding and certain mood disorders, including anxiety and depression. Both the peptides are abundantly present in CNS, especially in the hypothalamus and amygdala. Although they are known to exert opposite effects, particularly with reference to anxiety, the underlying mechanisms are not known. We were interested in studying the interaction between these two peptides in the regulation of anxiety, within the framework of amygdala. We administered agents like NPY, alpha-MSH, selective melanocortin-4 receptor (MC4-R) antagonist HS014 and NPY Y1 receptor agonist [Leu(31), Pro(34)]-NPY, alone and in combinations, unilaterally in right amygdala of rats and measured the response using elevated plus maze test. While NPY and [Leu(31), Pro(34)]-NPY increased the time spent and number of entries in the open arms suggesting anxiolytic-like effects, alpha-MSH resulted in opposite responses. Anxiolytic-like effect of NPY (10 nM) or [Leu(31), Pro(34)]-NPY (5 nM) was significantly reduced following prior alpha-MSH (250 ng) administration. Co-administration of HS014 (1 nM) and NPY (5 nM) or [Leu(31), Pro(34)]-NPY (1 nM) at subeffective doses evoked synergistic anxiolysis. Since the closed arm entries displayed by animals of all the groups were in a similar range, the effects might not be ascribed to the changes in general locomotor activity. These results suggest that endogenous alpha-MSH and NPY containing systems may interact in the amygdala and regulate exploratory behavior in an animal model of anxiety.

Involvement of α-MSH in the social isolation induced anxiety- and depression-like behaviors in rat

Although physical isolation of rats is known to cause anxiety- and depression-like symptoms, the underlying mechanisms are not fully understood. We have attempted to define the role of endogenous melanocortins (MC) in the manifestation of these symptoms. Weaning rats were socially isolated for 6 weeks and subjected to behavioral paradigms like elevated plus maze (EPM), social interaction, and forced swim test (FST). While socially isolated rats spent less time in social interaction, and showed significantly decreased activity in the open arms of the EPM, the immobility time in FST was significantly increased thus reflecting anxiety- and depression-like phenotypes. Intracerebroventricular injection of HS014 (5 or 10 nmol/rat), selective antagonist of MC4 receptors, attenuated these symptoms. This suggested the involvement of endogenous alpha-melanocyte stimulating hormone (alpha-MSH) in anxiety and depression. With a view to determining the neuroanatomical substrates in which the endogenous alpha-MSH may process the related information, profile of the peptide in paraventricular (PVN), arcuate (ARC), dorsomedial hypothalamic-dorsal (DMNd) and -ventral (DMNv) nuclei, and central nucleus of amygdala (CeA) was investigated with immunohistochemistry. While social isolation significantly reduced alpha-MSH-immunoreactivity profile in all these components, re-socialization of the socially isolated rats, over a period of 72 h, resulted in full recovery of the alpha-MSH-immunoreactivity profile; the symptoms of anxiety- and depression-like behaviors were also fully attenuated. We suggest that alpha-MSH in the PVN, ARC, DMNd, DMNv and CeA, acting via MC4 receptors, are involved in manifestation of affective disorders like anxiety and depression.

Importance of cocaine- and amphetamine-regulated transcript peptide in the central nucleus of amygdala in anxiogenic responses induced by ethanol withdrawal.

We studied the involvement of cocaine- and amphetamine-regulated transcript peptide (CART) in the central nucleus of amygdala (CeA), lateral bed nucleus of the stria terminalis (BNSTl) and nucleus accumbens shell (AcbSh) in generation of ethanol withdrawal symptoms, with particular focus on anxiety-like behavior using a social interaction test. Administration of CART (54–102) into the lateral ventricle (50 and 100 ng) and bilaterally in the CeA (10 and 20 ng) caused a significant reduction in social interaction, suggesting an anxiogenic action of the peptide. Chronic ethanol treatment for 15 days followed by withdrawal precipitated an anxiogenic response at 24 h that was attenuated by intracerebroventricular (5 μl) and intra-CeA (1 μl) administration of antibodies against CART (1 : 500 dilution). An immunocytochemistry protocol was employed to study the response of the endogenous CART system in the CeA following chronic ethanol withdrawal. At 0 h ethanol withdrawal, CART immunoreactivity was apparent in few fibers and the profile was similar to that in the pair-fed control rats. Twenty-four hours following ethanol withdrawal, a highly significant increase (P<0.001) in CART immunoreactivity was noticed in the CeA, which returned to normal 48 and 72 h post-withdrawal. Similar doses of CART or CART antibody injected bilaterally into the BNSTl or AcbSh produced no response in the social interaction test. Furthermore, the CART immunoreactivity profile did not change at the post-withdrawal time points in each of these brain sites. We suggest that CART may mediate the early signs of anxiety-like behavior induced by ethanol withdrawal within the neuroanatomical framework of the CeA.

Cocaine- and Amphetamine-Regulated Transcript Peptide Plays a Role in the Manifestation of Depression: Social Isolation and Olfactory Bulbectomy Models Reveal Unifying Principles

We investigated the effect of cocaine- and amphetamine-regulated transcript (CART) peptide on depression-like behavior in socially isolated and olfactory bulbectomized (OBX) rats. Administration of CART (54–102) into the lateral ventricle (50–100 ng) or central nucleus of amygdala (CeA) (10–20 ng) caused significant decrease in immobility time in the forced swim test (FST) without influencing locomotion, suggesting antidepressant-like effect. Social isolation as well as OBX models were undertaken to produce depression-like conditions. Although isolation reared (6 weeks) rats showed significant increase in immobility time in FST, OBX animals exhibited hyperactivity (increase in the ambulation, rearing, grooming, and defecation scores) on day 14 in the open-field test. The isolation- or OBX-induced depression-like phenotypes were reversed following acute or subchronic treatment of CART, respectively, given via intracerebroventricular and intra-CeA routes. Drastic reduction in CART-immunoreactivity was observed in most cells in the paraventricular (PVN), arcuate and Edinger–Westphal nuclei of the socially isolated and OBX animals. Although the fibers in the PVN showed variable response, those in ARC and prefrontal cortex did not change. The CART-immunoreactive fibers in the locus coeruleus also showed highly significant reduction. However, dramatic increase in CART-immunoreactive fibers was noticed in the CeA in both the experimental models. The response by the cells and fibers in the periventricular area and perifornical nucleus in the OBX and socially isolated rats was variable. The study underscores the possibility that endogenous CART system might play a major role in mediating symptoms of depression.

GABAergic agents prevent alpha-melanocyte stimulating hormone induced anxiety and anorexia in rats

Alpha-melanocyte stimulating hormone (alpha-MSH) is a hypothalamic peptide believed to play a tonic inhibitory role in feeding and energy homeostasis. Systemic administration of alpha-MSH is known to produce anorexia and anxiety. Since synaptic contacts between gamma-aminobutyric acid (GABA)ergic terminals and alpha-MSH neurons in the hypothalamus have been reported, the present work was undertaken to refine our knowledge on the role of GABAergic systems in anxiety and anorexia induced by intracerebroventricular (icv) administration of alpha-MSH in rats. The anxiety was assessed by elevated plus maze, and spontaneous food consumption was monitored during dark cycle. Prior administration of diazepam and muscimol that promote the function of GABA(A) receptors reversed the anxiogenic response and decreased food intake elicited by alpha-MSH. In contrast, bicuculline, the GABA(A) receptor antagonist, not only enhanced the effects of alpha-MSH but also prevented the influence of GABAergic drugs on alpha-MSH-induced anorexia and anxiety. These findings suggest that alpha-MSH-induced anxiety and anorexia are due to its negative influence on GABAergic system.

Participation of α-melanocyte stimulating hormone in ethanol-induced anxiolysis and withdrawal anxiety in rats

Although recent reports underscore a close association between the ethanol consumption and the central melanocortin (MC) system in rats, neurobehavioral component of this association has not been explored. In this study, we investigated the role of alpha-melanocyte stimulating hormone (alpha-MSH) in ethanol (1.5-2 g/kg, i.p.) induced anxiolysis and anxiety-like behavior following withdrawal from prolonged ethanol (9% v/v ethanol, 15 days) consumption, using elevated plus maze (EPM) test in rats. While alpha-MSH (1-5 microg/rat, i.c.v.) showed dose-dependent anxiogenic-like effect, the MC4 receptor antagonist HS014 (1-10 nM/rat, i.c.v.) or antiserum against alpha-MSH (1:500-1:50 dilution, 5 microl/rat, i.c.v.) failed to produce any effect in the EPM test. The anxiolytic-like effect of ethanol was suppressed by central administration of alpha-MSH (0.5 microg/rat, i.c.v.). On the other hand, pretreatment with either HS014 (5 nM/rat, i.c.v.) or antiserum against alpha-MSH (1:100 dilution, 5 microl/rat, i.c.v.) enhanced anxiolytic action of ethanol. Moreover, ethanol withdrawal anxiety was markedly blocked by HS014 (1-10 nM/rat, i.c.v.). These results suggest that alpha-MSH may be implicated in ethanol-induced anxiolysis and withdrawal anxiety. These findings also suggest MC4 receptors as possible therapeutic target for development of drugs to address the ethanol withdrawal-related conditions.

CART in the brain of vertebrates: circuits, functions and evolution.

Cocaine- and amphetamine-regulated transcript peptide (CART) with its wide distribution in the brain of mammals has been the focus of considerable research in recent years. Last two decades have witnessed a steady rise in the information on the genes that encode this neuropeptide and regulation of its transcription and translation. CART is highly enriched in the hypothalamic nuclei and its relevance to energy homeostasis and neuroendocrine control has been understood in great details. However, the occurrence of this peptide in a range of diverse circuitries for sensory, motor, vegetative, limbic and higher cortical areas has been confounding. Evidence that CART peptide may have role in addiction, pain, reward, learning and memory, cognition, sleep, reproduction and development, modulation of behavior and regulation of autonomic nervous system are accumulating, but an integration has been missing. A steady stream of papers has been pointing at the therapeutic potentials of CART. The current review is an attempt at piecing together the fragments of available information, and seeks meaning out of the CART elements in their anatomical niche. We try to put together the CART containing neuronal circuitries that have been conclusively demonstrated as well as those which have been proposed, but need confirmation. With a view to finding out the evolutionary antecedents, we visit the CART systems in sub-mammalian vertebrates and seek the answer why the system is shaped the way it is. We enquire into the conservation of the CART system and appreciate its functional diversity across the phyla.

Central administration of selective melanocortin 4 receptor antagonist HS014 prevents morphine tolerance and withdrawal hyperalgesia

Major problem involved in treatment of chronic pain with morphine is the development of tolerance and dependence. Previous studies have demonstrated the participation of melanocortin (MC) system in the development of tolerance to antinociceptive effect of morphine. However, the impact of supraspinal MC4 receptors (MC4 R) modulation on this phenomenon and morphine withdrawal hyperalgesia remained unexplored. We investigated the role of central MC4 R in acute, chronic effects and withdrawal reactions of morphine using tail flick test. Acute intracerebroventricular (icv) administration of morphine (2-20 microg/rat) exhibited antinociceptive activity, which was antagonized by subeffective dose of nonselective MC R agonist NDP-MSH (0.04 ng/rat, icv), and potentiated by subeffective dose of MC4 R antagonist HS014 (0.008 ng/rat, icv). Isobolographic analysis revealed antagonistic interaction between NDP-MSH and morphine, and additive interaction between HS014 and morphine combinations. While chronic icv infusion of morphine (20 ng/microl/h) via osmotic pump for 7 days developed tolerance to its antinociceptive effect, its discontinuation produced hyperalgesia. Co-administration of HS014 (0.008 ng/rat, icv) with chronic morphine not only delayed the development of tolerance but also prevented withdrawal hyperalgesia. Furthermore, acute treatment with HS014 (0.008 and 0.04 ng/rat, icv) dose dependently attenuated the withdrawal hyperalgesia. This suggests the involvement of central MC4 R in the mechanism of development of tolerance and dependence following chronic morphine administration. We speculate that targeting this receptor may be a novel strategy to improve the effectiveness of morphine in the treatment of chronic pain.

A simple and inexpensive method to fabricate a cannula system for intracranial injections in rats and mice

INTRODUCTION Stereotaxic administration of neuroactive agents, either in ventricles, or targeted at specific intracranial sites, is a widely employed strategy for neurological studies in rodents. Surgical implantation of cannula on the skull is particularly useful in chronic treatments. We describe a simple, inexpensive and reliable method to fabricate a cannula system for delivery of drugs at the targeted sites in the brain of rat or mouse. METHODS The system consists of a guide cannula made from a hypodermic needle (24 gauge), a stainless steel wire (30 gauge) that serves as a dummy cannula, and an internal cannula made of stainless steel needle (30 gauge) taken from a hypodermic syringe. The cannula can be implanted by routine stereotaxic procedure and used for acute or chronic drug administration to conscious, free moving animals. RESULTS With a view to test the system for accuracy, the guide cannula was stereotaxically implanted, and neuropeptide Y was directly delivered into the lateral ventricle. These rats showed a significant increase in food intake. Another set of rats were cannulated for chronic protocol, wherein ethanol was delivered directly into the ventral tegmental area. In operant chamber, these rats showed increased ethanol self-administration. The proposed cannula takes around 5 min to fabricate and costs less than a dollar. CONCLUSION We feel that it may serve as an economical and reliable tool in neuropharmacological and neurobehavioral studies.

Involvement of alpha-melanocyte stimulating hormone (α-MSH) in differential ethanol exposure and withdrawal related depression in rat: Neuroanatomical–behavioral correlates

We investigated the involvement of alpha-melanocyte stimulating hormone (alpha-MSH) following acute, chronic and withdrawal treatments of ethanol with reference to depression. The degree of depression was evaluated using Porsolts forced swim test. While intracerebroventricular (i.c.v.) alpha-MSH (100-400 ng/rat) dose-dependently increased the immobility, opposite response was observed following administration of selective MC4 receptor antagonist HS014 (0.01-0.07 ng/rat, i.c.v.). The anti-immobility effect of acute ethanol (1-2 g/kg), injected via intra-peritoneal route (i.p.), was suppressed by central administration of alpha-MSH (100 ng/rat, i.c.v.), but was enhanced following pretreatment with HS014 (0.01 ng/rat, i.c.v.). Chronic ethanol resulted in increased immobility time, while further augmentation in immobility was noticed following ethanol withdrawal. However, concomitant HS014 (0.01 ng/rat, i.c.v.) treatment prevented tolerance as well as attenuated enhanced immobility in ethanol-withdrawn rats. Acute administration of HS014 (0.01-0.03 ng/rat, i.c.v.), at 24h post-withdrawal time point, also antagonized the ethanol withdrawal immobility in rats. The profile of alpha-MSH-immunoreactivity in the paraventricular (PVN), arcuate (ARC), paraventricular thalamic (PVT), dorsomedial hypothalamic-dorsal (DMNd) and -ventral (DMNv) nuclei, lateral hypothalamus (LH) and central nucleus of amygdala (CeA) was investigated with immunocytochemistry. Acute ethanol significantly reduced the alpha-MSH-immunoreactivity in the cells and fibers of ARC, and fibers in the PVN, DMNd, DMNv and CeA. While chronic ethanol treatment significantly increased the alpha-MSH-immunoreactivity as compared to the pair-fed control group, further augmentation was noticed following 24 h ethanol withdrawal. However, the alpha-MSH-immunoreactive profile in the PVT and LH did not respond. alpha-MSH in discrete areas may play a role in ethanol-induced antidepressant-like response and withdrawal-induced depression.