Praful S. Singru

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.

Elucidating the role of disulfide bond on amyloid formation and fibril reversibility of somatostatin-14: Relevance to its storage and secretion

Background: Peptide/protein hormones are stored as amyloids within endocrine secretory granules. Results: Disulfide bond cleavage enhances conformational dynamics and aggregation kinetics in somatostatin-14, resulting in amyloid fibrils with increased resistance to denaturing conditions and decreased reversibility. Conclusion: Disulfide bond could be a key modulating factor in somatostatin-14 amyloid formation associated with secretory granule biogenesis. Significance: Defective disulfide bonding might cause dysregulation of hormone storage/secretion. The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys3–Cys14) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion.

Hypothalamic cocaine- and amphetamine-regulated transcript peptide is reduced and fails to modulate feeding behavior in rats with chemically-induced mammary carcinogenesis.

Cocaine- and amphetamine-regulated transcript peptide (CART) is a major anorectic agent present in the hypothalamus. We investigated the possible role of CART in mammary cancer-induced anorexia and body weight loss in rats. Mammary carcinogenesis was induced in the female Sprague-Dawley rats by intraperitoneal injection of N-methyl-N-nitrosourea (MNU). Following administration of MNU, rats progressively showed a reduction in food intake and body weight. Fourteen weeks after MNU treatment, rats were injected daily with CART or CART-antibody intracerebroventricularly for 5days, and food intake and body weight were monitored (g) before the next injection time-point. In normal rats, while a distinct anorexia and weight loss was observed following CART administration, injection of CART-antibody produced opposite effects. However, both the agents failed to produce any significant alterations in food intake and body weight of mammary tumor-bearing animals. An immunohistochemical application of antibodies against CART to the brain sections of cancerous rats showed a reduced immunoreactivity in the hypothalamic dorsomedial, ventromedial, lateral, paraventricular and arcuate nuclei. The results suggest that, cancerous condition might down-regulate the CART system in the hypothalamus. Alternatively, reduction in hypothalamic CART activity might be a counter-regulatory strategy to reverse food under-consumption or body mass erosion.

Cocaine- and amphetamine-regulated transcript peptide (CART) in the telencephalon of the catfish, Clarias gariepinus: Distribution and response to fasting, 2-deoxy-D-glucose, glucose, insulin, and leptin treatments

The cocaine‐ and amphetamine‐regulated transcript peptide (CART)‐containing system in the forebrain of Clarias gariepinus was studied with immunocytochemistry. While the immunoreactivity was prominently seen in the neurons of the entopeduncular nucleus (EN) located in the ventral telencephalon, CART‐immunoreactive fibers were widely distributed in the dorsal and ventral telencephalon. In view of the established role of CART in energy metabolism, we investigated the response of the CART immunoreactive system to positive and negative nutritional conditions. Neurons of the EN and fibers in the different areas of the telencephalon showed significant reduction in CART immunoreactivity following 48 hours food deprivation, or 2 hours following intracranial administration of 2‐deoxy‐D‐glucose (2DG, 100 ng/g body weight, a metabolic antagonist of glucose). However, intracranial injection of glucose (100 ng/g body weight) resulted in a distinct increase in CART immunoreactivity in these components. In mammals, insulin and leptin have been recognized as adiposity agents that convey peripheral energy status‐related information to brain. Intracranial administration of insulin (3 mU/fish) and leptin (10 ng/g body weight) significantly increased CART immunoreactivity in the EN neurons and in the fiber network within 2 hours. Superfusion of the EN‐containing tissue fragments in the medium enriched in glucose, insulin, or leptin evoked a significant increase in CART immunoreactivity in the EN neurons, but 2DG reduced the immunoreactivity. We suggest that CART‐containing neurons of the EN, and fibers in the telencephalon, may process the energy status‐related information and contribute to satiety. J. Comp. Neurol. 519:1281–1300, 2011.

Alpha-melanocyte stimulating hormone modulates ethanol self-administration in posterior ventral tegmental area through melanocortin-4 receptors

Although the role of alpha‐melanocyte stimulating hormone (α‐MSH) in alcohol seeking behaviour in rats has been demonstrated, the underlying mechanisms are not understood. Herein, we test the hypothesis that α‐MSH might have a permissive effect in promoting the reward action of ethanol. Rats were implanted with cannulae targeted at the posterior ventral tegmental area (pVTA), because the site is sensitive to reinforcing effects of ethanol. These rats were trained to self‐administer ethanol in standard two‐lever (active/inactive) operant chamber test. Each active lever press resulted in self‐administration of 100 nl of ethanol (100–300 mg%) containing solution. Over a period of 7 days, ethanol significantly increased the number of lever presses, which was considered as a measure of reward. Because ethanol at 200 mg% resulted in maximum number of lever presses (∼18–20 lever presses/30‐minute session), the dose was employed in further studies. While prior administration of melanocortin (MC) agonists, α‐MSH or [Nle4,D‐Phe7]‐alpha‐MSH into pVTA, resulted in an 89% increase in lever presses, the response was attenuated following pre‐treatment with MC4 receptors (MC4R) antagonist, HS014. In an immunohistochemical study, the brains of rats that were trained to self‐infuse ethanol showed significantly increased α‐MSH immunoreactivity in the nucleus accumbens shell, bed nucleus of stria terminalis and arcuate nucleus of the hypothalamus. In the pVTA, α‐MSH fibres were found to run close to the dopamine cells, labelled with tyrosine hydroxylase antibodies. We suggest that α‐MSH‐MC4R system in the pVTA might be a part of the neuroadaptive mechanism underlying ethanol addiction.

Involvement of cocaine- and amphetamine-regulated transcript peptide in the hyperphagic and body weight promoting effects of allopregnanolone in rats.

Allopregnanolone (ALLO), a gamma-aminobutyric acid (GABA) type A receptor active neurosteroid, elicits hyperphagic response in rodents. Since GABA-A receptors are present on the peptidergic neurons in the hypothalamus, we were interested in finding out if ALLO and neuropeptide cocaine- and amphetamine-regulated transcript (CART) interact and influence feeding behavior. While subcutaneous ALLO treatment, for a period of 7 days, produced a significant increase in food intake and body weight, pretreatment with subthreshold dose of CART (intracerebroventricular) attenuated both the effects. On the other hand, subcutaneous administration of dehydroepiandrosterone sulfate (DHEAS; GABA-A inhibitor neurosteroid) for a period of 7 days resulted in a significant reduction in food intake and body weight. These effects of DHEAS were potentiated by intracerebroventricular pretreatment with subeffective dose of CART. The brains of ALLO-treated rats were processed for the immunohistochemical analysis of CART immunoreactive elements. ALLO treatment resulted in a significant reduction in CART immunoreactivity in the hypothalamic arcuate, paraventricular and lateral nuclei, and nucleus accumbens shell. The results of the present study suggest that ALLO and CART might interact in the brain, and influence food intake and body weight. However, further investigations are needed to clarify the precise mechanisms by which ALLO modulate feeding behavior.

Interaction between Dopamine- and Isotocin-Containing Neurones in the Preoptic Area of the Catfish, Clarias batrachus: Role in the Regulation of Luteinising Hormone Cells

Apart from gonadotrophin‐releasing hormone (GnRH) and dopamine (DA), oxytocin has emerged as an important endogenous agent that regulates reproduction. Although the interaction between these factors has been extensively studied in mammals, parallel information in teleosts is much limited. We studied the organisation of tyrosine hydroxylase (TH; a marker for dopamine) and isotocin neurones in the preoptic area (POA) and hypothalamus of the catfish, Clarias batrachus and its implication in the regulation of luteinising hormone (LH) cells in the pituitary. Nucleus preopticus periventricularis (NPP), a major dopaminergic centre in the brain, consists of anterior (NPPa) and posterior (NPPp) subdivisions. Using retrograde neuronal tracing, we found that majority of the DA neurones in NPPa, but none from NPPp, project to the pituitary. The nucleus preopticus (NPO) of C. batrachus contains a conspicuous assemblage of large isotocin‐positive neurones. It consists of a paraventricular subdivision (NPOpv) located on either side of the third ventricle and lies roughly sandwiched between the dopaminergic neurones of NPPa and NPPp. An additional subset of isotocin neurones was located above the optic chiasm in the supraoptic subdivision of the NPO (NPOso). Isotocin‐containing neurones in both the subdivisions of NPO were densely innervated by DA fibres. Superfusion of the POA‐containing brain slices with DA D1‐like receptor agonist (SKF‐38393) resulted in significant increase in isotocin immunoreactivity in the NPOpv neurones; NPOso neurones did not respond. However, treatment with DA D2‐like receptor agonist (quinpirole) reduced isotocin immunoreactivity in the NPOso, but not in the NPOpv. Thus, DA appears to differentially regulate the components of isotocinergic system. Isotocin fibres extend to the pituitary and terminate on LH cells and the superfused pituitary slices treated with isotocin caused significant reduction in LHβ‐immunoreactivity. An elaborate interplay between the DA and isotocin systems appears to be an important component of the LH regulatory system.

Evidence for the participation of cocaine- and amphetamine-regulated transcript peptide (CART) in the fluoxetine-induced anti-hyperalgesia in neuropathic rats

Cocaine- and amphetamine-regulated transcript peptide (CART) has a role in chronic pain, and also in the actions of selective serotonin reuptake inhibitors (SSRIs) employed in the treatment of neuropathic pain. Herein, we test the hypothesis that CART may mediate the anti-hyperalgesic effect of the SSRI, fluoxetine, in neuropathic rats. Sciatic nerve in the right hind paw of rat was ligated to induce neuropathic pain, and the paw withdrawal latency was evaluated using Hargreaves apparatus. Fluoxetine [5-25mg/kg, intraperitoneal (ip)] or CART (54-102) [0.1-1.5μg/rat, intracerebroventricular (icv)] dose-dependently attenuated the hyperalgesic response observed in neuropathic rats, indicating anti-nociceptive properties of each agent. The anti-hyperalgesic effect of fluoxetine was potentiated by the subeffective dose of CART, and attenuated by CART-antibody (1:500 dilution; 5μl/rat, icv); CART-antibody had no effect per se. Isobolographic analysis showed a significant synergism between fluoxetine and CART, and antagonism between fluoxetine and CART-antibody. Immunocytochemical labeling with monoclonal antibodies against CART showed drastic increase in CART-immunoreactive fibers in the ventrolateral periaqueductal gray (VLPAG; 116%), dorsal subdivision of dorsal raphe nucleus (DRD; 176%), and locus coeruleus (LC; 733%) of neuropathic animals. Fluoxetine treatment significantly reduced the immunoreactivity in these areas. However, CART-immunoreactive cells and fibers in the arcuate nucleus did not respond to neuropathy or fluoxetine treatments. We suggest that the CART innervation of DRD, LC and VLPAG may be involved in the (i) central processing of neuropathic pain and (ii) fluoxetine-induced anti-hyperalgesic effect in neuropathic pain.

Tyrosine Hydroxylase in the Olfactory System, Forebrain and Pituitary of the Indian Major Carp, Cirrhinus cirrhosus: Organisation and Interaction with Neuropeptide Y in the Preoptic Area

Dopamine (DA) inhibits, whereas gonadotrophin‐releasing hormone (GnRH) stimulates, luteinisiing (LH) cells in the pituitary of some but not all teleosts. A reduction in the hypophysiotropic dopaminergic tone is necessary for the stimulatory effect of GnRH on LH cells. Neuropeptide Y (NPY) has emerged as one of the potent, endogenous agent that modulates LH secretion directly or indirectly via GnRH. Involvement of NPY in the regulation of hypophysiotropic DA neurones, however, is not known, but there is good evidence suggesting an interaction in the mammalian hypothalamus. DA neurones, identified by tyrosine hydroxylase (TH)‐immunoreactivity, were observed widely throughout the brain of the Indian major carp, Cirrhinus cirrhosus. The granule cells and ganglion cells of terminal nerve in the olfactory bulb, and cells in ventral telencephalon and preoptic area (POA) showed conspicuous TH immunoreactivity. In the POA, the nucleus preopticus periventricularis (NPP), divisible into anterior (NPPa) and posterior (NPPp) components, showed prominent TH‐immunoreactivity. The majority of TH neurones in NPPa showed axonal extensions to the pituitary and were closely associated with LH cells. The NPPa also appeared to be the site for intense interaction between NPY and DA because it contains a rich network of NPY fibres and few immunoreactive cells. Approximately 89.7 ± 1.5% TH neurones in NPPa were contacted by NPY fibres. Superfused POA slices treated with a NPY Y2‐receptor agonist, NPY 13‐36 resulted in a significant (P < 0.001) reduction in TH‐immunoreactivity in NPPa. TH neurones in NPPa did not respond to NPY Y1‐receptor agonist, [Leu31, Pro34] Neuropeptide Y treatment. We suggest that, by inhibiting DAergic neurones in NPPa via Y2‐receptors, NPY may contribute to the up‐regulation of the GnRH–LH cells axis. The microcircuitry of DA and NPY and their interaction in NPPa might be a crucial component in the central regulation of LH secretion in the teleosts.