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NEUROPHARMACOLOGY
Division of Neuroscience, Yerkes National Primate Research Center of Emory University, Atlanta, Georgia
Received September 28, 2005; accepted November 30, 2005.
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Abstract |
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), response to fear and stress (Vicentic et al., 2004), endocrine regulation (Kuriyama et al., 2004), and the reward/reinforcing effects of psychostimulants (Douglass et al., 1995; Brenz Verca et al., 2001; Jaworski et al., 2003; Couceyro et al., 2005). Expressed throughout the central nervous system, CART peptides are partly located in the ventral tegmental area and nucleus accumbens (NAc), regions of the brain included in the mesolimbic dopamine (DA) pathway and associated with reward and reinforcement (Dallvechia-Adams et al., 2002). Interestingly, CART modulates mesolimbic dopaminergic activity (Yang et al., 2004) and attenuates the locomotor effects of cocaine (Jaworski et al., 2003; Kim et al., 2003) and feeding (Yang et al., 2005), suggesting a complex relationship among CART, mesolimbic DA, and the brain's reward/reinforcement pathways.
Because of the unique anatomical distribution of CART peptides and the nature and variety of the stimuli that influence changes in CART levels, CART expression must be highly regulated. CART levels in the NAc and other brain regions follow a diurnal rhythm (Vicentic et al., 2004), which may influence the diurnal variation in cocaine sensitization and reward. In addition, CART expression appears to be under hormonal regulation because CART levels are affected by glucocorticoids (Vicentic et al., 2004; Hunter et al., 2005, Hunter et al., 2005), which, similar to CART, follow diurnal rhythms and are involved in the modulation of rewarding behaviors, including food and drug intake (Goeders, 2002). The mechanisms underlying the regulation of the CART gene are not fully understood and, thus, are the focus of the current study. Several genes associated with reward and reinforcement are regulated via cAMP-response-binding protein (CREB). The CART promoter contains a cAMP-response element (CRE) binding site (Dominguez et al., 2002); therefore, it was hypothesized that the CART gene may be regulated by CREB. Indeed, in vitro studies suggest that CREB, a transcription factor activated via phosphorylation by protein kinase A (PKA), is involved in the regulation of the CART gene. For instance, activation of adenylyl cyclase (AC) increases CART mRNA levels in a PKA-dependent manner in cell culture (Lakatos et al., 2002). Moreover, mutations of the CRE binding site on the CART promoter decrease promoter activity (Barrett et al., 2002; Dominguez and Kuhar, 2004) and, thus, likely decrease CART mRNA levels.
Both mesolimbic DA and CART peptides are involved in processes associated with reward and reinforcement, including feeding, stress, and psychostimulant addiction (Dallvechia-Adams et al., 2002). For instance, cocaine targets the DA system, activating G-proteins, cAMP signal transduction pathways, and, ultimately, pCREB. Thus, it is feasible that cocaine, which increases mesolimbic DA, may indirectly affect CART expression through the stimulation of some DA receptors and the downstream activation of CREB. Indeed, under binge-dosing regimes, cocaine reliably increases CART expression (Fagergren and Hurd, 1999; Brenz Verca et al., 2001; Hunter et al., 2005, Hunter et al., 2005). In addition, pCREB, which, in part, is activated via the stimulation of DA receptors, has been implicated in the rewarding effects of food intake and drugs of abuse, including opiates and cocaine (Carlezon et al., 1998; Nestler, 2001). Cocaine increases the phosphorylation of CREB in the NAc (Walters et al., 2003), which up-regulates several genes associated with reward and reinforcement (Carlezon et al., 2005). Interestingly, cocaine overdose victims demonstrate increased levels of pCREB and CART mRNA, suggesting a possible relationship between the transcription factor and neuropeptide (Tang et al., 2003).
Therefore, we hypothesize that in vivo regulation of CART is, in part, mediated by cAMP signaling and CREB. The activation of DA receptors subsequently activates G-proteins and affects cAMP signaling, which, in turn, increases pCREB levels and modulates CART expression. Using intra-accumbal administration of forskolin, we examined the role of the AC/cAMP/PKA/CREB pathway (Fig. 1) on CART mRNA and peptide levels in vivo in the rat NAc. Because a potential role for cocaine in CART regulation has been suggested and pCREB expression is increased in cocaine overdose victims, we examined the effect of cocaine on forskolin-induced (cAMP-mediated) CART mRNA levels. Understanding the regulation of CART peptide and its role in addiction will not only allow us to better understand the function of neuropeptides but will also contribute to our understanding of the addiction process.
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Materials and Methods |
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Animals. Male Sprague-Dawley rats (Charles River Laboratories Inc., Wilmington, MA) weighing 
300 g were used in all experiments. Animals were group-housed prior to surgery and individually housed thereafter. Animals were allowed access to food and water ad libitum and maintained on a 12-h light/dark cycle. All experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
Bilateral Guide Cannula Surgery. Cannula surgeries were performed according to Jaworski et al. (2003) with modifications. Rats were anesthetized with a mixture of ketamine HCL (75 mg/kg i.p.) and medetomidine HCL (0.5 mg/kg i.p.). A bilateral stainless steel guide cannula assembly (22 gauge with a center to center distance of 3.0 mm; Plastics One, Roanoke, VA) was implanted directly over the NAc in both hemispheres via aseptic stereotaxic surgery. Stereotaxic coordinates for the guide cannula (relative to bregma) were A/P + 1.7 mm, M/l ± 1.5 mm, D/V –5.7 mm (Paxinos and Watson, 1998). Guide cannulas were anchored to the skull using dental acrylic and three stainless steel screws driven into the skull. A dummy cannula was inserted to prevent blockage, and a dust cap was screwed onto the top of the assembly. The rats were allowed to recover for 7 to 10 days. Cannulas were successfully implanted into the NAc approximately 98% of the time, consequently minimizing the number of animals eliminated from analysis.
Intra-Accumbal Infusions. Stainless steel injector cannulas (28 gauge; Plastics One), which extend 2 mm beyond the tips of the guide cannulas and thus were centered over the shell/core junction of the NAc, were used for all drug infusions. Although there could be differences in CART regulation between the shell and core of the NAc, the shell/core junction was chosen as the target site for injections, because this is a novel experiment and CART expression in the whole NAc was examined and injections of CART peptide into the shell/core junction of the NAc attenuated the rewarding effects of cocaine (Jaworski et al., 2003). The injector cannulas were attached to 10-µl Hamilton syringes (Hamilton Co., Reno, NV) via polyethylene-10 tubing. Rats were placed in a polyethylene box (12 x 36 x 48 cm), and all infusions were conducted on freely moving rats. Left and right hemispheres were simultaneously infused with 0.5 µl volume/side over 30 s using an infusion pump (PHD 2000; Harvard Apparatus, Cambridge, MA). Infusions were conducted so that each rat received drug in one hemisphere and a vehicle in the other, thus permitting each rat to serve as its own control. Injector cannulas were left in place for an additional 30 s to permit drug diffusion and prevent backflow. To control for possible hemispheric differences, drug infusions were alternated between hemispheres so that some animals received drug in the left and others received drug in the right hemisphere. The location of cannula injection sites was determined histologically, and only animals with injector sites in the desired anatomical location, on or near the accumbal shell/core junction, were used for analysis (Fig. 2).
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, Hunter et al., 2005). Following infusions, rats were decapitated, the brains were removed, and 14-µm-thick sections encompassing the injection site were cut on a cryostat and slide-mounted. Sections were postfixed in 4% paraformaldehyde followed by consecutive washes in 2x SSC; triethanolamine/0.5% acetic anhydride (0.1 M); H20; 70, 95, and 100% ethanol; chloroform (5%); and 95 and 70% ethanol, and then air dried. Slides were incubated at 37°C in prehybridization buffer (50% deionized formamide, 4x SSC, 1x Denhardt's solution, 0.02 M NaPO4, pH 7.0, 1% N-lauroylsarcosine, and 10% dextran sulfate) in a humidifying chamber for 2 h. An oligodeoxynucleotide probe complementary to rat CART mRNA (nucleotides 223–270) was synthesized by the Emory Microchemical Facility (Emory University, Atlanta, GA). The probe was labeled on the 3' end with [35S]dATP (NEN, Boston, MA) to a specific activity of 5 x 109 cpm/µl using terminal deoxynucleotide transferase (Amersham Biotech) and then purified with a QIAquick Nucleotide removal kit (Qiagen Inc., Valencia, CA). Hybridization solution (prehybridization buffer plus 500 mg/l denatured salmon testis DNA and 200 mM dithiothreitol) containing the CART probe (5 x 105 cpm/slide) was applied to each section. Slides were hybridized overnight in a humidifying chamber at 42°. Posthybridization washing consisted of successive washes in 2x SSC, 50% ethanol/0.3 M ammonium acetate, 85% ethanol/0.3 M ammonium acetate, 100% ethanol, and H20. Sections were air-dried and exposed to Kodak BioMax MR autoradiography film for 10 to 12 days (Eastman Kodak, Rochester, NY).
Autoradiogram Image Analysis. Levels of CART mRNA in the NAc were quantified by capturing the autoradiograms with a Photometrics CoolSNAP camera (Photometrics, Roper Scientific Inc., Tucson, AZ) with the illuminating light adjusted so that the optical densities (ODs) of the probe signal fell within the linear portion of a standard curve generated from C14 microscales (American Radiolabeled Chemicals Inc., St. Louis, MO). Analysis was conducted using MCID Basic imaging software (Imaging Research Inc., Ontario, Canada). Relative ODs of the autoradioactive regions corresponding to the NAc of each hemisphere were measured using an outline with a consistent area (60 x 80 pixels) centered over the NAc shell/core junction. Magnification was held constant throughout the analysis. Measurements were taken through the injection site and without knowledge of treatment in brain slices at 2.2, 1.7, 1.6, and 1.2 mm from bregma (Fig. 2), which represents a major portion of the NAc.
Western Immunoblot Analysis. Western blot analysis for CART peptide (Vicentic et al., 2004) and CREB/pCREB (Walters et al., 2003) expression was carried out as previously described with modifications. Briefly, following infusions, rats were decapitated, and the NAc was dissected out and frozen. Total protein was extracted in 50 µl of lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM 
-glycerophosphate, 1 mM Na3VO4, 5 nM acidic acid, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Fifteen to 20 µg of total protein was loaded in 1x sample buffer [62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% glycerol, and 0.01% (w/v) bromphenol blue] onto 16% Novex precast SDS-Tris-glycine gel (Invitrogen, Carlsbad, CA). Proteins were transferred at 30 V at 4°C overnight. Membranes were blocked with 5% nonfat milk in 1x Tris-buffered saline (0.1% Tween 20, pH 7.6) for 1 h and then incubated with primary antibodies, either polyclonal antiserum to C4 peptide (CART nucleotides 61–102; 1:5000) or polyclonal antiserum to pCREB (1:1000) at 4° overnight. Chemiluminescent signal was detected by using horseradish peroxidase-conjugated chicken anti-rabbit (1:1000) secondary antibody and an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL). For CREB detection, pCREB membranes were stripped with Restore Western Blot Stripping Buffer (Pierce, Rockford IL) and reprobed with a polyclonal antiserum against CREB (1:1000). Quantitative analysis was conducted using Scion Image (Scion Corporation, Frederick, MD) and measuring the relative OD of each band.
Statistical Analyses. MCID, Scion Image, and GraphPad Prism (GraphPad Software Inc., San Diego, CA) were used for data analysis. Data are presented as mean ± S.E.M. Statistical analyses were carried out by either a Student's t test or a one-way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant. Comparisons made and specific analyses conducted for each experiment are detailed in the figure legends.
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), were unaffected by intra-accumbal forskolin injection (data not shown), indicating a local effect. To confirm the effect of forskolin on CART mRNA levels, a second experiment was conducted, infusing forskolin into one hemisphere and the vehicle or an inactive forskolin analog, INAF, into the other and sacrificing the animals 60 min following treatment. Forskolin (1.0 µg) significantly increased CART mRNA levels in the NAc approximately 2-fold over saline and INAF (Fig. 4B). Finally, as a positive control for forskolin-induced pCREB activity, preprodynorphin (PPD) mRNA levels, which are known to increase in response to forskolin and are regulated by pCREB (Simpson and McGinity, 1994), were measured with in situ hybridization using an oligonucleotide targeting rat PPD mRNA. As expected, intra-accumbal forskolin injections increased PPD mRNA levels in a PKA-dependent manner (data not shown).
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Inhibition of PKA Activity Attenuated Forskolin-Induced Increase in CART mRNA. Forskolin-induced stimulation of adenylyl cyclase increases cAMP levels, activates PKA, and increases pCREB levels (Simpson and McGinity, 1994). Phosphorylation of CREB by PKA is a necessary step in the activation of CREB, permitting it to regulate transcriptional activity. Consequently, inhibition of PKA activity should block forskolin's cAMP-mediated activation of CREB and, subsequently, should attenuate forskolin-induced CART mRNA expression. H89 or Rp-cAMPS, PKA inhibitors with different mechanisms of action, were injected into one hemisphere 20 min prior to the administration of forskolin (1.0 µg) into both hemispheres. The doses chosen for intra-accumbal administration of H89 (2 µg) and Rp-cAMPS (2 µg) were based on previous studies (Cervo et al., 1997; Sutton et al., 2000). Both H89 and Rp-cAMPS attenuated forskolin-induced CART mRNA expression by approximately 30 and 40%, respectively (Fig. 5), indicating the involvement of PKA in CART gene regulation. Interestingly, hemispheres treated with Rp-cAMPS alone demonstrated a significant decrease in CART mRNA expression compared with control levels (Fig. 5), suggesting that CART regulation may be under the tonic regulation of PKA. Representative autoradiograms of forskolin-induced (and PKA-attenuated) CART mRNA expression in the rat NAc are shown in Fig. 6.
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; Jaworski et al., 2003; McClung and Nestler, 2003; Couceyro et al., 2005; Hunter et al., 2005, Hunter et al., 2005). Consequently, it was hypothesized that, if pCREB modulates the effects of cocaine and regulates the expression of CART, then cocaine may potentiate forskolin-induced CART mRNA expression. Indeed, acute systemic administration of cocaine (20 mg/kg i.p.) immediately following intraaccumbal infusions of forskolin (0.5 µg) significantly increased CART mRNA expression in the rat NAc compared with forskolin or cocaine alone (Fig. 7). A dose of 0.5 µg of forskolin (rather than a near maximal dose of 1.0 µg) was used because it produces a minimal effect on CART expression and thus should show clear potentiation with cocaine. Consistent with other reports (Vrang et al., 2002; Marie-Claire et al., 2003; Hunter et al., 2005, Hunter et al., 2005), a single dose of cocaine failed to increase CART expression. However, in combination with forskolin, CART mRNA levels increased by approximately 42% compared with forskolin alone, suggesting that, under conditions of enhanced cAMP signaling, cocaine may influence CART levels.
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Intra-Accumbal Forskolin Administration Increased CART Peptide Levels in the Rat NAc. Because the CART peptide, not the mRNA, is functionally active, it is important to understand not only the regulation of mRNA but also the regulation of CART peptide. Therefore, we measured CART peptide levels in the NAc following intra-accumbal forskolin administration. Western immunoblot analysis showed that intra-accumbal forskolin administration significantly increased CART peptide levels in the rat NAc (Fig. 8). Accumbi treated with forskolin had CART peptide levels 2.5-fold greater than those of saline (control)-treated accumbi, suggesting that changes in CART mRNA expression lead to changes in CART peptide levels. Inhibition of PKA with H89 attenuated forskolin-induced CART peptide expression. Therefore, it appears that, similar to CART mRNA, CART peptide levels in the rat NAc are regulated, at least in part, by AC/cAMP/PKA-mediated signaling.
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Intra-Accumbal Forskolin Administration Increased pCREB Levels. We hypothesized that cAMP/PKA-mediated phosphorylation and activation of CREB are key mediators in the regulation of CART expression in the rat NAc. This hypothesis was examined by using forskolin to activate and PKA inhibitors to block the cAMP/PKA second messenger system. To confirm that forskolin is indeed stimulating the phosphorylation of CREB in our model, accumbal tissue from forskolin-treated animals was subjected to Western blot analysis with antibodies targeting pCREB and CREB. Consistent with other reports (Simpson and McGinity, 1994), central forskolin administration significantly increased pCREB levels while simultaneously decreasing the amount of CREB protein in the rat NAc (Fig. 9). Inhibition of PKA with H89 attenuated pCREB expression. Although this finding does not directly implicate pCREB in CART regulation, it provides evidence indicating that intra-accumbal forskolin injections increase the phosphorylation of CREB and, hence, may be involved in regulating CART mRNA and peptide levels.
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Discussion |
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; Kim et al., 2003; Couceyro et al., 2005; Kuhar et al., 2005). The regulatory pathways involved in CART expression in the brain have yet to be elucidated. Our hypothesis states that pCREB ultimately regulates expression of the CART gene via AC/cAMP/PKA-mediated signaling (Fig. 1). Consequently, manipulations of this pathway should affect CART mRNA and peptide levels. Accordingly, various stimulators and inhibitors of the AC/cAMP/PKA pathway were centrally administered, and CART mRNA and peptide, as well as pCREB, levels in the rat NAc were measured. As predicted by the model, forskolin increased CART mRNA levels, an effect attenuated by the inhibition of PKA. Interestingly, Rp-cAMPS alone reduced mRNA levels compared with controls, indicating a tonic regulation by PKA on CART expression. Forskolin significantly increased CART peptide levels in a manner consistent with the observed increase in CART mRNA levels. Finally, cocaine potentiated forskolin-induced CART mRNA expression in the accumbens, suggesting that, under conditions of enhanced cAMP signaling, cocaine may affect CART-containing neurons. Forskolin increases CART mRNA expression in cell culture (Barrett et al., 2002; Dominguez et al., 2002; Lakatos et al., 2002), and mutations of the CRE binding site in the CART promoter decrease CART promoter activity (Dominguez and Kuhar, 2004). To our knowledge, these findings are the first to provide evidence demonstrating CART regulation via AC/cAMP/PKA-mediated signaling in vivo in the rat NAc, probably via the transcriptional activity of pCREB.
Inhibition of PKA attenuated forskolin-induced CART expression. Inhibition of forskolin-induced CART mRNA expression appears greater with Rp-cAMPS than with H89. Reasons for this difference are unclear; however, differences in the drugs mechanism of action could provide some insight. H89 inhibits the phosphorylation process by blocking ATP binding sites on PKA. Thus, like most ATP site inhibitors, H89 may have effects on other protein kinase pathways and ATP receptors within the cell. Rp-cAMPS, on the other hand, is highly specific for PKA, working a step earlier in the activation process by inhibiting the dissociation of the catalytic and regulatory units, thus preventing any possible physiological actions by the regulatory units. Indeed, numerous differences between the inhibitory actions of H89 and Rp-cAMPS have been reported (Biolog Life Sci Institute; http://www.biolog.de/ti1003.html). Regardless of mechanism, the present results indicate that both PKA inhibitors significantly attenuated forskolin induced CART mRNA expression, providing strong evidence for the involvement of PKA in the regulation of the CART gene.
Interestingly, in addition to attenuating forskolin-induced CART expression, Rp-cAMPS alone significantly reduced CART mRNA levels compared with controls, suggesting that the expression of CART may be, in part, tonically regulated by PKA. Although treatment with forskolin stimulated CART expression, CART mRNA was also detected in control samples (vehicles and INAF; Fig. 4), indicating basal expression of CART in the NAc. Consequently, perhaps CART expression is under the tonic regulation of PKA. Receptor-mediated signal transduction pathways are often under a continuous basal level of signaling independent of receptor stimulation. Protein kinases and phosphatases, for example, may regulate basal level expression of multiple proteins by spontaneously activating (phosphorylation) and deactivating (dephosphorylation) targeted proteins. The steady-state level of expression in unstimulated cells, therefore, is the consequence of a balance between positive and negative regulators of a given pathway. Receptor stimulation produces an increase in expression over basal levels. The finding that inhibition of PKA decreases CART expression compared with basal levels suggests that CART expression in the NAc may be tonically regulated by PKA. Consequently, other secondary signaling pathways that ultimately converge on PKA and CREB may also have a role in CART regulation. CREB is activated by multiple cell surface receptors and intracellular signaling cascades, including neurotrophin, glutamate, NMDA, G-protein-coupled receptors, and L-type Ca2+ channels (Carlezon et al., 2005). For instance, Ca2+ mediates multiple signaling pathways that result in the phosphorylation of CREB (Carrion et al., 1999) and the subsequent expression of proteins associated with addiction, including dynorphin, fos, corticotropin-releasing factor, and brain-derived neurotrophic factor (Carlezon et al., 2005). Therefore, Ca2+-mediated signaling may participate in CART regulation. Interestingly, several of the biological processes that CART is associated with, including feeding, anxiety/fear, and psychostimulant addiction, have also been linked to increases in pCREB, supporting the contention that CREB is associated with CART.
Although CREB is regulated by multiple signaling pathways and, in turn, regulates several genes associated with reward and reinforcement, the present study focused on a single CREB regulatory pathway (i.e., cAMP/PKA) because in the NAc, this pathway has been closely linked to the addictive properties of drugs of abuse. Adenylyl cyclase proteins are plasma membrane-bound and coupled upstream to G-proteins. DA, which activates multiple G-protein-coupled receptors ultimately affecting cAMP signaling and pCREB-mediated gene transcription, modulates the effects of many drugs of abuse, including cocaine (Ikegami and Duvauchelle, 2004). Therefore, because the rewarding effects of cocaine are partially modulated by pCREB (Carlezon et al., 1998; McClung and Nestler, 2003), and cocaine increases CART mRNA levels (Fagergren and Hurd, 1999; Brenz Verca et al., 2001; Hunter et al., 2005, Hunter et al., 2005), it is plausible to suggest that DA receptors may regulate CART expression. Indeed, CART expression in the NAc may be partially regulated via DA D3 receptors (Beaudry et al., 2004; Hunter et al., 2005, Hunter et al., 2005). Moreover, dopaminergic nerve terminals in the NAc synapse on CART-containing neurons (Koylu et al., 1998; Smith et al., 1999) and CART and DA receptor mRNAs are colocalized (Beaudry et al., 2004), providing the proximity required for neurotransmitter signaling. Finally, CART levels are affected by glucocorticoids (Vicentic et al., 2004; Hunter et al., 2005, Hunter et al., 2005), which appear to modulate the dopaminergic system (Czyrak et al., 2003). These studies suggest that dopamine may play a role in regulating CART expression. This is relevant because drugs of abuse, including cocaine, ultimately lead to the stimulation of DA receptors; thus, cocaine may indirectly impact the CART gene.
Studies of the relationship between cocaine and CART have not been without controversy. Douglass et al. (1995) reported that cocaine increased CART mRNA levels. Although this finding has been difficult to replicate (Vrang et al., 2002; Marie-Claire et al., 2003), it appears that a binge-dosing regime, rather than acute administration of cocaine, more reliably increases CART mRNA levels (Fagergren and Hurd, 1999; Brenz Verca et al., 2001; Hunter et al., 2005, Hunter et al., 2005). The effect of acute cocaine on forskolin-induced CART expression was examined. Cocaine significantly potentiated forskolin-induced CART mRNA expression in the rat NAc compared with either of the drugs administered independently. Therefore, cocaine appears to impact the regulation of the CART gene under conditions of enhanced cAMP signaling, which, interestingly, is increased in the ventral tegmental area of human cocaine overdose victims (Tang et al., 2003). Increased cAMP/CREB expression in the addiction process has been associated with the development of dependence and tolerance to several common drugs of abuse (Nestler, 2004). Thus, it is plausible to suggest that perhaps CART levels are regulated by cocaine in a chronic abuser, whose basal cAMP signaling is enhanced due to constant cocaine use. Moreover, both manipulations of the cAMP pathway (Carlezon et al., 1998; Knapp et al., 2001) and central administration of CART peptide (Jaworski et al., 2003; Kim et al., 2003) modulate some of the behavioral effects of cocaine. These findings are compatible with the view that CART peptides are involved in the rewarding properties of cocaine. Interestingly, because CART peptides appear to oppose some of cocaine's effects, the stimulatory effect of cocaine on CART expression suggests the existence of an endogenous feedback mechanism meant to decrease the behavioral effects of cocaine (i.e., cocaine increases CART, which in turn attenuates the effects of cocaine).
CART peptides are involved in the modulation of the brain's reward circuitry, appearing to oppose the reinforcing properties of several processes, including feeding and drug addiction. Therefore, because CART may serve as a therapeutic target for obesity and drug addiction, understanding its regulation is important. The present results demonstrate the in vivo regulation of the CART gene. CART mRNA and peptide levels in the rat NAc were elevated following intraaccumbal activation of adenylyl cyclase, an effect attenuated by PKA inhibitors, suggesting that CART gene expression in the rat NAc is regulated, in part, via cAMP signaling and, most likely, through pCREB. Cocaine potentiated forskolin-induced CART expression, suggesting that cocaine may participate in the regulation of the CART gene, probably via its effects on DA receptors and cAMP/PKA-mediated signaling. These results provide a basis for future studies on the mechanisms underlying the in vivo regulation of CART expression and its complex relationship with cocaine, feeding, anxiety/fear, and other processes associated with the brain's reward and reinforcement pathways.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: CART, cocaine-amphetamine-regulated transcript; NAc, nucleus accumbens; DA, dopamine; CREB, cAMP-response element-binding protein; CRE, cAMP-response element; PKA, protein kinase A; AC, adenylyl cyclase; pCREB, phospho-cAMP-response element-binding protein; NIDA, National Institute on Drug Abuse; INAF, 7-deacetyl-7-[O-(N-methylpiperazino)-g-butyryl]-,dihydrochloride; H89, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline-sulfonamide hydrochloride; Rp-cAMPS, adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer; SSC, standard saline citrate; OD, optical density; ANOVA, analysis of variance; PPD, preprodynorphin; HCL, hydrochloric acid.
Address correspondence to: Douglas C. Jones, Division of Neuroscience, Yerkes National Primate Research Center of Emory University, Atlanta, GA 30329. E-mail: douglas.jones{at}emory.edu
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, p < 0.01).
