Manoj A. Upadhya

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.

CART peptide in the nucleus accumbens shell acts downstream to dopamine and mediates the reward and reinforcement actions of morphine.

The opioid-mesolimbic-dopamine circuitry operates between ventral tegmental area (VTA) and nucleus accumbens (Acb) and serves as a major reward pathway. We hypothesized that the neuropeptide cocaine- and amphetamine-regulated transcript (CART) is involved in the natural reward action mediated by the circuitry. Therefore, the modulation of opioid-mesolimbic-dopamine reward circuitry by CART was investigated using pellet self-administration paradigm in operant chamber. Morphine administered bilaterally in shell region of Acb (AcbSh) significantly increased active lever pressings and pellet self-administration. While CART given bilaterally in the AcbSh significantly increased pellet self-administration, CART antibody produced no effect. Morphine induced pellet self-administration was potentiated by CART, and antagonized by CART antibody administered in AcbSh. A close interaction between dopamine and CART systems was observed. Several tyrosine hydroxylase (marker for dopamine) immunoreactive fibers were seen contacting CART neurons in the AcbSh. Intraperitoneal administration of pramipexole, a dopamine agonist, increased pellet self-administration. The effect was blocked by prior treatment with CART antibody targeted at AcbSh. CART-immunoreactive cells and fibers in the AcbSh, and cells but not fibers in hypothalamic paraventricular nucleus (PVN), were significantly increased in the animals trained in operant chamber. However, CART-immunoreactive profile in the medial forebrain bundle, VTA and arcuate nucleus of hypothalamus did not respond. We suggest that CART, released from the axonal terminals in the framework of AcbSh, may serve as the final output of the endogenous opioid-mesolimbic-dopamine circuitry that processes natural reward.

Cocaine- and amphetamine-regulated transcript peptide increases spatial learning and memory in rats

AIM We investigated the involvement of cocaine- and amphetamine-regulated transcript peptide (CART) in spatial learning and memory. MAIN METHODS Rats were intracerebroventricularly injected with CART or CART-antibody, with or without intraperitoneal scopolamine, for a period of 4 days, during which they were subjected to the acquisition protocol in Morris water maze (MWM). In retrieval protocols, at 24 h and 15 days post-acquisition time points similar treatments were given to trained rats and subjected to MWM. The response of endogenous CART system to the training as well as retrieval sessions in MWM was evaluated with immunohistochemistry. KEY FINDINGS CART-administered rats showed a significant reduction in escape latency from day 1 through 4 days of acquisition; the rats spent more time in the platform quadrant in MWM during the retrieval protocol. CART-antibody or scopolamine produced an opposite effect. The effects of scopolamine were attenuated by CART, and potentiated by CART-antibody. CART-immunoreactivity in the arcuate and paraventricular nuclei, central nucleus of amygdala, bed nucleus of stria terminalis, accumbens shell, dentate gyrus (DG), and thalamic paraventricular nucleus (PVT), but not in the cornu ammonis 1-3 of hippocampus, was significantly increased following 4 days of training, and at 24 h retrieval time point in MWM. The changes were blocked by scopolamine. At 15 days retrieval time point, the immunoreactivity profiles resembled those in naïve control. SIGNIFICANCE While CART seems to promote spatial learning and memory, navigational experiences in MWM up regulates the endogenous CART systems in extended amygdala, hypothalamus, DG and PVT.

Neuropeptide Y Y1 receptors in the central nucleus of amygdala mediate the anxiolytic-like effect of allopregnanolone in mice: Behavioral and immunocytochemical evidences.

Since allopregnanolone (ALLO) elicits anxiolytic-like action and increases neuropeptide Y Y1 (NPY Y1) receptors gene expression in the amygdala, we were interested in studying the involvement of NPY Y1 receptors in the anxiolytic-like actions of ALLO. The anxiety-like behavior was evaluated in mice using Vogels conflict test (VCT), in which number of shocks were measured. ALLO and NPYergic agents, alone or in combinations, were administered bilaterally into the central nucleus of amygdala (CeA). The intra-CeA administration of ALLO, NPY or NPY Y1/Y5 receptors agonist [Leu(31), Pro(34)]-NPY resulted in dose-dependent increase in the number of shocks in VCT, indicating anxiolytic-like effect. However, opposite effect was observed following administration of selective NPY Y1 receptors antagonist BIBP3226. While prior administration with NPY or [Leu(31), Pro(34)]-NPY, at the subeffective dose, potentiated the ALLO-induced anxiolytic-like effect, the same was antagonized by BIBP3226. Further, the effect of acute ALLO (30 mg/kg, intraperitoneal) on the endogenous NPY system in the CeA, ventral part of lateral division of bed nucleus of the stria terminalis (BSTLV), nucleus accumbens core (AcbC) and arcuate nucleus (ARC) was studied with immunocytochemistry. Acute ALLO treatment significantly decreased the population of NPY-immunoreactive cells in the CeA and also in the ARC. While NPY-immunoreactive fibers were slightly increased in the AcbC and BSTLV, the cells in AcbC and fibers in ARC did not respond. We suggest that NPY may mediate ALLO induced anxiolytic-like behavior in the neuroanatomical framework of the CeA, possibly via NPY Y1 receptors.

Neuropeptide Y modulates the antidepressant activity of imipramine in olfactory bulbectomized rats: Involvement of NPY Y1 receptors

Since long-term treatment with imipramine increases the neuropeptide Y (NPY) levels in the frontal cortex and hypothalamus, the possibility exists that the antidepressant action of imipramine may be mediated via the NPY Y1 receptors. Bilateral olfactory bulbectomy (OBX) resulted in hyperactivity (increased number of ambulation, rearing and grooming episodes) in open field test (OFT) suggesting a depression-like condition. Chronic (14 days) administration of NPY, NPY Y1/Y5 receptor agonist [Leu(31), Pro(34)]-NPY (intracerebroventricular, i.c.v.) or tricyclic antidepressant imipramine (intraperitoneal) to OBX rats dose-dependently resulted in decreased hyperactivity in OFT, while selective NPY Y1 receptor antagonist BIBP3226 (i.c.v.) produced opposite effects. The antidepressant actions of imipramine were enhanced by co-administration of NPY or [Leu(31), Pro(34)]-NPY, and antagonized by BIBP3226 given at sub-effective doses. The data suggest that NPY, acting via NPY Y1 receptors, may be involved in antidepressant action of imipramine in OBX rats.

Involvement of neuropeptide Y in the acute, chronic and withdrawal responses of morphine in nociception in neuropathic rats: Behavioral and neuroanatomical correlates

Although morphine is a potent antinociceptive agent, its chronic use developed tolerance in neuropathic pain (NP). Furthermore, opioid antagonist naloxone attenuated the antinociceptive effect of neuropeptide Y (NPY). The present study investigated the role of NPY and NPY Y1/Y5 receptors in acute and chronic actions of morphine in neuropathic rats using thermal paw withdrawal test and immunocytochemistry. In acute study, intracerebroventricular (icv) administration of morphine, NPY or NPY Y1/Y5 receptors agonist [Leu(31),Pro(34)]-NPY produced antinociception, whereas selective NPY Y1 receptors antagonist BIBP3226 caused hyperalgesia. While NPY or [Leu(31),Pro(34)]-NPY potentiated, BIBP3226 attenuated morphine induced antinociception. Chronic icv infusion of morphine via osmotic minipumps developed tolerance to its antinociceptive effect, and produced hyperalgesia following withdrawal. However, co-administration of NPY or [Leu(31),Pro(34)]-NPY prevented the development of tolerance and withdrawal hyperalgesia. Sciatic nerve ligation resulted in significant increase in the NPY-immunoreactive (NPY-ir) fibers in ventrolateral periaqueductal gray (VLPAG) and locus coeruleus (LC); fibers in the dorsal part of dorsal raphe nucleus (DRD) did not respond. While chronic morphine treatment significantly reduced NPY-ir fibers in VLPAG and DRD, morphine withdrawal triggered significant augmentation in NPY-immunoreactivity in the VLPAG. NPY-immunoreactivity profile of LC remained unchanged in all the morphine treatment conditions. Furthermore, removal of sciatic nerve ligation reversed the effects of NP, increased pain threshold and restored NPY-ir fiber population in VLPAG. NPY, perhaps acting via Y1/Y5 receptors, might profoundly influence the processing of NP information and interact with the endogenous opioid system primarily within the framework of the VLPAG.

NPY mediates reward activity of morphine, via NPY Y1 receptors, in the nucleus accumbens shell

Although the interaction between endogenous neuropeptide Y (NPY) and opioidergic systems in processing of reward has been speculated, experimental evidence is lacking. We investigated the role of NPY, and its Y1 receptors, in the nucleus accumbens shell (AcbSh) in morphine induced reward and reinforcement behavior. Rats were implanted with cannulae targeted at AcbSh for drug administration, and with stimulating electrode in the medial forebrain bundle (MFB). The rats were then conditioned in an operant conditioning chamber for electrical self-stimulation of the MFB. Increased rate of lever pressings was evaluated against the frequency of the stimulating current. Increase in rate of lever presses was considered as a measure of reward and reinforcement. About 30-70% increase in self-stimulation was observed following bilateral intra-AcbSh treatment with morphine, NPY or [Leu(31), Pro(34)]-NPY (NPY Y1/Y5 receptors agonist), however, BIBP3226 (selective NPY Y1 receptors antagonist) produced opposite effect. The reward effect of morphine was significantly potentiated by NPY or [Leu(31), Pro(34)]-NPY, but antagonized by BIBP3226. NPY-immunoreactivity in the AcbSh, arcuate nucleus (ARC) and lateral part of bed nucleus of stria terminalis (BNSTl) was significantly more in the operant conditioned rats than in naïve control. However, morphine administration to the conditioned rats resulted in significant decrease in the NPY-immunoreactivity in all these anatomical regions. Since the role of morphine in modulation of mesolimbic-dopaminergic pathway is well established, we suggest that NPY system in AcbSh, ARC and BNSTl, perhaps acting via Y1-receptor system, may be an important component of the mesolimbic-AcbSh reward circuitry triggered by endogenous opioids.

Effect of alpha-melanocyte stimulating hormone on locomotor recovery following spinal cord injury in mice: Role of serotonergic system

The present study underscores the effect of serotonergic antagonist on alpha-melanocyte stimulating hormone (α-MSH) induced neuronal regeneration. Swiss-albino mice were subjected to experimental spinal cord injury (ESCI) and treated with serotonergic antagonist, ritanserin, alone or in combination with α-MSH, and the locomotor recovery was investigated. ESCI was induced at thoracic T(10-12) level by compression method. Motor function score (0-10) of each mouse was monitored prior to, and on days 1, 4, 7, 10 and 14 following ESCI. Untreated ESCI animals showed almost normal hind limb motor function by 14days. Similar degree of recovery was observed on day 10 in animals given α-MSH or ritanserin. However, in animals treated with both agents, comparable recovery was observed on day 4. While histological examination of the spinal cord following ESCI showed demyelination, necrosis and cyst formation, treatment with ritanserin, alone and in combination with α-MSH, significantly prevented the tissue damage. We suggest that early antagonism of serotonergic 5-HT(2a/2c) receptors may potentiate the neurotropic and locomotor recovery activity of α-MSH.

Cocaine- and amphetamine-regulated transcript peptide (CART) in the central nucleus of amygdala potentiates behavioral and hormonal responses of the rat exposed to its predator

Since cocaine- and amphetamine-regulated transcript peptide (CART) regulates anxiety and stress in amygdala, we hypothesized that the peptide may also process negative psychological experience like fear. During acute exposure to a cat, the rat showed freezing behavior and subsequently, profound signs of anxiety in social interaction test, and elevated serum cortisol concentration. While these behavioral effects were potentiated by the intracerebroventricular (icv) and intra-central nucleus of amygdala (intra-CeA) administration of CART peptide, they were blocked by CART antibody. On the other hand, chronic exposure for 7 days resulted in a steady reduction in freezing, increase in social interaction index and restored cortisol levels. In these rats, the behavior resembled with that of the time matched control rats suggesting habituation. However, CART peptide treatment, via the icv or intra-CeA route, chronically for 7 days, prevented habituation; significant freezing behavior and anxiety were noticed in these rats. The results suggest that CART peptide, in the framework of CeA, may process predator triggered innate fear in acute time scale, while chronic exposure might down-regulate the system and produce habituation.