Neurochemistry International 75 (2014) 105–111

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Microinjection of CART (cocaine- and amphetamine-regulated transcript) peptide into the nucleus accumbens inhibits the cocaine-induced upregulation of dopamine receptors and locomotor sensitization Qinghua Peng a,1, Xi Sun b,1, Ziyong Liu c, Jianghua Yang b, Ki-Wan Oh d, Zhenzhen Hu e,⇑ a

Department of Anesthesiology, The First Affiliated Hospital, Nanchang University, Nanchang, Jiangxi 33006, China Evidence Identification Center, Department of Jiangxi Provincial Public Security, Nanchang, Jiangxi 33006, China c Department of Physiology, College of Medicine, Nanchang University, Nanchang, Jiangxi 33006, China d College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea e Department of Pathophysiology, College of Medicine, Nanchang University, Nanchang, Jiangxi 33006, China b

a r t i c l e

i n f o

Article history: Received 11 April 2014 Received in revised form 6 June 2014 Accepted 10 June 2014 Available online 19 June 2014 Keywords: Cocaine CART Nucleus accumbens CREB Dopamine receptor signaling

a b s t r a c t Repeated exposure to addictive drugs enhances dopamine receptor (DR) signaling and the ultimate phosphorylation of the cyclic adenosine 50 -monophosphate (cAMP)-response element-binding protein (CREB)-regulated cocaine- and amphetamine-regulated transcript (CART) expression in the nucleus accumbens (NAcc). These effects are known to contribute to the expression of behavioral sensitization. CART peptides are neuropeptides that modulate drug reward and reinforcement. The present experiments investigated the effects of CART 55–102 microinjection into the NAcc on (1) the phosphorylation of CREB, (2) cAMP/protein kinase A (PKA) signaling and (3) extracellular signal-regulated kinase (ERK) phosphorylated kinase signaling. Here, we show that repeated microinjections into the NAcc of CART 55–102 peptides (1.0 or 2.5 lg, 0.5 ll/side) attenuates cocaine-induced enhancements of D1R, D2R and D3R phosphorylation in this sites. Furthermore, the microinjection of CART 55–102 followed by repeated injections of cocaine (15 mg/kg) dose-dependently blocked the enhancement of cAMP levels, PKA activity and pERK and pCREB levels on the fifth day of cocaine administration. The cocaine-induced locomotor activity and behavioral sensitization in rats were also inhibited by the 5-day-microinjection of CART peptides. These results suggest that the phosphorylation of CREB by cocaine in the NAcc was blocked by the CART 55–102 peptide via the inhibition of D1R and D2R stimulation, D3R phosphorylation, cAMP/PKA signaling and ERK phosphorylated kinase signaling. These effects may have played a compensatory inhibitory role in the behavioral sensitization of rats that received microinjections of CART 55–102. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Cyclic AMP-response element-binding protein (CREB) is a common phosphoprotein target of multiple kinase cascades activated by psychostimulants, such as protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) (DiRocco et al., 2009; Hollander et al., 2010). Moreover, CREB is known to be an important contributor to sensitization-dependent neuronal genes and behavioral sensitization by psychostimulants (Carlezon et al., 1998, 2005;

⇑ Corresponding author. Tel.: +86 13755752400; fax: +86 79186360556. 1

E-mail address: [email protected] (Z. Hu). These two authors contributed equally to this work.

http://dx.doi.org/10.1016/j.neuint.2014.06.005 0197-0186/Ó 2014 Elsevier Ltd. All rights reserved.

Dong et al., 2006). Pharmacological studies have indicated that the inhibition of PKA and ERK was accompanied by the attenuation of CREB activation and psychostimulant induced-behavioral sensitization (Lynch and Taylor, 2005; Valjent et al., 2006). Elevations of CREB in the NAcc of rats by virus-mediated gene transfer has been shown to increase the rewarding effects of cocaine (Larson et al., 2011), whereas CREB antisense increased the threshold dose of cocaine required for reinstating cocaine’s sensitization (Choi et al., 2006). Psychostimulant-induced ERK and CREB phosphorylation is mediated, in part, by D1 and D3 receptors (D1Rs and D3Rs) that stimulate PKA signaling (Zhang et al., 2004). However, D2 agonists can phosphorylate ERK and CREB via Gq-coupled phospholipase Cb signaling (Schierberl et al., 2012). In a previous study,

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a greater increase in striatal ERK and CREB phosphorylation in the striatum of cocaine-sensitized rats was observed than in salinepretreated rats (Marin et al., 2009). Cocaine- and amphetamine-regulated transcript (CART) peptides are highly expressed neuropeptides within the nucleus accumbens (NAcc), hypothalamus and ventral tegmental area (VTA) that modulate drug reward and reinforcement (Jaworski and Jones, 2006). CREB binding sites in the CART promoter sequence are involved in the expression of the CART gene (Lakatos et al., 2002). Mutations of the CRE binding site on the CART promoter decrease promoter activity (Dominguez and Kuhar, 2004). Intra-accumbal activation of cAMP/PKA signaling stimulated the phosphorylation of CREB and increased CART mRNA and peptide levels in the rat NAcc (Jones and Kuhar, 2006). These findings demonstrate that CART is regulated by dopamine in the NAcc and is at least partially regulated by D3R (Hunter et al., 2006). CART peptides reduce the effects of cocaine by modulating the simultaneous activation of both D1R and D2R, suggesting dopaminergic involvement (Moffett et al., 2011). In addition, microinjections of CART 55–102 peptide into either the VTA or NAcc, a region known as the neuronal substrate for induction of behavioral sensitization, attenuated the locomotor effects of cocaine (Jaworski et al., 2003, 2007; Moffett et al., 2011). These results suggest that CART 55–102 may play a negative regulatory role to psychostimulants. However, direct evidence of a connection between a negative regulatory role of CART 55–102 and intracellular changes in the NAcc has not been reported. Therefore, the present study examined whether CART 55–102 peptide regulates DRs, cAMP, PKA, pERK and pCREB levels in the rat NAcc during repeated cocaine-induced behavioral sensitization. 2. Materials and methods 2.1. Animals and chemicals Sprague Dawley (SD) male rats weighing between 260 and 280 g were used. Animals were housed one per cage with water and food available ad libitum under an artificial 12:12 (h) light/dark cycle (light at 07:00) and constant temperature (22 ± 2 °C). All of the animals used in this study were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23, revised 1985). The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Nanchang (Permit Number: 2010–0002). Surgery was performed under sodium pentobarbital or hypothermia anesthesia, and all efforts were made to minimize suffering. Cocaine and CART 55–102 peptides were purchased from Sigma Chemical Company (St. Louis, MO). In general, the phrase ‘‘CART peptides’’ refers to rlCART 55–102 and/or rlCART 66–102, both of which are tested for biological activity by examining their effects on locomotor activity (Kimmel et al., 2002). As previously reported, intra-NAcc rlCART 55–102 inhibited cocaine inducedlocomotor activity, while rlCART 1–27 were not active (Jaworski et al., 2003). Unless specified otherwise, CART peptide or CART 55–102 in this paper refers to the peptide rlCART55–102. All chemicals were dissolved immediately before use in physiological saline. 2.2. Surgical and infusion procedures At least 1 week after arrival, rats were anesthetized with pentobarbital sodium (42 mg/kg, i.p.; Sigma Co., St. Louis, MO). A bilateral stainless steel guide cannula assembly (22-gauge; Plastics One, Roanoke, VA) was implanted above the nucleus accumbens using a motorized stereotaxic stereodrive (Neurostar Co.,

Sindelfingen, Germany). Target coordinates relative to bregma were A/P + 1.7, L/M ± 1.6, and D/V – 7.5 (Paxinos and Watson, 2005). Guide cannulae were anchored in place using dental acrylic and two stainless steel screws into the skull. Dummy cannulae extending 0.5 mm past the tip of the cannulae were inserted to prevent blockage, and a dust cap was attached to the top of the cannula assembly. The rats were allowed to recover from surgery for at least 2 weeks before the start of the experiment. Stainless steel injector cannulae (28-gauge; Plastics One), which projected 2 mm past the tip of the guide cannulae, were used for infusions. These cannulae were connected to 5-ll syringes (Neurostar Co., Sindelfingen, Germany) via polyethylene-5 tubing. Microinjection systems (Neurostar Co., Sindelfingen, Germany) were used for fluid delivery. For each infusion, rats were confined to a small polyethylene box. Bilateral infusions of 0.5 ll per side were given into the NAcc over 30 s. After the infusion, the injector cannulae were removed, the dummy cannulae were put back in place, and the dust cap was secured. 2.3. Measurement of locomotor activity All of the experiments were performed using a randomized, balanced repeated-measures design, such that each rat received all of the treatments (control and treatment). A separate group of rats was used for each experiment. Thus, each rat received four bilateral accumbal infusions (two saline infusions and 1.0 and 2.5 lg/ side CART 55–102 peptide infusions). The infusions of saline or peptide were immediately followed by an i.p. injection of either saline or 15 mg/kg cocaine, respectively, for 5 consecutive days according to our previous methods to induce behavioral sensitization (Cai et al., 2014). The control rats were treated with saline under the same conditions as the treatment rats. The locomotor activity of each rat was measured using a tilting-type ambulometer (AMB-10, O’Hara, Tokyo, Japan). Each rat was placed in the activity cage (20 cm in diameter, 18 cm in height). Chemicals were administered after an adaptation period of 10 min. The rats were allowed to perambulate for 10 min in the activity cages followed by a 1 h test period immediately after the cocaine administration. The development of sensitization on the 5th day was evidenced by an increased locomotor activity response to cocaine compared with that on the 1st day. 2.4. Western blotting Nucleus accumbens tissue was dissected in ice-cold saline and homogenized in protein extraction solution. Supernatants were collected and were frozen for storage until use. The protein concentration of the supernatant was determined using the Bradford method with bovine serum albumin as the standard. Equal amounts of proteins were separated on a SDS/16%-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in 0.5% not-fat milk with TBS-T [10 mM Tris (pH 8.0) containing 0.05% tween-20], followed by 3 washes in TBS-T. Membranes were bound with specific antibodies using the SNAP i.d. system (Millipore Co., Bedford, MA). The anti-D1R (1:150, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-D2R (1:150, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-ERK (1:100, Abcam, Cambridge, MA, USA), anti-phospho-ERK (1:100, Abcam, Cambridge, MA, USA), anti-CREB (1:250, Abcam, Cambridge, MA, USA) and anti phospho-CREB (1:167, Abcam, Cambridge, MA, USA) antibodies were used in this study. The membrane was then incubated with the corresponding conjugated immunoglobulin G-horseradish peroxidase. Immunoreactivity was revealed by incubating with ECL-Plus chemiluminescent substrate. Chemiluminescence was detected by the imaging system FUSION-FX7 (Vilber Lourmat, Co., Cedex, France). The

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quantification of the bands was performed by densitometry (OD) using the analysis software FUSION-CAPI.

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3. Results 3.1. CART peptides block DR activation by cocaine in rat NAcc

2.5. In vitro binding assay The D3R protein was separated from the total protein (300 lg) by immunoprecipitation using Protein G Sepharose 4 Fast Flow (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and the D3R antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The D3R immunoprecipitate was used for Western blot analysis with anti-phospho-serine (1:67 dilutions, Santa Cruz Biotechnology) antibodies. 2.6. Cyclic AMP analysis Rats were sacrificed by decapitation 22 h after the last administration of cocaine or saline. The NAcc was extracted at the coronal level at +1.6 and +1.0 mm from the bregma, according to the stereotaxic atlas (Paxinos and Watson, 2005). The tissue was dissected in ice-cold saline and homogenized in protein extraction solution. The cAMP levels were determined using a competition enzymelinked immunoassay (ELISA). The absorbance of the developed color was measured by a spectrofluorometer (BMG Co., Ortenberg, Germany) at an emission wavelength of 450 nm. Measurement of absorbance using a cAMP standard allows for the calculation of an absolute amount of cAMP in a sample of interest. The amount of tissue was 5.0 mg, and the results were expressed as nmol (nM) cAMP/mg tissue. 2.7. Radiometric analysis Rats were sacrificed by decapitation 22 h after the last administration of the chemicals or saline. The PKA protein was purified from the total protein (300 lg) using immunoprecipitation with an antibody to PKA. The immunoprecipitate of PKA was dissolved in assay dilution buffer for radiometric analysis. Equal amounts of the PKA immunoprecipitate were added to incubation tubes containing 1.67 lM cAMP, 0.08 mM kemptide, 0.33 lM PKC/CaMK inhibitor cocktail and 1.67 lCi of the [c-32P]ATP/Magnesium/ATP cocktail and brought to a total volume of 60 ll. The solution was incubated with shaking at 30 °C for 10 min. A 25 ll aliquot was spotted on phosphocellulose 81 paper, washed 3 times with 0.75% phosphoric acid and once with acetone. The result was read in a Wallac 1450 Microbeta Trilux liquid scintillation counter (Cardinal Health, Co., Dublin, OH) and calculated by comparing the counts per minute (CPM) of enzyme PKA to CPM of the control samples that contained no enzyme. The results are expressed as pmol of phosphate incorporated into kemptide/min/lg protein. 2.8. Histology After the completion of the experiments, each rat was given ethyl ether and decapitated. After each brain was removed, it was immediately frozen for slicing on a cryostat. Each brain was sliced in 50-lm-thick coronal sections through the area of the guide cannulae. These sections were mounted onto slides, stained with toluidine blue, and examined under a microscope to localize the tip of the injector cannula. 2.9. Data analysis The results are presented as the mean ± SEM. The significance of the effects of the CART 55–102 peptides in various doses and at different response times were assessed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Statistical significance was set at p < 0.05.

The Western blot analysis revealed that the 5-day-injection of cocaine (15 mg/kg) increased the protein levels of the D1Rs (n = 4, p < 0.001, Fig. 1A) and D2Rs (n = 4, p < 0.01, Fig. 1B) in the NAcc on the 5th day when compared with the control (saline, n = 4) group, which was consistent with the result of previously published findings (Lee et al., 2006). However, cocaine inducedsignificant increases in the protein level of the D1Rs and D2Rs was inhibited by the repeated-microinjection into the NAcc of CART 55–102 (1.0 or 2.5 lg/side) on the 5th day (n = 4, p < 0.01 and p < 0.05, Fig. 1 A and B). A series of coimmunoprecipitation and Western blot analysis experiments were carried out to measure the D3R phosphorylation in the NAcc. Cocaine was found to increase serine phosphorylation, as detected by an antibody selective for phosphoserine (n = 4, p > 0.001, Fig. 1C) in basal D3R precipitates. This increase did not occurred after CART 55–102 (1.0 or 2.5 lg/side) microinjection into the NAcc of on the 5th day (n = 4, p < 0.01, Fig. 1C), indicating that the CART peptide promotes the dephosphorylation of D3Rs. 3.2. CART peptides block the activation of cAMP/PKA activity by cocaine in rat NAcc In ELISA and radiometric experiments, the repeated i.p. injection of 15 mg/kg cocaine following microinjections of saline increased the levels of cAMP (n = 4, p < 0.001, Fig. 2A) and PKA activity (n = 4, p < 0.001, Fig. 2B) in NAcc at the 5th day, compared with the control group. However, these effects were completely blocked by CART 55–102 (1.0 or 2.5 lg/side) microinjection into this site prior to the i.p. injection of cocaine (Fig. 2A and B). 3.3. CART peptides block the phosphorylation of ERK and CREB in response to cocaine in rat NAcc Western blot analysis revealed that the repeated injection of cocaine following the microinjection of saline increased the levels of CREB and ERK phosphorylation in the NAcc to approximately 1.3 and 2.0 times those obtained by saline treatment. However, the injection of CART 55–102 (1.0 or 2.5 lg/side) completely blocked the CREB and ERK phosphorylation effects of cocaine (n = 4, p < 0.01 or p < 0.001, Fig. 3D and F) in this site. Moreover, the levels of CREB and ERK in the NAcc showed no significant changes after administration of cocaine with CART 55–102 peptides, and it appeared almost the same as control levels (n = 4, Fig. 3E and G). 3.4. CART peptides block cocaine-induced behavioral sensitization in rats All injections were found in the accumbal area at or near the shell as desired, and no animals were eliminated based on improper injector location. In experiments using a tilting-type ambulometer, the repeated administration of cocaine following the microinjection of saline increased locomotor activity at the 1st day (F(3,12) = 60.6, p < 0.001, Fig. 4A) and continued to the 5th day (F(3,12) = 158.3, p < 0.001, Fig. 4A), compared with the control group. Moreover, the cocaine-treated rats exhibited more locomotor activity at the 2th day than at the 1st day, indicating that behavioral sensitization was induced by cocaine at the 2th day (n = 5, p < 0.05, Fig. 4A and B). This effect, however, was dose-dependently reduced by the microinjection of CART 55–102 into the NAcc. The microinjection of CART 55–102 (1.0 lg/side) into the NAcc following the administration

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Fig. 1. Repeated microinjections of CART 55–102 peptide into rat nucleus accumbens (NAcc) reduce the protein and phosphorylation level of dopamine receptor (DR) to cocaine at the 5th day. (A) A representative Western blot labeled with antibodies to D1R and GAPDH. (B) A representative Western blot labeled with antibodies to D2R and GAPDH. (C) Immunoblot analysis of phosphoserine was performed on D3R precipitates. Blots were scanned and the band intensities were quantified by relative densitometry and expressed as arbitrary relative OD values (i.e., D1R band intensity relative to the GAPDH band intensity). (D) The NAcc tissue region, where the caudal surface of coronal section (1.0 mm thick) extending 1.6–2.6 mm from bregma (Paxinos and Watson, 2005) were punched out, is shown. (E) Groups were treated with either saline/saline (h), saline/cocaine (15 mg/kg) ( ), CART (55–102) peptide (1.0 lg/0.5 ll/one side)/saline ( ), or CART (2.5 lg/0.5 ll/one side)/cocaine ( ). Data are presented as the mean ± SEM of each group (n/group = 5). ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001, respectively, compared with that of the control group. #p < 0.05 and ##p < 0.01, compared with that of the cocaine group (one-way ANOVA followed by Dunnett’s post hoc test).

of cocaine also increased locomotor activity from the 1st day to the 5th day (n = 4, p < 0.001, Fig. 4A and B), but decreased the locomotor activity compared with animals that received cocaine with a saline microinjection (n = 4, p < 0.01 and p < 0.001, Fig. 4A and B) and blocked cocaine-induced behavioral sensitization. Further, the animals that received cocaine with CART 55–102 (2.5 lg/side) microinjection save the similar activity in locomotor to the control group, indicating that the cocaine-induced locomotor and sensitization effects were completely blocked by the dose 2.5 lg/side of CART 55–102.

4. Discussion The present study revealed that the intra-accumbal injection of CART 55–102 peptides (1.0 or 2.5 lg, 0.5 ll/side) in the NAcc inhibited the phosphorylation of CREB via the decreased activation of DR protein and phosphorylation levels, cAMP/PKA signaling and ERK signaling in response to cocaine and inhibited the expression of locomotor activity and behavioral sensitization to cocaine. This narrow dose range (1 or 2.5 lg) implies that CART is acting as a

physiological antagonist that is acting in an ‘‘all or nothing’’ manner to inhibit the cocaine-induced increase in DR production rate and/or decrease in receptor degradation rate constant. This is in addition to, or a consequence of, the changes in DR phosphorylation. CART peptides are highly expressed neuropeptides in the regions that modulate drug reward and reinforcement (Jaworski and Jones, 2006). In addition to the anatomical distribution of CART mRNA and peptides in the major brain regions, a large amount of behavioral evidence in human and animals strongly supports its role in drug reward and reinforcement. A report from Busto (Busto et al., 2010) suggests that the CART gene plays a role in the vulnerability to alcohol and nicotine abuse in persons with schizophrenia. Taken together, CART peptides play an important modulatory role in the psychostimulant effects of abuse drugs. Our data provide further evidence of the effects of CART peptides on psychostimulant actions in the NAcc. The repeated administration of cocaine elicits specific behaviors referred to as sensitization, rewards, reinforcement and drug induced psychosis in animals and humans (Kuribara, 1995; McGregor et al., 1992; Nazarian et al., 2004; Woolverton and Ranaldi, 2002). Cocaine increases dopamine neurotransmission

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Fig. 2. Repeated microinjection of the CART 55–102 peptide into rat NAcc dose-dependently attenuates the enhancement of cyclic adenosine 50 -monophosphate (cAMP) levels and protein kinase A (PKA) activity to cocaine at the 5th day. (A) Cocaine (15 mg/kg) following the microinjection of saline, induced an increase in the level of cAMP in rat NAcc as shown by the competition enzyme-linked immunoassay (ELISA). These effects were inhibited by the microinjection of CART 55–102 peptide (1.0 or 2.5 lg/side). (B) After PKA protein was purified by immunoprecipitation, the activity of PKA was measured using the radiometric assay. The cocaine-induced PKA activity was blocked by microinjection of CART 55–102 peptide. (C) the NAcc tissue region, where the caudal surface of the coronal section (1.0 mm thick) extending 1.6–2.6 mm from bregma (Paxinos and Watson, 2005) were punched out, is shown. Groups were treated with either saline/saline (h), saline/cocaine (15 mg/kg) ( ), CART (55–102) peptide (1.0 lg/ 0.5 ll/one side)/saline ( ), or CART (2.5 lg/0.5 ll/one side)/cocaine ( ). Data are presented as the mean ± SEM of group (n/group = 4). ⁄⁄⁄p < 0.001, compared with the control group (one-way ANOVA followed Dunnett’s post hoc test). #p < 0.05, ##p < 0.01 and ###p < 0.001, respectively, compared with that of the cocaine group (one-way ANOVA followed Bonferroni’s post hoc test).

Fig. 3. Microinjection of CART 55–102 peptides into the NAc blocks the increase of cocaine-induced phosphorylation levels of extracellular signal-regulated kinase (ERK) and cAMP-response element-binding protein (CREB) in this site. (A) A representative Western blots analysis labeled with antibodies against phosphorylated and total ERK, phosphorylated and total CREB and GAPDH. Blots were scanned and the band intensities were quantified by relative densitometry and expressed as arbitrary relative OD values (i.e., pERK band intensity relative to the GAPDH band intensity). (B) The NAcc tissue region, where the caudal surface of the coronal section (1.0 mm thick) extending 1.6–2.6 mm from bregma (Paxinos and Watson, 2005), were punched out is shown. (C) Groups were treated with either saline/saline (h), saline/cocaine (15 mg/kg) ( ), CART (55–102) peptide (1.0 lg/0.5 ll/one side)/saline ( ), or CART(2.5 lg/0.5 ll/one side)/cocaine ( ). (D–G) A graph of the data obtained from (A). Data are presented as the mean ± SEM of the group (n/group = 4). ⁄⁄⁄p < 0.001, compared with that of the control group. ##p < 0.01 and ###p < 0.001, respectively compared with that of the cocaine group (one-way ANOVA followed Dunnett’s post hoc test).

and stimulates postsynaptic dopaminergic (D) receptors (R). Dopamine exerts its pleiotropic actions through interactions with two classes of dopamine receptors, which belong to the following G-protein-coupled receptors: the D1 class (D1 and D5 subtypes)

and the D2 class (D2, D3 and D4 subtypes) (Doherty et al., 2008; Neve et al., 2004). Cocaine activate cAMP/PKA and ERK signal transduction pathways, and, ultimately, pCREB by upregulating dopamine receptors in the NAcc (Ferre, 2010). Phosphorylation of CREB

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Fig. 4. Microinjection into the NAcc of CART 55–102 peptide inhibits the expression of locomotor and sensitization effects by cocaine. Groups indicate microinjection of either saline or CART 55–102 (1.0 lg or 2.5 lg) followed by either saline or i.p. injection of cocaine. (A) Day-course data are shown as the group mean (+SEM) locomotor activity counts obtained during the 1 h test period following the treatment for 7 days. Rats microinjected with saline showed a significantly greater locomotor response to cocaine compared to saline groups (⁄⁄⁄p < 0.001) for 7 days and behavioral sensitization responses to cocaine for 6 days compared to the first day (+++p < 0.001). The sensitized response was absent when CART 55–102 was microinjected followed by cocaine injection (##p < 0.01 and ###p < 0.001). (B) The location of the injection cannula tips were included in the data analyses. The line drawing is from work by Paxinos and Watson (2005). The numbers to the right indicate millimeters from bregma. Data are shown as group mean (+SEM) locomotor activity counts obtained during the 1 h test period following the treatment on different test days. Symbols indicate significant differences as revealed by one-way ANOVA followed Dunnett’s post hoc test. Symbols represent the different groups: h/s, saline (intra-NAc)–saline (i.p.); /d, saline–cocaine; /., CART 55–102 (1.0 lg/side)-cocaine; /N, CART 55–102 (2.5 lg/side)-cocaine.

is mainly due to the activation of adenylyl cyclase linked to G-protein-coupled receptors and the consequent upregulation of cAMP-PKA signaling and ERK signaling by psychostimulants. The high expression of CREB in forebrain sites, such as the NAcc, contributes to both the induction and expression of sensitization by psychostimulants (Cahill et al., 2014; Dong et al., 2006). In the present experiments, we found that the cocaine-induced increase of DR protein and phosphorylation levels, cAMP levels, PKA activity, ERK phosphorylation and CREB phosphorylation in the NAcc was blocked by active CART 55–102 peptide microinjected into this site. These results suggest that the inhibitory effect of CART 55–102 on behavioral sensitization, as shown in the present study, is possibly modulated by the CART 55–102 peptide-induced blockade of CREB activation in the NAcc. Whether the blockade of CART 55–102 on the cocaine-induced increase of pCREB levels is modulated by direct interactions with CREB and involves signal pathways in the NAcc is not known. We have found that the active CART 55–102 peptide directly inhibited the enhancement of D1R, D2R, and D3R phosphorylation, cAMP/PKA signaling and ERK signaling induced by cocaine in rat NAcc neurons in a dose-dependent manner. Previous data has shown that DA receptors were found on CART neurons (Hubert and Kuhar, 2006); moreover, increases in CART expression in the NAcc may be partially regulated by D3 receptors (Hunter et al., 2006). A D1 or D2/D3 antagonists blocked the over-expression of CART induced by ethanol in rat NAcc (Salinas et al., 2006). Moreover, the cAMP/PKA/pCREB signal pathway is involved in the regulation of CART expression in the rat NAcc (Jones and Kuhar, 2006). More recently, studies have shown that the CART 55–102 peptide reduces the behavioral sensitization of psychostimulants by modulating the simultaneous activation of both D1 and D2 dopamine receptors. The increase of ERK1/2 phosphorylation levels in the NAcc induced by cocaine was completely blocked by microinjecting CART 55–102 in this site (Yoon et al., 2007). These results suggest that the phosphorylated CREB may be a downstream nuclear target of cAMP/PKA signaling and that ERK signaling in the NAcc plays a role in the sensitization to cocaine. In light of our present finding, that direct microinjection of the CART 55–102 peptide into the NAcc inhibits the increase of cAMP/PKA-ERK-CREB activity and behavioral sensiti-

zation induced by cocaine in this site, it will be important to explore the contribution of NAcc CART peptide in the regulation of cAMP/ PKA-ERK-CREB signaling and the molecular mechanism involved in the expression of behavioral sensitization. To our knowledge, this is the first time that cocaine-induced CREB activation in the NAcc has been shown to be inhibited by direct, repeated microinjections of CART 55–102 in this site. These results further suggest that the opposing effect of CART 55–102 in the NAcc on the expression of cocaine-induced behavioral sensitization may be modulated by interrupting cAMP/PKA and ERK signaling, resulting in the inhibition of CREB phosphorylation in this site. The detailed molecular mechanisms by which CART regulates cAMP/PKA-ERK-CREB signaling, however, remain unknown. In recent years, several streams of research have shown that the anatomical distribution, terminal synapses formation, and expression of CART peptides are involved in the development of behavioral sensitization by abuse drugs, particularly by psychostimulants. These studies have shown that CART-containing axons and nerve terminals were found in the NAcc, hypothalamus and VTA and were associated with the rewarding effects of psychostimulants. CART-containing axons and nerve terminals, active dopaminergic neurons, and some CART peptide-positive terminals form inhibitory synapses onto GABAergic interneurons in the VTA and substantial nigra (SNr) (Dallvechia-Adams et al., 2001, 2002). In addition, CART peptides are present in a subset of GABAergic projection neurons that express dynorphin, which inhibit the effects of dopamine by activating the j-receptor in the NAcc (Dallvechia-Adams et al., 2002; Hubert and Kuhar, 2006). Coincidentally, a previous report from Hubert et al. (2010) showed that CART-containing terminals that originate in the NAcc and form symmetric synapses onto inhibitory GABAergic synapses inhibit cocaine-induced locomotion in the ventral pallidum. In this manner, the mechanism of the negative regulation of CART peptides on psychostimulant effects in the striatum may be associated with the formation of inhibitory dopaminergic neurons, indirectly of dynorphin and substance P release in the NAcc. In conclusion, the results demonstrate that the repeatedmicroinjection of CART 55–102 peptide in the NAcc inhibited

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