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1. Abstract The discovery of the CART transcript and peptides implicated CART peptide (CARTp) as a neurotransmitter involved in the action of psychostimulants and several other physiological processes, buttressing the importance of the CARTp system. While there is evidence that a receptor(s) for CARTp exists, the receptor(s) has/have not been cloned. To understand how CARTp functions, it is important to identify its receptor(s). Given the evidence that CARTp receptor is a GPCR, a reasonable approach to searching for a CARTp receptor is to closely examine GPCR orphan receptors, receptors for which there are no known neurotransmitters. One of such GPCR orphan receptors that may be linked directly to CARTp function is GPR160. While studying neuropathic pain, Yosten et al (2020) identified CARTp effects were related to GPR160 expression. While studying food and water intake, Haddock et al (2021) also determined that the effects of CARTp were blocked by immuno-neutralization of GPR160. These findings suggest that the orphan receptor GPR160 may be the elusive CARTp receptor, a welcome observation after so many years of searching. This important discovery will advance the understanding of the CARTp system, including facilitating drug screening for small molecule agonists and antagonists and the identification of therapeutic compounds.

In this study, we reexamined the effect of Cocaine-and-Amphetamine-Regulated-Transcript (CART) peptide on psychostimulant (PS)-induced locomotor activity (LMA) in individual rats. The Methods utilized were as previously published. The PS-induced LMA was defined as the distance traveled after PS administration (intraperitoneal), and the CART peptide effect was defined as the change in the PS-induced activity after bilateral intra-NAc administration of CART peptide. The experiments included both male and female Sprague-Dawley rats, and varying the CART peptide dose and the PS dose. While the average effect of CART peptide was to inhibit PS-induced LMA, the effect of CART peptide on individual PS-treated animals was not always inhibitory and sometimes even produced an increase or no change in PS-induced LMA. Upon further analysis, we observed a linear correlation, reported for the first time, between the magnitude of PS-induced LMA and the CART peptide effect. Because CART peptide inhibits PS-induced LMA when it is large, and increases PS-induced LMA when it is small, the peptide can be considered a homeostatic regulator of dopamine-induced LMA, which supports our earlier homeostatic hypothesis.

Earlier studies suggesting an involvement of cocaine and amphetamine regulated transcript peptide (CARTp) in the actions of drugs of abuse are confirmed in the most recent publications. This seems especially true for the psychostimulants where CARTp in the nucleus accumbens inhibits or regulates the actions of these drugs; the regulation is lost after repeated drug use which may be an important mechanism in addiction. The other drugs, including nicotine, alcohol, opiates, and perhaps caffeine can affect CARTp or CART mRNA levels. While the exact mechanism is not always clear, the hope is that these findings may provide some insight for the development of medications. While binding studies indicate the existence of specific G-protein coupled receptors (GPCR) receptors for CARTp, major work to be done is the cloning of these receptors.

Since the identification of CART peptides in the 1990s [1], evidence has been steadily accumulating that they are involved in a great variety of physiologic processes. These include feeding and body weight, depression and anxiety, stress, hormonal control, psychostimulant (PS) action [2, 3, 4, 5, 6, 7, 8], and others. With regard to PS action, CART peptides appear to regulate the action of cocaine, amphetamine, and dopamine.

CART peptide is known for having an inhibitory effect on cocaine- and dopamine-mediated actions after acute administration of cocaine and dopamine. In this regard, it is postulated to be a homeostatic, regulatory factor on dopaminergic activity in the nucleus accumbens (NAc). However, there is no data on the effect of CART peptide after chronic administration of cocaine, and this study addresses this. It was found that CART peptide blunted cocaine-induced locomotion (LMA) after acute administration of cocaine, as expected, but it did not affect cocaine-mediated LMA after chronic administration of cocaine. The loss of CART peptide's inhibitory effect did not return for up to 9 weeks after stopping the repeated cocaine administration. It may not be surprising that homeostatic regulatory mechanisms in the NAc are lost after repeated cocaine administration, and that this may be a mechanism in the development of addiction.

Photoaffinity labeling, a usefulin vivobiochemical tool, is limited when appliedin vivobecause of the poor tissue penetration by ultraviolet (UV) photons. This study investigates affinity labeling using tissue-penetrating radiation to overcome the tissue attenuation and irreversibly label membrane receptor proteins. Using X-ray (115 kVp) at low doses (

WOS: 000310414300006, PubMed ID: 22960117, 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. (C) 2012 Elsevier B.V. All rights reserved., NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [3 R01 DA010732-05S1]; National Center for Research ResourcesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Center for Research Resources (NCRR) [P51RR165]; Office of Research Infrastructure Programs/OD [P51OD11132], This study was supported by NIH Grant no. 3 R01 DA010732-05S1 and by the National Center for Research Resources (P51RR165) and is currently supported by the Office of Research Infrastructure Programs/OD (P51OD11132).

Aims/hypothesis: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart−/− mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. Methods: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca2+ oscillation patterns and exocytosis were studied in mouse islets. Results: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca2+ signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. Conclusions/interpretation: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.

The goal of collegial ethics is to actively support our colleagues and to develop the skills needed to do so. While collegial interactions are key for our careers, there is little or no training in this. Many of our actions and reactions with our colleagues are instinctive. Human nature has evolved to be self-protective, but many evolved and automatic responses to others are not always in the best interests of our society or of us. Developing courage and a style of supportive language, avoiding destructive acts, and adhering to the golden rule will improve our relationships and provide a more positive environment for all.

CART mRNA and peptides are highly expressed in the anatomical structures composing the hypothalamo-pituitary-adrenal (HPA) axis and sympatho-adrenal system. Anatomical and functional studies suggest that CART peptides may have a role in the regulation of the neuroendocrine and autonomic responses during stress. Our previous study showed that CART peptides increased significantly in the male hypothalamus and amygdala 10 min after the forced swim stress. The present study aimed to examine the effect of forced swim stress on CART peptide expression in selected brain regions, including those where CART peptide expression has not been reported before (frontal cortex, pons, medulla oblongata), as well as in endocrine glands related to stress in male Sprague Dawley rats. A total of 16 (n = 8) animals were used, including control groups. Rats were subjected to forced swim on two consecutive days, and sacrificed on the second day, 2 h after the termination of the stress procedure. Frontal cortex, pons, medulla oblongata, hypothalamus, pituitary and adrenal glands were dissected and homogenized. CART peptide expression in these tissues was measured by Western Blotting and six different CART peptide fragments were identified. Our results showed that forced swim stress elicited region-specific changes in CART peptide expression. CART was upregulated in the frontal cortex, hypothalamus, medulla oblongata and adrenal gland while there was no change in the pons and pituitary. Enhanced CART peptide fragments in these brain regions and adrenal glands may have a role in the regulation of the HPA and sympatho-adrenal axis activity during stress response.

IDARS is an acronym for the International Drug Abuse Research Society. Apart from our scientific and educational purposes, we communicate information to the general and scientific community about substance abuse and addiction science and treatment potential. Members of IDARS are research scientists and clinicians from around the world, with scheduled meetings across the globe. IDARS is developing a vibrant and exciting international mechanism not only for scientific interactions in the domain of addiction between countries but also ultimately as a resource for informing public policy across nations. Nonetheless, a lot more research needs to be done to better understand the neurobiological basis of drug addiction – A challenge for IDARS scientists.

Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action.

Discoveries in basic science have helped us understand the drug abuse/dependence/addiction brain disorder. One can view this brain disorder as a long-lasting, relapsing pattern of drug seeking and taking with adverse consequences. Drug self-administration studies in animals have revealed brain circuits and neurotransmitters that underlie drug-induced reward and reinforcement. Moreover, studies of effects of drugs on receptors have shown us how drugs can change gene expression and how drugs can change the biochemical makeup of the brain. Drug-induced changes in the brain are very long lasting, which presumably can explain why drug addiction is a chronic and relapsing disease. Also, drugs exert their actions at least partly through evolved brain circuits that serve functions critical for survival such as feeding and sex. Thus, drugs can harness our strongest instincts and the desire to use them can become very powerful. These findings should influence research, treatment and policy towards the disorder of drug addiction and abuse.

Cocaine- and amphetamine-regulated-transcript (CART) peptides are associated with multiple physiological processes, including, feeding, body weight, and the response to drugs of abuse. CART mRNA and peptide levels and the expression of the CART gene appears to be under the control of a number of extra- and intra-cellular factors including the transcription factor, cAMP response element binding protein (CREB). Similar to the effects of CART, Ca(2+) signaling leads to the phosphorylation of CREB and has been associated with both feeding and the actions of psychostimulants; therefore, we hypothesized that Ca(2+) may play a role in CART gene regulation. We used real-time PCR (rtPCR) and GH3 cells to examine the effect of ionomycin, which increases intracellular Ca(2+), on CART mRNA levels. Ionomycin increased CART mRNA in a dose- and time-dependent manner. The effect of ionomycin appeared transient as CART mRNA had returned to control levels 3 h following treatment. Calmidazolium and KN93, inhibitors of calmodulin and Ca(2+)-modulated protein (CaM) kinases respectively, attenuated the effect of ionomycin (10 microM) on CART mRNA levels suggesting a calmodulin-dependent mechanism. Western immunoblotting indicated that ionomycin increased phosphorylated cAMP response element binding protein (pCREB) levels and electrophoretic mobility shift assay/supershift assay using antibodies against pCREB demonstrated increased levels of a CART oligo/pCREB protein complex. Finally, we showed that injection of ionomycin into the rat nucleus accumbens increases CART mRNA levels. To our knowledge, this is the first study providing evidence that the CART gene is, in part, regulated by Ca(2+)/CaM/CREB-dependent cell signaling.

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.

Over the past decade or so, CART (cocaine- and amphetamine-regulated transcript) peptides have emerged as major neurotransmitters and hormones. CART peptides are widely distributed in the CNS and are involved in regulating many processes, including food intake and the maintenance of body weight, reward and endocrine functions. Recent studies have produced a wealth of information about the location, regulation, processing and functions of CART peptides, but additional studies aimed at elucidating the physiological effects of the peptides and at characterizing the CART receptor(s) are needed to take advantage of possible therapeutic applications.

Because licking and grooming behavior of dams with pups can influence some behaviors of pups when they are adults, we tested if licking and grooming scores in a maternal separation protocol correlated with cocaine or ethanol self-administration in the pups as adults. The protocol produced litters that were separated from dams for 0 (MS0), 15 (MS15) or 180 (MS180) min, and a nonhandled (NH) group as well. Self-administration of both drugs as shown in earlier studies was lowest in the MS15 group, highest in the NH group and intermediate in the other groups. Licking and grooming scores correlated negatively with drug intake and suggests that maternal care of pups can influence drug use in pups when they are adults.

Cocaine- and amphetamine-regulated transcript (CART) peptides (CART 55-102 and CART 62-102) are peptidergic neurotransmitters that are widely but specifically distributed throughout the brain, gut and other parts of the body. They are found in many brain regions associated with drug addiction including the nucleus accumbens, ventral tegmental area and ventral pallidum. Injections of CART 55-102 into the nucleus accumbens have no effect on basal locomotor activity. However, an injection of CART just before an i.p. injection of cocaine reduces the locomotor activating effects of cocaine. These and other data suggest that CART in the accumbens blunts the effects of cocaine. A hypothesis is that CART is homeostatic in the accumbens and tends to oppose large increases in dopamine signaling. These actions would therefore be able to regulate the effects of some abused drugs such as the psychostimulants.

Aims: Cocaine- and amphetamine-regulated transcript (CART)-peptide is found in the brain and participates in the control of feeding behavior. It is also expressed in the peripheral nervous system and is suggested to have neuromodulatory and/or neurotrophic effects in rat intestine. The aims of this study were to investigate the presence of CART-peptide in the normal ganglionic as well as aganglionic intestine from patients with Hirschsprung's disease and the pepticle's possible coexistence with other neurotransmitters. Methods: Intestinal specimens from nine patients with Hirschsprung's disease were examined using immunohistochemistry. A double immunostaining technique was used in order to elucidate the presence of CART-peptide in NOS and VIP-containing enteric neurons. Results: in ganglionic intestine, CART-peptide was found in numerous nerve fibers, predominantly within the smooth muscle layers and in myenteric nerve cell bodies. A high degree of co-localization of CART with NOS and VIP was seen. Only very few CART immunoreactive nerve fibers and no nerve cell bodies were found in the aganglionic intestine. Conclusions: This is the first report on the presence of CART-peptide in the human intestine. In the ganglionic intestine CART was detected mainly in myenteric neurons, while only very few CART-IR nerve fibers were found in the aganglionic intestine. This, together with the coexistence of CART with NOS and VIP, indicates an intrinsic origin of the CART-containing neurons and suggests that CART may influence NO and VIP-induced effects. (Less)

CART (cocaine- and amphetamine-regulated transcript) peptides appear to be mediators or modulators of psychostimulant drugs. An interesting result in the nucleus accumbens has been that injection of CART peptide has no effect by itself on locomotor activity, but it reduces the locomotor activity induced by cocaine or amphetamine. However, in the ventral tegmental area (VTA), injections of CART peptide have been shown to increase locomotor activity, although to a lesser degree [Kimmel, H.L., Gong, W., Vechia, S.D., Hunter, R.G., Kuhar, M.J., 2000. Intra-ventral tegmental area injection of rat cocaine and amphetamine-regulated transcript peptide 55-102 induces locomotor activity and promotes conditioned place preference. J. Pharmacol. Exp. Ther. 294, 784-792]. This study was carried out to clarify the interaction of intra-VTA CART 55-102 and systemic cocaine on locomotor activity. The CART-cocaine interaction has been examined using the rigorous isobolographic approach. This type of analysis permits an assessment of additivity, subadditivity, or synergism of two substances. By measuring locomotor activity and using a range of doses of CART peptide and cocaine, both alone and together, with different dosing strategies, clear evidence of subadditivity was found. CART reduced the locomotor activating effects of systemic cocaine, especially at higher doses of CART. These results imply that intra-VTA CART is not simply acting in the same manner as cocaine, and is likely to oppose the action of cocaine. This has implications for the physiological significance of CART-DA (dopamine) interactions and for medications development.

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 125I-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 Kd of 1.43 ± 0.25 nM and a Bmax 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, γ-aminobutyric acid, or cocaine showed no affinity for the receptor at 10 μM 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. Synapse 62:122–127, 2008. © 2007 Wiley-Liss, Inc.

The apparent molecular masses of photoaffinity-labeled dopamine transporters (DATs) from rat, human, dog, and primate kidney COS cells expressing the rat DAT1 cDNA differ. Sequences predicted from cDNA cloning reveal only one amino acid difference between the length of the rat and human DAT but one less site for potential N-linked glycosylation in the human DAT. Possible posttranslational and postmortem bases for species differences in DAT molecular mass were explored. Rat DAT proteins from striata subjected to approximately 5 h of postmortem delay modeled after the human postmortem delay process revealed small but consistent losses in apparent molecular mass and in cocaine analogue binding; the DAT molecular mass displayed no further losses for up to 30 h of model postmortem treatment. Degradative postmortem changes could thus contribute to molecular mass differences between rat and human DATs. Neuraminidase treatment reduced the apparent molecular mass of native rat DAT but not that of the rat DAT expressed in COS cells, suggesting that the sugars added to the DAT expressed in COS cells were different than those added to the rat brain striatal transporter. These differences could account for the somewhat higher Km values for expressed DAT cDNA in COS cells when compared with the wild-type striatal transporter. These results are in accord with the differences in number of predicted N-linked glycosylation sites between rat and human DATs and with cell-type specificity in transporter posttranslational processing.

CART is expressed abundantly in the hypothalamic paraventricular nucleus and locus coeruleus, major corticotropin releasing factor (CRF) and noradrenaline sources, respectively. There is a bidirectional relation between CART and hypothalamo-pituitary-adrenal axis activity. CART stimulates CRF, adrenocorticotropic hormone and glucocorticoid secretion, whereas CRF and glucocorticoids increase the transcriptional activity of the CART gene; adrenalectomy declines CART expression in the hypothalamus. Stress exposure modulates CART expression in hypothalamus and amygdala in rat brain in a region and sex specific manner. CART may be a mediator peptide in the interaction between stress, drug abuse, and feeding. The review discusses the established role of CART as it relates to the stress response.

A variety of studies indicate that CART in the nucleus accumbens (NAcc) is involved in the action of psychostimulants. In order to understand in more detail if and how dopamine is involved in the regulation of CART mRNA in the NAcc, the present studies of individual receptors were performed. The D1 agonist, dihydrexidine, and the D1 antagonist, SCH23,390, were administered separately and in combination to adult male rats; however, no changes were found in CART mRNA as measured by in situ hybridization. The D2/3 agonist, quinpirole, was administered either separately or in combination with the D2 selective antagonist, L741,626, or the D3 selective antagonist, GR103,691. Quinpirole produced a decrease in CART mRNA of up to 43%. This effect was blocked by pretreatment with the D3 antagonist GR103, 691, but not by the D2 antagonist, L741,626. CART peptide levels showed a similar decrement after acute quinpirole. CART mRNA levels in the NAcc of D3 mutant mice were found to be higher than that in wild-type animals, but treating the mutants with quinpirole failed to produce a decrease in CART expression like that observed in wild-type rodents. These findings demonstrate that CART is regulated by dopamine in the NAcc, at least partly by D3 dopamine receptors.

The discovery and preclinical development of selective dopamine reuptake inhibitors as potential pharmacotherapies for treating cocaine addiction are presented. The studies are based on the hypothesis that a dopamine reuptake inhibitor is expected to partially substitute for cocaine, thus decreasing cocaine self-administration and minimizing the craving for cocaine. This type of indirect agonist therapy has been highly effective for treating smoking addiction (nicotine replacement therapy) and heroin addiction (methadone). To be an effective pharmacotherapy for cocaine addiction, the potential drug must be safe, long-acting, and have minimal abuse potential. We have developed several 3-phenyltropane analogs that are potent dopamine uptake inhibitors, and some are selective for the dopamine transporter relative to the serotonin and norepinephrine transporters. In animal studies, these compounds substitute for cocaine, reduce the intake of cocaine in rats and rhesus monkeys trained to self-administer cocaine, and have demonstrated a slow onset and long duration of action and lack of sensitization. The 3-phenyltropane analogs were also tested in a rhesus monkey self-administration model to define their abuse potential relative to cocaine. Based on these studies, 3beta-(4-chlorophenyl)-2beta-[3-(4'-methylphenyl)isoxazol-5-yl]tropane (RTI-336) has been selected for preclinical development.

Chronic cocaine and withdrawal induce significant alterations in nucleus accumbens (NAc) glutamatergic function in humans and rodent models of cocaine addiction. Dysregulation of glutamatergic function of the prefrontal cortical-NAc pathway has been proposed as a critical substrate for unmanageable drug seeking. Previously, we demonstrated significant up-regulation of NMDA, (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptor subunit mRNAs and protein levels in the ventral tegmental area (VTA), but not the substantia nigra, of cocaine overdose victims (COD). The present study was undertaken to examine the extent of altered ionotropic glutamate receptor (iGluR) subunit expression in the NAc and the putamen in cocaine overdose victims. Results revealed statistically significant increases in the NAc, but not in the putamen, of NMDA receptor subunit (NR)1 and glutamate receptor subunit (GluR)2/3 wit trends in GluR1 and GluR5 in COD. These results extend our previous finding and indicate pathway-specific alterations in iGluRs in COD. In order to determine that changes were related to cocaine intake and not to other factors in the COD victims, we examined the effects of cocaine intravenous self-administration in rhesus monkeys for 18 months (unit dose of 0.1 mg/kg/injection and daily drug intake of 0.5 mg/kg/session). Total drug intake for the group of four monkeys was 37.9 +/- 4.6 mg/kg. Statistically significant elevations were observed for NR1, GluR1, GluR2/3 and GluR5 (p < 0.05) and a trend towards increased NR1 phosphorylated at serine 896 (p = 0.07) in the NAc but not putamen of monkeys self-administering cocaine compared with controls. These results extend previous results by demonstrating an up-regulation of NR1, GluR2/3 and GluR5 in the NAc and suggest these alterations are pathway specific. Furthermore, these changes may mediate persistent drug intake and craving in the human cocaine abuser.

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.

CART (Cocaine- and Amphetamine-Regulated Transcript) was initially described as an mRNA which had increased expression in the rat striatum following administration of acute cocaine or amphetamine but not saline. However, not all subsequent studies confirmed this. The present study aimed to repeat experiments with conflicting results and to reexamine and extend the original finding of acute regulation of nucleus accumbens CART mRNA by cocaine. Acute administration of cocaine failed to produce any change in levels of CART mRNA or peptide. Chronic administration of cocaine, as well as unilateral 6-hydroxydopamine lesions, also failed to alter CART mRNA levels in the accumbens. However, binge administration of cocaine, which also caused some seizures, did cause a significant increase in CART message. Given the involvement of corticosteroids with both stress and the effects of psychostimulants, we examined the possible effects of corticosteroids. We acutely administered ascending doses of corticosterone and found an increase in CART message. Similar effects were seen on CART peptides after acute corticosterone administration, and acute metyrapone administration was found to reduce CART peptide levels in the accumbens. This suggests that CART mRNA may be regulated by cocaine under certain conditions, such as binge administration, and this may at least partly involve corticosterone.

Maternal separation (MS) in neonatal rats affects ethanol self-administration (SA) in adulthood; however, the conditions and mechanisms need to be clarified.The goal of this study was to determine the effect of MS on ethanol SA in adulthood in different groups of rats, which control for time of separation, handling, and rearing conditions and, for mechanistic assessment, to examine GABA-A receptors in the central nucleus of the amygdala (CeA) and levels of liver metabolizing enzymes.Newborn, male Long-Evans rats were randomly assigned to different groups and treated over postnatal days 2-14. The rats were picked up by their tails and put back down with no separation (MS0), separated from their mother for 15 min/day (MS15), separated from their mother for 180 min/day (MS180), handled once for a bedding change (NH), or were animal facility reared (AFR). In adulthood, these rats were allowed 5-day continuous access to ethanol, and GABA-A receptors and liver enzymes were measured.The MS15 group consumed and preferred significantly less ethanol (about one third) than the MS180 group; however, neither group was different from the MS0 or the AFR group. The NH group consumed and preferred significantly more ethanol than all other groups, at least twice that of the MS180s. GABA-A receptors were increased in the CeA in MS15s, which could help explain the effects. Alcohol dehydrogenase may have been altered in the AFRs.Various treatments in neonates affect ethanol intake and GABA-A receptors, and possibly ethanol metabolism, in adulthood. These changes were not simply related to time of separation but were also due to the degree of handling.

Cocaine- and amphetamine-regulated transcript (CART) peptides (55 to 102 and 62 to 102) are neurotransmitters with important roles in a number of physiologic processes. They have a role in drug abuse by virtue of the fact that they are modulators of mesolimbic function. Key findings supporting a role in drug abuse are as follows. First, high densities of CART-containing nerve terminals are localized in mesolimbic areas. Second, CART 55 to 102 blunts some of the behavioral effects of cocaine and dopamine (DA). This functional antagonism suggests that CART peptides be considered as targets for medications development. Third, CREB in the nucleus accumbens has been shown to have an opposing effect on cocaine self-administration. CREB may activate CART expression in that region, and, if so, CART may mediate at least some of the effects of CREB. Fourth, in addition to the effects of CART on DA, DA can influence CART in the accumbens. Thus a complex interacting circuitry likely exists. Fifth, in humans, CART is altered in the ventral tegmental area of cocaine overdose victims, and a mutation in the CART gene associates with alcoholism. Overall, it is clear that there are functional interactions among CART, DA, and cocaine and that plausible cellular mechanisms exist to explain some of these actions. Future studies will clarify and extend these findings.

Cocaine-and-amphetamine-regulated-transcript peptides play a role in the modulation of feeding and psychomotor stimulant-like behaviors. The ventral tegmental area and the lateral hypothalamus are likely structures where cocaine-and-amphetamine-regulated-transcript peptides mediate both of these functions. Although lateral hypothalamus inputs to the ventral tegmental area have long been known, the chemical nature of this pathway remains poorly understood. To address this issue, we tested the possibility that cocaine-and-amphetamine-regulated-transcript peptide-containing neurons in the lateral hypothalamus project to the ventral tegmental area using the retrograde transport of cholera toxin subunit B combined with cocaine-and-amphetamine-regulated-transcript peptide immunostaining. The largest density of retrogradely-labeled neurons in the hypothalamus after cholera toxin subunit B injection in the ventral tegmental area was found, ipsi- and contralaterally, in the lateral hypothalamus/perifornical area, although substantial numbers of retrogradely-labeled cells were also found in the medial preoptic area, lateral preoptic area, paraventricular nucleus, dorsomedial hypothalamus and ventromedial hypothalamus. More than 80% of the retrogradely-labeled cocaine-and-amphetamine-regulated-transcript peptide-immunoreactive neurons in the hypothalamus were found in the lateral hypothalamus/perifornical area both ipsilateral and contralateral to the injection sites. Although retrogradely-labeled neurons were seen in the amygdala, locus coeruleus, and raphe nucleus, none of them displayed cocaine-and-amphetamine-regulated-transcript peptide immunoreactivity. Therefore, the hypothalamic projection to the ventral tegmental area provides a substrate whereby cocaine-and-amphetamine-regulated-transcript peptides could mediate the rewarding aspects of feeding and psychomotor stimulant-like behaviors. These findings, combined with the fact that the lateral hypothalamus receives strong inputs from the shell of the nucleus accumbens and ventral pallidum, suggest that these structures are part of integrative functional loops that control reward and appetitive behaviors.

We have recently shown that CART peptides exhibit a diurnal rhythm in blood that is affected by food intake and glucocorticoids. In the present study, we extend our observations by demonstrating that CART peptides also exhibit a diurnal rhythm in several brain regions, notably the nucleus accumbens, hypothalamus and amygdala, but not in the midbrain. To examine whether the CART peptide rhythm was dependent on food intake, animals were food-deprived for 24 h. In regular-fed animals, CART peptide levels were lower in the morning compared to evening hours. However, this diurnal variation of CART peptide was not apparent in fasted animals, and CART peptide levels were reduced. The diurnal variation of CART mRNA in the nucleus accumbens paralleled the variation of CART peptide in this region. Similar to the peptide, the mRNA did not change in midbrain. These results show that CART peptide levels and gene expression undergo a diurnal variation in some brain regions, and the variation is altered by fasting. These findings suggest a variety of regulatory mechanisms for CART and additional considerations for CART's role in brain.

CART (cocaine- and amphetamine-regulated transcript) peptides are neurotransmitters that have received much attention as mediators of feeding behavior and body-weight regulation in mammals. CART peptides and their mRNAs are found in many brain regions and in peripheral tissues that are involved in feeding, and many animal studies implicate CART as an inhibitor of feeding. Animal studies also demonstrate that CART expression is regulated by both leptin and glucocorticoids, two hormones known to be associated with the regulation of body weight. A recent study also links a mutation in the CART gene to obesity in humans. These peptides might become targets for drug development in the area of obesity.

CART peptides have been shown to be peptide neurotransmitters and endocrine factors in a series of cumulative studies over the past eight years or so. This brief review touches on three aspects of CART: CART as a mediator or modulator of mesolimbic dopamine, CART's regulation by glucocorticoids, and CART as a regulator of feeding, satiety, and body weight. There have been several recent reviews and publications on various aspects of CART peptides. These aspects include the sequence and numbering of the peptides, and their structure, processing, and roles in various physiologic processes.

Cocaine- and amphetamine-regulated transcript (CART) peptides are novel neurotransmitters that are implicated in several physiological functions such as control of feeding behavior, drug reward, sensory processing, stress, and development. Although a majority of studies have examined the role of CART in the brain, less is known about its function in the periphery. Therefore, the goals of this study were to examine the levels and species of CART peptides in blood, to determine whether they undergo diurnal rhythms, and to elucidate their sources and regulatory factors. RIA showed that CART peptides are present in the blood of rats and monkeys and that they exhibit a diurnal variation. Western blotting confirmed the pattern of diurnal variation in rats and, additionally, showed that CART immunoreactivity was due to a single predominant fragment with an apparent molecular weight in the range of the active CART 55-102 peptide. Adrenalectomy caused a 70% reduction in CART peptide levels in rat blood, and this was reversed by corticosterone replacement. CART levels paralleled glucocorticoid levels in rat and monkey blood. Control of CART levels by corticosterone suggests the possibility that CART peptides in blood may be influenced by hypothalamic-pituitary-adrenal interactions and that they may play a role in glucocorticoid-related processes such as stress.

Changes in Cocaine- and Amphetamine-Regulated Transcript (CART) mRNA levels have been observed in brain as a result of various physiologic stimuli including feeding, drugs of abuse, stress and glucocorticoids, and activators of the cyclic AMP (cAMP) and protein kinase A (PKA) pathway. Accordingly, we are interested in identifying factors involved in CART gene regulation. CATH.a cells, derived from the locus coeruleus (LC), express a 213-bp CART mRNA species that is translated and processed. The promoter activity of three CART-LUC constructs containing 3451, 641, and 102 bp of 5′ upstream sequence, respectively, were tested in CATH.a cells. cAMP regulation was detected in the construct containing 641 bp of CART promoter sequence which contains a consensus CRE site. Mutation of the CRE site within −641CART-LUC significantly reduced basal and forskolin-induced promoter activity. Additionally, forskolin-induced transcription was inhibited by a dominant-negative mutant of CRE-binding protein (CREB) in CATH.a cells. Finally, tropin-releasing factor (CRF), an endogenously occurring activator of the cAMP/PKA pathway in CATH.a cells, was shown to increase transcriptional activity that was inhibited by a CRF receptor antagonist and a PKA inhibitor. This study provides evidence that the CRE site in the CART proximal promoter is involved in cAMP/PKA/CREB regulation in cells having a neuronal phenotype. Also, given the evidence for involvement of CREB in reward and reinforcement, these results are compatible with a role for CART in these processes as well.

Cocaine- and amphetamine-regulated transcript (CART) mRNA and CART peptide are abundant in the hypothalamic nuclei that control anterior pituitary function. CART peptide has also been localized in the anterior pituitary gland itself, although its role in pituitary function has not as yet been elucidated. In the present study, we investigated the localization and function of CART peptide in the anterior pituitary gland. Immunohistochemical observations revealed that CART peptide colocalized with FSH and LH in gonadotroph cells but that it was absent from the other hormone-producing cells. Immunoelectronmicroscopy suggested that CART peptide and gonadotropin were colocalized in the same secretory granules. CART peptide suppressed prolactin release from dispersed anterior pituitary cells 15 min after its addition into the media [basal production, 234.9 +/- 14.6 ng/ml vs. CART 55-102 peptide 100 nm, 125.0 +/- 18.2 ng/ml (P < 0.01, n = 5)]. Prolactin release was suppressed by CART in a dose-related manner; on the other hand, CART peptide did not affect the secretion of other anterior pituitary hormones. CART peptide synthesis by these cells was elevated 15 min after the addition of leptin to the media (100 nm), as determined by immunoblotting, but LHRH (10 nm) did not significantly affect CART peptide expression. These findings suggest that CART synthesis in the anterior pituitary may be stimulated by leptin and that CART peptide may play a role in the regulation of anterior pituitary hormone secretion in the rat.

Cocaine- and amphetamine-regulated transcript (CART) is an anorexigenic peptide widely expressed in the central and peripheral, including the enteric, nervous systems. CART is also expressed in pituitary endocrine cells, adrenomedullary cells, islet somatostatin cells, and in rat antral gastrin cells. We used immunocytochemistry (IHC) and in situ hybridization (ISH) to study CART expression in developing rat pancreas. We also examined co-expression of CART and islet hormones and developmental markers and the effect of CART on proliferation using clonal insulin cells (INS-1 832/13). A major portion of each of the islet cell types, except the ghrelin cells, expressed CART during a period before and around birth. Two weeks postnatally, CART expression was restricted to somatostatin cells. Pre- and early postnatally, many of the CART-expressing cells co-expressed cytokeratin 20 (CK20), a marker of duct cells and islet precursor cells, the trophic hormone gastrin, and a smaller subpopulation also harbored the proliferation marker Ki67. CART was also expressed in pancreatic nerve fibers, both sensory and autonomic, and in ganglion nerve cell bodies. Although highly expressed in the developing islets, CART did not affect proliferation of INS-1 cells. We have demonstrated that CART is expressed in several islet cell types during rat development but is restricted to somatostatin cells and neurons in the adult rat.

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.

In order to test for glucocorticoid regulation of CART in the arcuate nucleus, adrenalectomies (ADX) and hormone replacements (HRs) were carried out in groups of rats. CART mRNA levels were determined by in situ hybridization and CART peptide levels by immunocytochemistry. ADX caused a lowering of CART mRNA and peptides levels in the arcuate and this was reversed by HR. These results indicate a glucocorticoid regulation of CART in the arcuate. The regulation could be direct through an action of glucocorticoid receptors or indirectly through ADX-induced changes in leptin levels. These findings suggest a role for CART in the stress response.

CART peptides are relatively novel neuropeptides involved in feeding, drug reward and stress. They are formed from a proCART polypeptide that is 89 amino acids in length in the human version. Fragments 42-89 and 49-89 are behaviorally active in feeding and locomotion as well and other functions. These peptides are highly abundant and widely but discretely distributed in the brain, gut, pituitary, adrenals and pancreas. The presence of CART immunoreactivity in specific nuclei of the hypothalamus has led to an examination of icv-injected CART peptides effects on feeding, which have proven to be significantly anorectic. Studies of transgenic animals and humans have also demonstrated a linkage to both obesity and anorexia. Similarly, the localization of CART to sub-regions of the mesolimbic dopamine system has led to demonstration of the effects of CART peptides on locomotor activity and conditioned place preference when injected into the ventral tegmental area (VTA), which are psychostimulant-like in quality. These findings also suggest that CART has the capacity to modulate mesolimbic dopamine, which could have implications for the treatment not only of psychostimulant abuse but also for the treatment of other disorders with mesolimbic dopamine involvement, such as schizophrenia. Other lines of evidence also show that CART peptides are involved in fear and startle behaviors which may have implications for understanding anxiety and stress. An important part of the development of CART mimetics and related drugs would be the identification of CART receptors. At the present time such receptors have not been identified, and much effort should be directed at this problem. Nonetheless, CART peptides offer interesting targets for new drug development for obesity and, potentially, a number of other disorders.

Dopamine transporters (DATs) from the caudate nucleus of four species (rat, mouse, dog, and human) and four regions of rat brain (striatum, nucleus accumbens, prefrontal cortex, and midbrain) were photoaffinity labeled and analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis for cross-reactivity to four epitope-specific rat antipeptide antibodies. Each of these antibodies varied in its efficiency at recognizing DAT. The DATs from the rat brain regions exhibited the same degree of recognition by each of the four sera, a result compatible with these proteins being the product of a single gene. The DATs from the different species were recognized by all four sera but with different efficiencies, possibly relating to amino acid sequence differences within the immunizing epitope. All of the photolabeled, immunoprecipitated DATs migrated with a molecular mass of approximately 80 kDa, and no lower molecular mass forms were found. The DATs from all species and brain regions tested were shown by enzymatic deglycosylation to contain N-linked carbohydrates and sialic acids in amounts comparable with rat striatal DATs. The finding that no photolabeled DAT forms < 80 kDa were isolated from membranes indicates that partially or incompletely glycosylated forms are not present, even in the midbrain cell bodies where immature forms might be expected to be found. These findings verify the utility of these anti-rat antibodies as biochemical tools for studying DATs from other species and extend our knowledge of biochemical characteristics of DATs from these species and brain regions.

Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport Vmax in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK1 cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport Vmax by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter Vmax suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.