Cocaine- And Amphetamine-regulated Transcript Peptide Expression Research Articles

Reproductive phase related variations in the expression of neuropeptide, cocaine- and amphetamine- regulated transcript (CART) in the brain and pituitary gland of adult male Microhyla ornata

Cocaine- and amphetamine-regulated transcript (CART) peptide is a multifaceted neuropeptide involved in several physiological functions including appetite and reproduction. While studies in mammals, aves and fishes suggest evolutionary conserved role of CART, the information in amphibian is scanty. We have investigated the reproductive phase related variations of CART in the brain of adult male Microhyla ornata. Seasonal changes in the expression of CART peptide were noticed in the brain and pituitary of M. ornata. Significant differences were observed in the nucleus infundibularis ventralis (NIV), epiphysis (E), anteroventral tegmental region (AV), raphe nucleus (Ra) of the brain and pars intermedia (PI), pars distalis (PD) of the pituitary. Compared to the pre-breeding and post-breeding seasons, increase in CART immunoreactivity was seen in E, NIV, AV, Ra of brain and PI, PD of pituitary gland of animals collected during breeding season. Similarly, highest mRNA levels of CART were also observed in the breeding season in the middle region of brain that includes hypothalamus and pituitary gland. Variation in the levels of CART peptide and mRNA in the brain of M. ornata suggests its conserved role in seasonal control of appetite and reproduction.

Hindbrain Actions of Cocaine‐ and Amphetamine‐Regulated transcript Peptide (CART) Revealed by Meal Pattern Analysis

CART is expressed in central nervous system (CNS) sites known to be important in the control of food intake, including the hypothalamus and hindbrain, as well as in the nodose ganglion of the vagus nerve whose central projections terminate in the nucleus tractus solitarius (NTS), an important site for peripheral feedback controlling appetite and associated motivated behaviors. Numerous groups, including ours, have demonstrated that CART inhibited food intake when administered i.c.v. in rats. In addition, others have demonstrated that central administration of antibodies to CART peptides increased feeding. These studies established the potential for endogenously produced CART peptides to be physiologically relevant regulators of food intake and overall glucose homeostasis. Those pharmacologically-based studies were corroborated by experiments using CART knockout animals and human studies which identified CART SNPs associated with metabolic dysfunction. Like other centrally-expressed anorexigenic peptides, CART exhibits functional changes in expression depending on metabolic status. In particular, CART expression is reduced in the fasted state, but returns to baseline levels upon refeeding, an effect that is conserved across multiple species. Combined, these findings suggest a critical role of CART in appetite regulation. Recently, we identified the orphan G protein-coupled receptor, GPR160, to be a cognate receptor for CART and defined the obligate role of CART signaling via GPR160 in nerve injury-induced pain sensation (PMID 31999650). Similarly, we have demonstrated the role of GPR160 in transmitting the anorexigenic action of CART (PMID 33206556) and described the localization of GPR160 on neurons and glia in hindbrain and forebrain sites known to be important in the control of ingestive behaviors including the dorsal vagal complex in the brainstem. In addition we now have established that CART administration in fourth cerebroventricle directly above the NTS exerts its anorexigenic action by not only limiting the amount of food consumed, but also by changing the patterning of the ingestive behavior. CART administration reduced the amount of food ingested per meal, the total number of food bouts, the number of food bouts per meal, and the duration of each meal, while increasing the interval between meals. Thus CART exerted significant satiation (meal bout based) and satiety (total meal amounts) effects. We have developed a transgenic rat line in which the gene for GPR160 is located within loxP sites and are examining the effects on meal patterning after compromise of receptor expression in selective brain areas, in an attempt to identify the critical sites of action of the peptide not only for the control of ingestive behaviors, but also CART's actions related to reward behavior and neuroendocrine function.

Increased expression of CART, nNOS, VIP, PACAP, SP and GAL in enteric neurons of the porcine stomach prepyloric region following hydrochloric acid infusion.

Stomach hyperacidity leads to damage of the mucus/bicarbonate barrier, ulcerations and the development of stomach cancer. Key regulators of the mucosal barrier/luminal acid balance are neurotransmitters secreted by intramural neurons. The aim of the current study was to determine the expression of gastric neuropeptides and nNOS in the porcine stomach following hydrochloric acid instillation. We report on increased expression of enteric neurotransmitters involved in adaptive reaction to an experimentally-induced hyperacidity state. The investigation was conducted on eight 12-18 kg pigs. The influence of intragastric infusion of hydrochloric acid on the expression of cocaine- and amphetamine-regulated transcript peptide (CART), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), substance P (SP) and galanin (GAL) in the submucous and myenteric gastric neurons of the pig has been studied with double immunofluorescence. A mimicked hyperacidity state significantly increased the proportion of enteric neurons immunoreactive to CART, nNOS, VIP, PACAP, SP and GAL in the submucous gastric neurons. In the myenteric plexus, a significant increase of the number of VIP-, CART- and GAL-immunoreactive (IR) neurons was found. Similarly, the percentage of myenteric nNOS-IR and PACAP-IR neurons tended to increase, while the fraction of SP-IR cells did not change. Stomach hyperacidity modifies the expression of the studied neurotransmitters in a specific way depending on the location of the neurons in particular plexuses of the stomach. Increased numbers of neurons expressing CART, nNOS, VIP, PACAP, SP and GAL clearly indicate their regulatory engagement in the restoration of the physiological gastric balance following hyperacidity.

Evaluation of the CART peptide expression in morphine sensitization in male rats

The importance of Cocaine- and amphetamine-regulated transcript (CART) peptide in reinforcing effects of addictive drugs specially alcohol and psychostimulants has been stablished. Involvement of CART peptide in rewarding effects of opioids in brain has recently been reported. Here we have studied the expression of CART mRNA and peptide in the reward pathway in morphine-induced sensitization phenomenon and also evaluated the peptide level fluctuations in CSF and plasma. Male Wistar rats received 7-day morphine injection (20mg/kg) and then after a 7-day washout period, a challenge dose of 10mg/kg morphine was administered and locomotor activity and oral stereotypical behaviors were recorded. Besides, the expression level of CART mRNA and peptide in four important areas of the mesocorticolimbic reward pathway including nucleus accumbens, striatum, prefrontal cortex, and hippocampus were measured by real-time PCR and western blotting, respectively. The level of the peptide in CSF and plasma was measured by Elisa method. The expression level of CART mRNA and protein in brain regions and also the peptide level in CSF and plasma were significantly down-regulated after 7-day morphine administration. These reduced levels returned to nearly normal rates after 7-day wash-out period. Administration of morphine challenge dose led to significant upregulation of CART gene expression (both mRNA and peptide) in the brain, and elevation of peptide level in CSF and plasma in morphine-sensitized rats. It can be concluded that CART is released in the framework of reward pathway and may serve as an important neurotransmitter in the process of morphine dependence and sensitization.

Nicotine regulates cocaine-amphetamine-Regulated Transcript (Cart) in the mesocorticolimbic system.

Cocaine-and-Amphetamine Regulated Transcript (CART) mRNA and peptides are intensely expressed in the brain regions comprising mesocorticolimbic system. Studies suggest that CART peptides may have a role in the regulation of reward circuitry. The present study aimed to examine the effect of nicotine on CART expression in the mesocorticolimbic system. Three different doses of nicotine (0.2, 0.4, 0.6 mg/kg free base) were injected subcutaneously for 5 days, and on day 6, rats were decapitated following a challenge dose. CART mRNA and peptide levels in medial prefrontal cortex (mPFC), nucleus accumbens (NAc), dorsal striatum (DST), amygdala (AMG), lateral hypothalamic area (LHA), and ventral tegmental area (VTA) were measured by quantitative real-time PCR (qPCR) and Western Blot analysis, respectively. In the mPFC, 0.4 and 0.6 mg/kg nicotine, decreased CART peptide levels whereas there was no effect on CART mRNA levels. In the VTA, a down-regulation of CART peptide expression was observed with 0.2 and 0.6 mg/kg nicotine. Conversely, 0.4 and 0.6 mg/kg nicotine increased CART mRNA levels in the AMG without affecting the CART peptide expression. Nicotine did not regulate CART mRNA or CART peptide expression in the NAc, DST, and LHA. We conclude that nicotine regulates CART expression in the mesocorticolimbic system and this regulation may play an important role in nicotine reward. Synapse 70:283-292, 2016. © 2016 Wiley Periodicals, Inc.

Differential expression of CART in feeding and reward circuits in binge eating rat model

Binge eating (BE) disrupts feeding and subverts reward mechanism. Since cocaine- and amphetamine-regulated transcript peptide (CART) mediates satiety as well as reward, its role in BE justifies investigation. To induce BE, rats were provided restricted access to high fat sweet palatable diet (HFSPD) for a period of 4 weeks. Immunoreactivity profile of the CART elements, and accompanying neuroplastic changes were studied in satiety- and reward-regulating brain nuclei. Further, we investigated the effects of CART, CART-antibody or rimonabant on the intake of normal chow or HFSPD. Rats fed on HFSPD showed development of BE-like phenotype as reflected by significant consumption of HFSPD in short time frame, suggestive of dysregulated satiety mechanisms. At the mid-point during BE, CART-immunoreactivity was significantly increased in hypothalamic arcuate (ARC), lateral (LH), nucleus accumbens shell (AcbSh) and paraventricular nucleus of thalamus (PVT). However, for next 22-h post-binge time-period, the animals showed no interest in food, and low CART expression. Pre-binge treatment with rimonabant, a drug recommended for the treatment of BE, produced anorexia, increased CART expression in ARC and LH, but not in AcbSh and PVT. Higher dose of CART was required to produce anorexia in binged rats. While neuronal tracing studies confirmed CART fiber connectivity from ARC and LH to AcbSh, increase in CART and synaptophysin immunostaining in this pathway in BE rats suggested strengthening of the CART connectivity. We conclude that CART bearing ARC-LH-PVT-AcbSh reward circuit may override the satiety signaling in ARC-PVN pathway in BE rats.

Immunodetection of cocaine- and amphetamine-regulated transcript in bovine pancreas

This study was aimed at identifying and determining the configuration of structures which contain the cocaine- and amphetamine-regulated transcript peptide (CART) in the bovine pancreas. The study material was collected from 20 animals. The distribution of CART in the bovine pancreas was investigated, by an immunohistochemical evaluation.CART peptide in the normal pancreas has been identified in intrapancreatic ganglia, nerve fibres and in endocrine cells of Langerhans islets and exocrine pancreas. CART immunoreactive nerve fibres innervate the exocrine and endocrine regions and the intrapancreatic ganglia, where they form a moderate number of networks, encircling the cell bodies. The few CART-immunoreactive endocrine cells, that appear in the bovine pancreas, are not limited to the islet cells, where they form a subpopulation of CART-containing cells, but are also individually distributed in the exocrine region. Furthermore, CART has been visualized in nerve fibres, innervating pancreatic outlet ducts and blood vessels.CART plays a physiological role in the integrated mechanisms that regulate both endocrine and exocrine pancreatic secretion.These results are consistent with the hypothesis that CART expression in nerve fibres and intrapancreatic ganglia is a common feature of the mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to single cells of the bovine pancreas.

Satiety and Reward: Dichotomy of Cocaine‐ and Amphetamine‐Regulated Transcript Peptide's Function During Binge Eating

Since cocaine‐ and amphetamine‐regulated transcript peptide (CART) mediates feeding as well as reward, we investigated its role in binge eating (BE), a clinical condition which disrupts satiety. BE was induced in rats by providing 2‐h access to high fat‐sweet palatable diet (HFSPD) on alternate days for 4 weeks. Effect of drug treatments on HFSPD intake and CART immunoreactive profile in reward and satiety regulating nuclei was studied. Rats fed on HFSPDshowed explosive BE. Higher doses of CART or rimonabant was required to reduce BE, as compared to that of normal chow intake in naïve rats. At the mid‐point during BE, CART immunoreactivity was significantly up‐regulated in the arcuate (ARC), lateral (LH) nucleus, paraventricular nucleus of thalamus (PVT), and nucleus accumbens shell (AcbSh) but not in the paraventricular (PVN) nucleus. At mid‐binge time‐point, the rimonabant treated rats showed increased CART expression in ARC and LH, while that in PVT and AcbSh was decreased. Increase in co‐expression of CART and synaptophysin in LH and cornu ammonis 1 of hippocampus suggested neuroplastic changes in CART system in BE rats. We suggests that BE enhanced the CARTergic signaling in the ARC/LH‐PVT‐AcbSh axis and suppressed the ARC‐PVN pathway. The net result may be the reward pathway overriding the satiety circuit. Opposite may happen following treatment with rimonabant. Further, CART seems to participate in the memory formation pertaining to HFSPD‐related reward.

Stability of CART peptide expression in the nucleus accumbens in aging.

Aging is accompanied by changes of several anorexigenic and orexigenic neuropeptides expressed in various brain areas that control food intake and these changes correlate with senescent anorexia. During aging expression of cocaine- and amphetamine-regulated transcript (CART) peptide was reported to be reduced in the hypothalamic nuclei related to food intake. Although CART peptide is abundant in the nucleus accumbens that also plays a crucial role in the food intake regulation, no data is available about the CART peptide expression in this region through aging. In the present study, CART peptide immunoreactivity was compared in the nucleus accumbens of young adult (4- and 7-month-old) middle-aged (15-month-old) and aging (25-32-month-old) Long-Evans rats. The density of CART-immunoreactive cells and axon terminals in the nucleus accumbens was measured with computer-aided densitometry. CART-immunodensity was similar in the old rats and in the younger animals without significant difference between age groups. In addition, no gender-difference was observed when CART-immunoreactivities in the nucleus accumbens of male and female animals were compared. Our results indicate that CART peptide expression in the nucleus accumbens is stable in adults and does not change with age.

P.1.029 DNase activity of immunoglobulins G in schizophrenia patients with tardive dyskinesia

Vagal afferent neurons (VAN) express the cholecystokinin (CCK) type 1 receptor (CCK1R) and, as predicted by the role of CCK in inducing satiation, CCK1R−/− mice ingest larger and longer meals. However, after a short fast, CCK1R−/− mice ingesting high fat (HF) diets initiate feeding earlier than wild-type mice. We hypothesized that the increased drive to eat in CCK1R−/− mice eating HF diet is mediated by ghrelin, a gut peptide that stimulates food intake. The decrease in time to first meal, and the increase in meal size and duration in CCK1R−/− compared to wild-type mice ingesting high fat (HF) diet were reversed by administration of GHSR1a antagonist D-(Lys3)-GHRP-6 (p < 0.05). Administration of the GHSR1a antagonist significantly increased expression of the neuropeptide cocaine and amphetamine-regulated transcript (CART) in VAN of HF-fed CCK1R−/− but not wild-type mice. Administration of the GHSR1a antagonist decreased neuronal activity measured by immunoreactivity for fos protein in the nucleus of the solitary tract (NTS) and the arcuate nucleus of both HF-fed wild-type and CCK1R−/− mice. The data show that hyperphagia in CCK1R−/− mice ingesting HF diet is reversed by blockade of the ghrelin receptor, suggesting that in the absence of the CCK1R, there is an increased ghrelin-dependent drive to feed. The site of action of ghrelin receptors is unclear, but may involve an increase in expression of CART peptide in VAN in HF-fed CCK1R−/− mice.

Cocaine-and-Amphetamine Regulated Transcript (CART) peptide attenuates dopamine- and cocaine-mediated locomotor activity in both male and female rats: Lack of sex differences

Cocaine-and-Amphetamine Regulated Transcript peptide (CART peptide) is known for having an inhibitory effect on dopamine (DA)- and cocaine-mediated actions and is postulated to be a homeostatic, regulatory factor in the nucleus accumbens (NAc). Some sex differences in cocaine-mediated locomotor activity (LMA) and in the expression and function of CART peptide have been reported. However, it is not known if the inhibitory effect of CART peptide on cocaine-mediated LMA is sexually dimorphic. In this study, the effects of CART 55-102 on LMA due to intra-NAc DA and i.p. cocaine were determined in male and female Sprague–Dawley rats. The results show that CART 55-102 blunted or reduced both the DA- and cocaine-induced LMA in both males and females. In conclusion, CART peptide is effective in blunting DA- and cocaine-mediated LMA in both males and females.

Region- and sex-specific changes in CART mRNA in rat hypothalamic nuclei induced by forced swim stress

Cocaine and amphetamine regulated transcript (CART) mRNA and peptides are highly expressed in the paraventricular (PVN), dorsomedial (DMH) and arcuate (ARC) nuclei of the hypothalamus. It has been suggested that these nuclei regulate the hypothalamic–pituitary–adrenal (HPA) axis, autonomic nervous system activity, and feeding behavior. Our previous studies showed that forced swim stress augmented CART peptide expression significantly in whole hypothalamus of male rats. In another study, forced swim stress increased the number of CART-immunoreactive cells in female PVN, whereas no effect was observed in male PVN or in the ARC nucleus of either sex. In the present study, we evaluated the effect of forced swim stress on CART mRNA expression in PVN, DMH and ARC nuclei in both male and female rats. Twelve male (stressed and controls, n=6 each) and 12 female (stressed and controls, n=6 each) Sprague-Dawley rats were used. Control animals were only handled, whereas forced swim stress procedure was applied to the stressed groups. Brains were dissected and brain sections containing PVN, DMH and ARC nuclei were prepared. CART mRNA levels were determined by in situ hybridization. In male rats, forced swim stress upregulated CART mRNA expression in DMH and downregulated it in the ARC. In female rats, forced swim stress increased CART mRNA expression in PVN and DMH, whereas a decrease was observed in the ARC nucleus. Our results show that forced swim stress elicits region- and sex-specific changes in CART mRNA expression in rat hypothalamus that may help in explaining some of the effects of stress.

CART peptide following social novelty in the prairie vole ( Microtus ochrogaster)

Prairie voles ( Microtus ochrogaster) are monogamous rodents that display high levels of affiliative behaviors, including pair-bonding, biparental care, and cooperative breeding. Species differences in basal cocaine- and amphetamine-regulated transcript (CART) mRNA and peptide expression have been found between prairie voles and polygamous meadow voles. Therefore, we hypothesized that the CART system may play a role in the regulation of social behavior in this species. Male and female adult prairie voles were placed in a cage either alone, or with a novel social partner of the same or opposite sex. After 45 min, subjects were sacrificed and CART peptide expression was examined using immunohistochemistry. We examined fifteen hypothalamic, limbic, and hindbrain regions of interest, focusing on areas that show species-specific patterns of expression. We found that subjects paired with a novel conspecific had lower levels of peptide in the bed nucleus of the stria terminalis (BNST) than isolated animals. This may reflect increased peptide release following increased dopaminergic activity in animals exposed to a novel conspecific. Additionally, CART peptide was higher in the nucleus accumbens (NAc) of subjects paired with an opposite sex partner compared to those paired with a same-sex conspecific, although there was no difference between isolated subjects and either socially housed group. These findings suggest that CART in the NAc is differentially responsive to the sex of adult conspecifics and that the social environment influences CART expression in the prairie vole in a region- and stimulus-specific manner.

Effect of ghrelin receptor antagonist on meal patterns in cholecystokinin type 1 receptor null mice

Vagal afferent neurons (VAN) express the cholecystokinin (CCK) type 1 receptor (CCK₁R) and, as predicted by the role of CCK in inducing satiation, CCK₁R⁻/⁻ mice ingest larger and longer meals. However, after a short fast, CCK₁R⁻/⁻ mice ingesting high fat (HF) diets initiate feeding earlier than wild-type mice. We hypothesized that the increased drive to eat in CCK₁R⁻/⁻ mice eating HF diet is mediated by ghrelin, a gut peptide that stimulates food intake. The decrease in time to first meal, and the increase in meal size and duration in CCK₁R⁻/⁻ compared to wild-type mice ingesting high fat (HF) diet were reversed by administration of GHSR1a antagonist D-(Lys3)-GHRP-6 (p<0.05). Administration of the GHSR1a antagonist significantly increased expression of the neuropeptide cocaine and amphetamine-regulated transcript (CART) in VAN of HF-fed CCK₁R⁻/⁻ but not wild-type mice. Administration of the GHSR1a antagonist decreased neuronal activity measured by immunoreactivity for fos protein in the nucleus of the solitary tract (NTS) and the arcuate nucleus of both HF-fed wild-type and CCK₁R⁻/⁻ mice. The data show that hyperphagia in CCK₁R⁻/⁻ mice ingesting HF diet is reversed by blockade of the ghrelin receptor, suggesting that in the absence of the CCK₁R, there is an increased ghrelin-dependent drive to feed. The site of action of ghrelin receptors is unclear, but may involve an increase in expression of CART peptide in VAN in HF-fed CCK₁R⁻/⁻ mice.

Cocaine- and amphetamine-regulated transcript peptide immunoreactivity in the brain of the CCK-1 receptor deficient obese OLETF rat

Cocaine- and amphetamine-regulated transcript (CART) peptide is expressed in brain areas involved in homeostatic regulation and reward. CART has been shown to reduce food intake, but the underlying mechanisms and the relevance of this effect on obesity yet remain unknown. Therefore, we used immunohistochemistry to investigate the expression of CART peptide in various brain regions of the obese Otsuka Long Evans Tokushima Fatty (OLETF) rats lacking the CCK-1 receptor. Analysis revealed that whereas the distribution of CART-peptide immunoreactive neurons and axonal networks was identical in OLETF rats and lean controls, the intensity of CART immunoreactivity was significantly reduced in the rostral part of the nucleus accumbens (p < 0.01), the basolateral complex of the amygdala (p < 0.05) and the rostro-medial nucleus of the solitary tract (p < 0.001) of the OLETF rats. These areas are involved in reward and integration of taste and viscerosensory information and have been previously associated with altered functions in this strain. The findings suggest that in addition to previously described deficits in peripheral satiety signals and augmented orexigenic regulation, the anorectic effect of CART peptide may also be diminished in OLETF rats.

Regulation of CART peptide expression by CREB in the rat nucleus accumbens in vivo

Production of mRNA from the cocaine- and amphetamine-regulated transcript (CART) gene is regulated by cocaine and other drugs of abuse in the nucleus accumbens (NAc), a brain reward region. Current hypotheses postulate that CART peptides there oppose the rewarding actions of cocaine by opposing the effects of dopaminergic transmission. Since over expression of CREB was shown to decrease cocaine-mediated reward, we hypothesized that CART could be a target gene for CREB in the NAc and that over expression of CREB would increase CART peptide levels. Transcription factor (TF) binding to DNA is influenced by sequences adjacent to consensus TF binding sites and other factors. We thus examined CREB binding to a 27mer oligonucleotide containing the CRE sequence from the CART gene proximal promoter. Using electrophoretic mobility shift assays and TF-antibody super shift assays, CREB was found to bind to the CRE sequence from the CART promoter. To test if over expression of CREB in the NAc affected CART peptide levels, Herpes simplex virus-1 vectors over expressing CREB (HSV-CREB), or a vector that expressed LacZ (HSV-LacZ) as a control, were injected into the NAc of rats. Western blotting and in situ hybridization showed that HSV-CREB injections increased CART mRNA and peptide levels. Injections of a dominant negative CREB mutant (HSV-mCREB) did not alter either CART mRNA or peptide levels. The finding that CREB can regulate the levels of CART mRNA and peptides in vivo in the NAc supports a role for CART peptides in psychostimulant-induced reward and reinforcement.

Unique responses of midbrain CART neurons in macaques to ovarian steroids

CART (cocaine and amphetamine regulated transcript) is a neuropeptide involved in the control of several physiological processes, such as response to psychostimulants, food intake, depressive diseases and neuroprotection. It is robustly expressed in the brain, mainly in regions that control emotional and stress responses and it is regulated by estrogen in the hypothalamus. There is a distinct population of CART neurons located in the vicinity of the Edinger–Westphal nucleus of the midbrain that also colocalize urocortin-1. The aims of this study were 1) to determine the distribution of CART immunoreactive neurons in the monkey midbrain, 2) to examine the effects of estrogen (E) and progesterone (P) on midbrain CART mRNA and peptide expression and 3) to determine whether midbrain CART neurons contain steroid receptors. Adult female rhesus monkeys ( Macaca mulatta) were spayed and either treated with placebo (OVX), estrogen alone (E), progesterone alone (P) or E+ P . Animals were prepared (a) for RNA extraction followed by microarray analysis and quantitative (q) RT-PCR ( n = 3/group); (b) for immunohistochemical analysis of CART and CART + tryptophan hydroxylase (TPH), CART + estrogen receptors (ER) or CART + progesterone receptors ( n = 5/group) and (c) for Western blots ( n = 3/group). Both E- and E + P-administration decreased CART gene expression on the microarray and with qRT-PCR. Stereological analysis of CART immunostaining at five levels of the Edinger–Westphal nucleus indicated little effect of E or E + P administration on the area of CART immunostaining. However, P administration increased CART-immunopositive area in comparison to the OVX control group with Student’s t-test, but not with ANOVA. CART 55–102 detection on Western blot was unchanged by hormone administration. ERβ and PR were detected in CART neurons and CART fibers appeared to innervate TPH-positive serotonin neurons in the dorsal raphe. In summary, E decreased CART mRNA, but this effect did not translate to the protein level. Moreover, P administration alone had a variable effect on CART mRNA, but it caused an increase in CART immunostaining. Together, the data suggest that CART neurons in the midbrain have a unique steroid response, which may be mediated by nuclear receptors, neuroactive steroids or interneurons.

Expression of cocaine- and amphetamine-regulated transcript (CART) peptides at climbing fibre-Purkinje cell synapses in the rat vestibular cerebellum

Cocaine- and amphetamine-regulated transcript (CART) peptides have a wide CNS distribution and appear to play an important role in a number of physiological processes including reward and reinforcement, feeding, locomotion, stress responses and perception of pain. We have further investigated the expression of CART peptides in rat cerebellum, using multiple fluorescent antibodies. Dense fibre-like immunofluorescence was observed in the molecular layer of the vestibular cerebellum (paraflocculus and lobes IX and X of the vermis). There was little or no immunofluorescence in any other region of the cerebellum (vermis or hemispheres). The immunofluorescence in lobes IX and X of the vermis showed a parasagittal banding pattern, with one medial and two lateral bands. We have provided several lines of evidence that CART peptides are expressed by climbing fibres: (1) There is CART peptide immunofluorescence in the inferior olivary complex, the source of climbing fibres. (2) No cerebellar cell bodies were labelled by the CART peptide antibody. (3) The developmental profile of CART peptide expression was consistent with climbing fibre development and finally (4) triple antibody labelling revealed co-expression of CART peptides with VGluT2 (the glutamate transporter at climbing fibre synapses) at boutons innervating calbindin D-28K labelled proximal Purkinje cell dendrites. Analysis of VGluT2 and CART peptide labelling in more detail showed that not all climbing fibres in the vestibular cerebellum expressed CART peptides but those fibres which did express CART peptides were immunofluorescent at all Purkinje cell synapses.

Ontogeny of cocaine- and amphetamine-regulated transcript (CART) peptide and calbindin immunoreactivity in granule cells of the dentate gyrus in the rat

Cocaine- and amphetamine-regulated transcript (CART) peptide was first discovered in the rat striatum following cocaine and amphetamine administration. However, even without psychostimulant treatment, many neuronal groups of the central nervous system, including granule cells of the dentate gyrus, express CART peptide. Earlier studies, based on the prenatal expression of CART peptide in the mesencephalon, suggest that it exerts neurotrophic effects. In the present study, ontogenetic expression of CART peptide in dentate gyrus granule cells was studied using immunohistochemistry in rats from 5 days to 3 months old. Expression was correlated with the expression of another neurochemical marker of granule cells, the calcium binding protein, calbindin.Calbindin was already present in granule cells at postnatal day 5 (P5), whereas CART peptide was first observed at P12. The first CART peptide- and calbindin-immunoreactive cells were localized to the lateral end of the dorsal blade, to the outer part of granule cell layer adjacent to the molecular layer, which agrees with the localization of the first-generated granule cells in the dentate gyrus. The first calbindin-immnunoreactive mossy fibers were seen at P9 in the stratum lucidum of CA3, and the entire projection path of mossy fibers expressed calbindin at P18. Mossy fibers were CART peptide-immunopositive at P12, and they were visible in the most distal part of CA3, in CA3a next to CA2. This localization fits with the known spatial organization of mossy fiber axon terminals. An adult-like expression of CART peptide and calbindin in the hippocampal formation was detectable at P30.The late postnatal appearance of CART peptide in dentate granule cells, and their axonal terminals, indicates that CART peptide may play a neurotrophic role in late developmental events, such as synaptogenesis. However, this does not exclude the possibility of a neuromodulatory role for this peptide.

Sex differences in the regulation of cocaine and amphetamine‐regulated transcript expression in hypothalamic nuclei of rats by forced swim stress

Cocaine and amphetamine-regulated transcript (CART) peptides are suggested to play a role in several physiological processes including feeding, reward, neuroendocrine modulation, and the stress response. Although some studies implicate the modulation of CART peptide expression by glucocorticoids, direct evidence relating CART to the stress response is limited. The present study was undertaken to evaluate the possible involvement of CART peptides during acute stress in male and female rats. Forced swim was used as the stress procedure. Following stress, serum adrenocorticotropic hormone (ACTH), and corticosterone (CORT) levels were determined, and CART immunocytochemistry was performed in the paraventricular (PVN) and arcuate (ARC) nuclei of the hypothalamus. Our results depict the following changes: (1) Serum ACTH and CORT levels were increased by stress and CORT levels were higher in female rats than males. (2) Stress modulated the number of CART expressing neurons. The degree and direction of this modulation varied according to the hypothalamic region and the sex of the subject. Forced swim stress increased CART peptide expression significantly in the PVN of female rats. In males, although there was a tendency for an increase in CART-immunoreactive cells by forced swim stress, the difference was not statistically significant. In the ARC nucleus, forced swim stress did not affect CART peptide expression in either sex. Our results suggest differential and sexually dimorphic modulation of CART expression in the PVN and ARC by forced swim stress.