Cocaine-amphetamine-regulated Transcript Peptide Research Articles

Injection of Cocaine-Amphetamine Regulated Transcript (CART) peptide into the nucleus accumbens does not inhibit caffeine-induced locomotor activity: Implications for CART peptide mechanism

Much evidence suggests that intra-nucleus accumbens (NAc) CART peptide (CART 55-102) injection inhibits locomotor activity (LMA) when there is an increase in the release and activity of dopamine (DA) in the NAc. However, this hypothesis has not been fully tested. One way to examine this is to determine if there is a lack of effect of intra-NAc CART peptide on LMA that does not involve increases in DA release in the NAc. Several studies have suggested that caffeine-induced LMA does not involve extracellular DA release in the NAc core. Therefore, in this study, we have examined the effect of injections of CART peptide (2.5μg) into the NAc core on the locomotor effects of caffeine in male Sprague-Dawley rats. Several LMA relevant doses of caffeine were used (0, 10, 20mg/kg i.p.), and an inverted U response curve was found as expected. We determined, in the same animals, that intra-NAc CART peptide had no effect on caffeine-induced LMA whereas it blunted cocaine-mediated LMA, as shown by other reports. We also extended a previous observation in mice by showing that at a LMA activating dose of caffeine there is no alteration of CART peptide levels in the NAc of rats. Our study supports the hypothesis that the inhibitory effects of CART peptide in the NAc may be exerted only under conditions of increased extracellular DA release and activity in this region. Our results also suggest that intra-NAc CART 55-102 does not generally inhibit increases in LMA due to all drugs, but has a more specific inhibitory effect on dopaminergic neurotransmission.

Up-regulation of orexigenic and down-regulation of anorexigenic neuropeptide gene expression in rat hypothalamus after partial lipectomy

Leptin down-regulates orexigenic and up-regulates anorexigenic neuropeptide gene expression in hypothalamus. Surgical removal of adipose tissue leads to decrease in circulating leptin concentrations in rats. In the present study, we tested: (a) regulation of neuropeptide gene expression in hypothalamus, (b) food intake, and (c) standard growth rate after removal of adipose tissue in rats. Partial lipectomy caused an approximately 10-fold reduction of subcutaneous, retroperitoneal and epididymal adipose tissue weight (at the end of experiments adipose tissue weight was 1.5±0.9 in lipectomy and 15±3.9g in control rats; statistically significant). Compared to control rats, the animals subjected to lipectomy presented increased food intake, standard growth rate, and decreased serum leptin concentrations (2.6±0.8 vs. 3.7±1.2ng/mL in the controls, statistically significant). These changes were associated with approximately twofold increase in neuropeptide Y, threefold increase in agouti-related peptide (orexigenic neuropeptides) and about 50% decrease in pro-opiomelanocortin and cocaine–amphetamine-regulated transcript peptide (anorexigenic neuropeptides) mRNA levels in the hypothalamus. These results suggest that partial lipectomy, leading to a decrease in circulating leptin concentrations, may exert an effect on hypothalamic orexigenic and anorexigenic neuropeptide gene expression, and consequently modulate food intake and standard growth rate in rats.

Gender differences in Nociceptin/Orphanin FQ-induced food intake in strains derived from rats prone (WOKW) and resistant (Dark Agouti) to metabolic syndrome: a possible involvement of the cocaine- and amphetamine-regulated transcript system

Our previous study found that when injected with Nociceptin/Orphanin FQ (N/OFQ) into the brain, male Dark Agouti (DA) rats, which are resistant to metabolic syndrome, have greater hyperphagia than male Wistar Ottawa Karlsburg W (WOKW) animals, which are prone to this disease. We attributed this difference to the fact that these two strains have different cocaine-amphetamine regulated transcript peptide (Cart) gene sequences and expression. In order to address this hypothesis, the present work focused on sex differences and analyzed not only male but also female N/OFQ-induced (0.25 and 0.5nmol/rat) food intake in terms of their Cart and N/OFQ receptor gene expression in the hypothalamic area. In N/OFQ-naive WOKW females, cart gene expression is extremely elevated compared to N/OFQ-naive WOKW males. When male and female WOKW littermates are stimulated with N/OFQ, the food intake of females is significantly lower than that of the males. Granted, the N/OFQ feeding behavior experiments were not performed on the animals measured for Cart gene expression, but nonetheless, the responses observed in littermates point to an interesting avenue for further inquiry.

CART receptor binding in primary cell cultures of the rat nucleus accumbens

Specific cocaine-amphetamine regulated transcript (CART) receptor binding has been reported in AtT20 and PC12 cells. In this study, receptor binding has been examined in primary cell cultures of several rat brain regions. Utilizing (125)I-CART 61-102 as a ligand and CART 55-102 as a displacer, significant levels of specific binding compared with nonspecific binding were found in the nucleus accumbens. Specific CART binding was substantially lower in hippocampal, hypothalamic, and pituitary cells. The specific binding was saturable with a K(d) of 1.43 +/- 0.25 nM and a B(max) of 49.03 +/- 2.33 fmol/mg protein. The binding was also highly specific for CART 55-102 as other inactive CART peptide fragments, unrelated peptides, gamma-aminobutyric acid, or cocaine showed no affinity for the receptor at 10 muM concentrations. Furthermore, addition of the GTP analog, Gpp(NH)p, significantly decreased specific binding but addition of an ATP analog did not, suggesting that the receptor is a g-protein coupled receptor. This is the first demonstration of CART peptide receptor binding in primary cell cultures of the rat brain.

Transcriptional activity by cocaine–amphetamine-regulated transcript

Cocaine–amphetamine-regulated transcript (CART) gene was identified in 1995 in a search for mRNAs in the striatum acutely upregulated by psychostimulants.1 CART is highest expressed in the hypothalamus and widely expressed in the central and peripheral nervous systems, as well as in endocrine cells.1, 2 CART peptides have received much attention as mediators of stress, reward and reinforcement, appetite control, addiction and neuroendocrine regulation.2 Recent studies link a mutation in the CART gene to obesity, type-II diabetes, anxiety and depression, and the mutation leads to CART deficiency or altered CART peptide levels, indicating a genetic cause for these human diseases or disorders.3, 4, 5, 6

Cocaine-Amphetamine-Regulated Transcript Expression in the Rat Nucleus Accumbens Is Regulated by Adenylyl Cyclase and the Cyclic Adenosine 5′-Monophosphate/Protein Kinase A Second Messenger System

Cocaine-amphetamine-regulated transcript (CART), a neuropeptide involved in the brain's reward/reinforcement circuit, modulates the effects of psychostimulants, including cocaine. The CART gene has been characterized, and binding sites for multiple transcription factors have been identified within the promoter region, including the cAMP-response element, which serves as a binding site for cAMP-response element-binding protein (CREB). CART expression appears to be regulated via cAMP/protein kinase A (PKA)/CREB-mediated signaling in cell culture. Therefore, the goal of these studies was to examine the involvement of cAMP/PKA/CREB-mediated signaling in CART mRNA and peptide expression in vivo in the rat nucleus accumbens. Intra-accumbal injections of forskolin, an adenylyl cyclase activator, stimulated the phosphorylation of CREB and increased both CART mRNA and peptide levels, an effect attenuated by inhibition of PKA with H89 [N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline-sulfonamide hydrochloride] and adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In addition, Rp-cAMPS alone decreased CART mRNA compared with saline-injected controls, suggesting that CART expression may be tonically regulated by PKA. Under certain conditions, cocaine increases CART mRNA levels; thus, we examined the effects of cocaine on forskolin-induced CART mRNA expression in the rat nucleus accumbens. Cocaine plus forskolin significantly increased CART mRNA over either of the drugs administered independently, suggesting that under conditions of heightened cAMP signaling, cocaine may impact CART gene expression. These results suggest that CART expression in vivo in the rat nucleus accumbens is regulated by adenylyl cyclase and cAMP/PKA-mediating signaling and, likely, through the activation of CREB.

Intra-accumbal injection of CART (cocaine-amphetamine regulated transcript) peptide reduces cocaine-induced locomotor activity.

Evidence suggests that CART (cocaine-amphetamine regulated transcript) peptides are mediators or modulators of the actions of psychostimulant drugs. In this study, the effects of intra-accumbal injections of rat long form (rl) CART 55-102 were examined. Injection of the peptide alone had no effect, but pretreatment with the peptide blunted or reduced the locomotor-inducing effects of cocaine after an i.p. injection. This effect was dose related and time limited, as expected. rlCART 1-27, a CART peptide fragment not active in other studies, was without effect on cocaine-induced locomotor activity. Because the actions of cocaine involve dopamine, the effect of rlCART 55-102 on dopamine-induced locomotor activity was examined. Intraaccumbal injection of dopamine produced a dose-related and time-limited increase in locomotor activity, as expected. Coinjection of rlCART 55-102 with dopamine blunted the effect. In summary, these data suggest that CART peptides in the nucleus accumbens would tend to oppose the actions of cocaine.

Fourth ventricular injection of CART peptide inhibits short-term sucrose intake in rats

Expression of CART (cocaine–amphetamine-regulated transcript) in the rat hypothalamus is modulated by nutritional status, and injection of synthetic CART peptide into the forebrain ventricular system suppresses food intake, indicating a possible role in hypothalamic control of energy homeostasis. Its recent identification in cell bodies and central terminals of vagal afferent neurons additionally suggests a role in brainstem mechanisms of meal termination and satiety. We demonstrate here that CART[55–102] (0.2 nmol) suppresses short-term sucrose intake and overnight chow intake in non-food-deprived rats even more when delivered into the fourth ventricle as compared to the lateral ventricle. At the threshold dose (0.02–0.08 nmol) no readily noticeable motor impairments were observed. The results are consistent, but do not prove a site of action within the brainstem, possibly in mediating vagal satiety signals at the level of the NTS.

Chronic intracerebroventricular administration of recombinant CART(42-89) peptide inhibits and causes weight loss in lean and obese Zucker (fa/fa) rats.

Hypothalamic neuropeptide CART (cocaine-amphetamine-regulated transcript) is a leptin-dependent endogenous satiety factor in the rat, and single central injections of recombinant CART(42-89) lowers food intake in rats and mice. To assess the potential role of CART as a long-term regulator of food intake, we investigated the effects of continuous infusion of recombinant CART(42-89) on food consumption and body weight. Two doses of CART(42-89) were tested: 12 or 4.8 microg/d. Adult male, both lean (+/?) and Zucker (fa/fa) obese, rats were equipped with intracerebroventricular cannulae in the right lateral ventricle. The cannulae were connected to subcutaneously placed osmotic mini-pumps. Pumps were filled with either CART(42-89) or vehicle (50 mM phosphate-buffered saline, pH 7.4). The pumps delivered a continuous infusion of CART(42-89) or vehicle, and food intake and body weight were followed for 10 days (12 microg/d) or 7 days (4.8 microg/d). Animals given the low dose had the pump removed on Day 7, and from half of the group, trunk blood was collected after decapitation, whereas the other half of the group had their mini-pumps removed and were followed for another 7 days before being decapitated. Animals receiving the high doses displayed overt motor disturbances, whereas the low dose was devoid of such behavioral side effects. Both doses significantly lowered food intake with maximal effect on days 3 to 5 of the infusion period. The high dose of CART decreased body weight of normal animals to 85% of initial weight at days 3 to 5, whereas the weight of Zucker (fa/fa) obese rats dropped to 95% of the initial weight. In animals receiving 4.8 microg/d, moderate effects on body weight were seen between days 4 and 6 of the treatment period, but soon after termination of the treatment animals regained lost weight. To assess the biological activity of the contents of the osmotic mini-pumps, the pumps were removed from the subcutaneous implantation site, and 5 microL of their contents were injected intracerebroventricularly to naive animals kept on a restricted feeding schedule. The content of pumps from animals receiving 4.8 microg/d of CART(42-89) potently inhibited food intake, confirming full biological activity despite being kept for 7 days at body temperature. Due to obvious effects on motor behavior, it is impossible with certainty to conclude that the observed effects on feeding and body weight are primary interference with satiety centers or secondary to effects on locomotor pathways. Also, the present experiments suggest that hypothalamic appetite-regulating neurons are subject to pharmacological desensitization upon prolonged exposure to CART peptide. The underlying mechanism of such desensitization is as yet unknown.

Recombinant CART peptide induces c-Fos expression in central areas involved in control of feeding behaviour

We have recently shown that the hypothalamic neuropeptide CART (cocaine-amphetamine-regulated-transcript) is a leptin dependent endogenous satiety factor in the rat. In the present study we confirm and extend our previous observations by showing that intracerebroventricular (i.c.v.) administered CART(42–89) dose-dependently inhibits 3-h food intake in food restricted rats with a lowest effective dose of 0.5 μg. CART also potently inhibits NPY-induced food intake in satiated rats as well as nighttime food intake in free feeding animals. To identify brain areas potentially involved in mediating the anorectic effects of CART, the temporal expression pattern of the immediate early gene c- fos was examined in the central nervous system by immunohistochemistry in rats receiving recombinant CART. Compared to vehicle, CART induced c-Fos expression in several hypothalamic and brainstem structures implicated in the central control of food intake. In the hypothalamus, high numbers of c-Fos immunoreactive (-ir) cells were observed in the medial parvocellular part of the paraventricular nucleus and in the posterior part of the dorsomedial nucleus. Lower numbers of c-Fos positive nuclei were found in the supraoptic and arcuate nuclei. A relatively high number of c-Fos-ir cells was found in the central nucleus of the amygdala. In the brainstem, c-Fos-positive nuclei were found in the parabrachial nucleus, and in the nucleus of the solitary tract. Notably both the area postrema and the dorsal motor nucleus of the vagus were virtually devoid of c-Fos-ir cells. The present experiments suggest that CART peptide exerts its inhibitory effects on appetite by activating hypothalamic and brainstem neurones implicated in the central control of feeding behaviour and metabolism.