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Addiction Biology

ORIGINAL ARTICLE

doi:10.1111/adb.12137

Serotonin2C receptors modulate dopamine transmission in the nucleus accumbens independently of dopamine release: behavioral, neurochemical and molecular studies with cocaine Adeline Cathala1,2, Céline Devroye1,2, Marlène Maitre1,2, Pier Vincenzo Piazza1,2, Djoher Nora Abrous1,3, Jean-Michel Revest1,2 & Umberto Spampinato1,2 Neurocentre Magendie, Physiopathology of Addiction Group, Inserm U862, France1, Université de Bordeaux, France2 and Neurocentre Magendie, Neurogenesis and Pathophysiology Group, Inserm U862, France3

ABSTRACT In keeping with its ability to control the mesoaccumbens dopamine (DA) pathway, the serotonin2C receptor (5-HT2CR) plays a key role in mediating the behavioral and neurochemical effects of drugs of abuse. Studies assessing the influence of 5-HT2CR agonists on cocaine-induced responses have suggested that 5-HT2CRs can modulate mesoaccumbens DA pathway activity independently of accumbal DA release, thereby controlling DA transmission in the nucleus accumbens (NAc). In the present study, we assessed this hypothesis by studying the influence of the 5-HT2CR agonist Ro 60-0175 on cocaine-induced behavioral, neurochemical and molecular responses. The i.p. administration of 1 mg/kg Ro 60-0175 inhibited hyperlocomotion induced by cocaine (15 mg/kg, i.p.), had no effect on cocaine-induced DA outflow in the shell, and increased it in the core subregion of the NAc. Furthermore, Ro 60-0175 inhibited the late-onset locomotion induced by the subcutaneous administration of the DA-D2R agonist quinpirole (0.5 mg/kg), as well as cocaine-induced increase in c-Fos immunoreactivity in NAc subregions. Finally, Ro 60-0175 inhibited cocaine-induced phosphorylation of the DA and c-AMP regulated phosphoprotein of Mr 32 kDa (DARPP-32) at threonine residues in the NAc core, this effect being reversed by the selective 5-HT2CR antagonist SB 242084 (0.5 mg/kg, i.p.). Altogether, these findings demonstrate that 5-HT2CRs are capable of modulating mesoaccumbens DA pathway activity at post-synaptic level by specifically controlling DA signaling in the NAc core subregion. In keeping with the tight relationship between locomotor activity and NAc DA function, this interaction could participate in the inhibitory control of cocaine-induced locomotor activity. Keywords

5-HT2C receptor, c-Fos, DARPP-32, dopamine release, locomotor activity, nucleus accumbens.

Correspondence to: Umberto Spampinato, Université Victor Segalen Bordeaux 2-Inserm U862, 146 rue Léo Saignat, Bordeaux Cedex 33076, France. E-mail: [email protected]

INTRODUCTION The mesoaccumbens dopaminergic (DA) pathway is known to play a major role in mediating the behavioral and neurochemical effects of drugs of abuse, such as cocaine (Kalivas & Volkow 2005; Di Chiara & Bassareo 2007). The central serotonin2C receptor (5-HT2CR), along with its dense localization in brain DA regions (Filip et al. 2012), is a well-known modulator of DA neuron activity in the mammalian brain. In keeping with its ability to control mesoaccumbens DA pathway activity and cocaine-induced behavioral responses, the 5-HT2CR is © 2014 Society for the Study of Addiction

currently considered as a promising target for improved treatments of cocaine abuse and dependence (Müller & Huston 2006; Filip et al. 2012; Devroye et al. 2013). Compelling evidence demonstrates that central 5-HT2CRs afford an overall inhibitory control on the behavioral and neurochemical effects of cocaine. Thus, peripheral administration of 5-HT2CR agonists and antagonists respectively inhibits and enhances the behavioral responses of cocaine, such as hyperlocomotive, discriminative stimulus, and reinforcing properties (Filip et al. 2012). On the other hand, previous studies in halothane-anesthetized rats have shown that Addiction Biology

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cocaine-increased accumbal DA release is enhanced by 5-HT2CR antagonists but unaltered by 5-HT2CR agonists (Navailles et al. 2004). In keeping with the tight relationship between nucleus accumbens (NAc) DA release and cocaine-induced behavioral responses (Dunnett & Robbins 1992), these findings led to the proposal that 5-HT2CR agonists may inhibit cocaine-induced DAdependent behaviors independently from an action on DA outflow itself, thereby controlling NAc DA transmission by acting downstream from DA neurons (Navailles et al. 2004, 2008). Combining behavioral, neurochemical and molecular approaches, the present study was therefore aimed at assessing this hypothesis by studying the ability of the 5-HT2CR agonist Ro 60-0175 (Martin et al. 1998) to modulate cocaine-induced changes of mesoaccumbens DA pathway activity at both pre- and post-synaptic levels. To this purpose, first we assessed the ability of Ro 60-0175 to inhibit cocaine-induced hyperlocomotion. Second, using in vivo microdialysis in freely moving animals to avoid possible anesthesia interference, we studied the influence of Ro 60-0175 on NAc DA outflow as an index of pre-synaptic DA neuron activity (Panin et al. 2012). Thereafter, to determine whether this interaction occurs at post-synaptic level, we studied the impact of Ro 60-0175 on the late-onset hyperlocomotion induced by the DA-D2R agonist quinpirole, whose effect, at variance with cocaine, occurs independently of changes of endogenous DA, and is related to direct stimulation of post-synaptic DA receptors (Koeltzow, Austin & Vezina 2003; Benaliouad et al. 2009). Finally, to characterize the role played by the NAc in this interaction, we evaluated possible changes of post-synaptic neuronal activity by assessing the effect of Ro 60-0175 on cocaine-induced changes of c-Fos immunoreactivity and phosphorylation states at threonine 34 and 75 residues of the DA and c-AMPregulated phosphoproteins Mr 32 kDa (DARPP-32). The specific involvement of 5-HT2CRs in the effect of Ro 60-0175 on cocaine-induced DARPP-32 phosphorylation was further studied in animals pretreated with the selective 5-HT2CR antagonist SB 242084 (Kennett et al. 1997). Specifically, in keeping with its location in dopaminoceptive neurons, DARPP-32 provides a useful tool for studying DA neurotransmission (Nishi et al. 2000) and is also known to participate in the mediation of reinforcing effects of cocaine by processes independent from changes of DA outflow (Svenningsson, Nairn & Greengard 2005). Neurochemical and molecular measurements were performed at the level of the shell and core subregions of the NAc, as these subregions are differentially involved in the behavioral effects of cocaine (Di Chiara 2002; Di Ciano 2008). © 2014 Society for the Study of Addiction

MATERIALS AND METHODS Animals Male Sprague-Dawley rats (IFFA CREDO, Lyon, France) weighing 320–350 g were used. Animals housed in individual plastic cages were kept at constant room temperature (21 ± 2°C) and relative humidity (60%) with a 12 hours of light/dark cycle (dark from 20:00 hours) and had free access to water and food. Animals were acclimated to the housing conditions for at least 1 week prior to the start of the experiments. All experiments were conducted during the light phase of the light-dark cycle, and animal use procedures conformed to International European Ethical Standards (86/609-EEC) and the French National Committee (décret 87/848) for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. Drugs The following compounds were used: cocaine hydrochloride purchased from Cooper (Melun, France), Ro 60-0175 fumarate (αS-6-chloro-5-fluoro-α-methyl-1H-indole-1ethanamine fumarate), and (–)-Quinpirole hydrochloride (trans-(–)-(4aR)-4, 4a,5,6,7,8,8a,9-octahydro-5-propyl1H-pyrazolo[3,4-g]quinoline monohydrochloride), purchased from Tocris Bioscience (Bristol, UK); SB 242084.2HCl {6-chloro-5-methyl-1-[6-(2-methylpiridin3-yloxy)pyridin-3-yl carbamoyl]indoline.dihydrochlo ride} purchased from Sigma-RBI (Saint Quentin Fallavier, France). All other chemicals and reagents were the purest commercially available (VWR, Strasbourg, France; Sigma, Illkirch, France). Pharmacological treatments Cocaine was diluted in NaCl 0.9%, and administered i.p. at 15 mg/kg. Quinpirole was diluted in NaCl 0.9%, and injected subcutaneously (s.c.) at 0.5 mg/kg. Ro 60-0175 dissolved in NaCl 0.9% was injected at 1 mg/kg, i.p., 15 minutes prior to cocaine or immediately before quinpirole administration. SB 242084 dissolved in NaCl 0.9% was injected at 0.5 mg/kg, i.p., 30 minutes prior to cocaine. All drug doses were calculated as the free base and injected in a volume of 1 ml/kg. The dose of cocaine was selected on the basis of previous studies reporting its ability to increase locomotor activity, as well as in vivo DA release and c-Fos expression in the rat NAc (Young, Porrino & Ladarola 1991; Grottick, Fletcher & Higgins 2000; Navailles et al. 2004). Dose and pretreatment administration time of Ro 60-0175 were chosen according to previous studies reporting its efficacy to modulate cocaine-induced DA-dependent behaviors including hyperlocomotion Addiction Biology

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through selective stimulation of central 5-HT2CRs (Grottick et al. 2000; De Deurwaerdère et al. 2004; Fletcher et al. 2008). Dose and pretreatment administration time of SB 242084 were chosen according to previous studies reporting its efficacy to block the effect of Ro 60-0175 on DA release (De Deurwaerdère et al. 2004; Di Matteo, Pierucci & Esposito 2004) and cocaine-induced DA-dependent behaviors (Grottick et al. 2000; Fletcher et al. 2008). The dose of quinpirole was chosen on the basis of previous studies (Koeltzow et al. 2003; Benaliouad et al. 2009) and internal laboratory data (unpublished results), showing its ability to elicit a lateonset robust hyperlocomotive response dependent on post-synaptic DA-D2R stimulation. For each experiment, a new batch of animals was used. On the test day, rats were divided into four groups according to the pharmacological treatments to be administered. In each experimental group, animals received either drugs or their appropriate vehicle according to a randomized design. Measurement of locomotor activity As described previously (Piazza et al. 1989), locomotor activity was measured in a circular corridor equipped with four photoelectric cells placed at the two perpendicular axes of the apparatus to automatically record horizontal locomotion. The apparatus was placed in a light- and sound-attenuated chamber. All rats were habituated to the test environment for 3 hours/day on each of the 3 days before the start of the experiment. In quinpirole experiments, 1 hour of habituation was additionally performed on the test day before drug administration. On the test day, rats were randomly assigned to four groups for cocaine (n = 7–8 animals/group) and quinpirole (n = 5–7 animals/group) experiments, respectively. Each group received a pretreatment with Ro 60-0175 or vehicle, and a treatment with vehicle or cocaine or quinpirole, according to the schedule reported in the ‘Pharmacological treatments’ section. Drug injections were performed outside the testing room. After the last injection, rats were placed into the circular corridor, and locomotor activity was recorded for 10-minute intervals over a period of 120 minutes (cocaine experiment) or 180 minutes (quinpirole experiment). Microdialysis and DA assay Surgery and perfusion procedures were performed as previously described (De Deurwaerdère et al. 2005) with minor modifications. Briefly, rats were anesthetized with a mixture of ketamine (100 mg/kg, i.p., Imalgéne, Merial, France) and xylazine (1 mg/kg, i.p., Rompun, Bayer, France). A siliconized stainless guide cannula © 2014 Society for the Study of Addiction

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(Carnegie Medicin, Phymep, Paris, France) was stereotaxically implanted just above the shell or the core subregions of the right NAc (coordinates of the lower extremity of the guide, in mm, relative to the interaural point: shell: anteroposterior = 10.6, lateral = 1, ventral = 4; core: anteroposterior = 10.6, lateral = 1.4, ventral = 3.8, Paxinos & Watson 1986), so that the tip of the probe (CMA/11, cuprophan, 240 μm outer diameter, 2 mm length, Carnegie Medicin, Phymep), once lowered through the guide cannula on the day of the experiment, reached a depth value of 2 mm above the interaural point. Experiments were performed in freely moving rats 5 to 7 days after surgery. During the experimental period, rats were randomly assigned to four groups (n = 4–6 animals/group). Each group received a pretreatment with Ro 60-0175 or vehicle, and a treatment with vehicle or cocaine, according to the schedule reported in the ‘Pharmacological treatments’ section. Dialysate samples (30 μl) were collected every 15 minutes and injected into a high-performance liquid chromatography apparatus with amperometric detection, as previously described (Auclair et al. 2010). Pharmacological treatments were performed 120 minutes after the beginning of the perfusion (stabilization period), and DA outflow was monitored 120 minutes after the last injection. At the end of each experiment, the animal was deeply anesthetized with a pentobarbital overdose (100 mg/kg, CEVA, Libourne, France), and its brain was removed and fixed in NaCl (0.9 %)/paraformaldehyde solution (10%). Probe location into the targeted subregions was determined histologically on serial coronal sections (60 μm) stained with cresyl violet, and only data obtained from rats with correctly implanted probes were included in the results. The DA content in each sample was expressed as the percentage of the average baseline level calculated from the three fractions preceding any treatment. The overall drug effect was calculated as the average of DA content from the eight dialysates collected after last injection. Immunohistochemistry On the test day, rats were randomly assigned to four groups (n = 5–7 animals/group). Each group received a pretreatment with Ro 60-0175 or vehicle, and a treatment with vehicle or cocaine, according to the schedule reported in the ‘Pharmacological treatments’ section. Two hours after the last vehicle or cocaine injection, animals were deeply anesthetized with a pentobarbital overdose (100 mg/kg) and transcardially perfused with a phosphate buffered solution of 4% paraformaldehyde. Brains were then cut on a vibratome and 50 μm coronal sections corresponding to the shell and core subdivisions of the NAc (Paxinos & Watson 1986, see Fig. 4a) were processed according to a standard immunohistochemical Addiction Biology

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procedure (Barrot et al. 1999). Antigenicity for c-Fos immunoreactivity was assessed with a rabbit polyclonal anti-c-Fos antibody [c-Fos (4): sc-52, Santa Cruz Biotechnology, Santa Cruz, CA, USA] at 1:5000 dilution in phosphate buffered saline containing 0.3% Triton-X100 and 1% normal goat serum. Following 72 hours of incubation at 4°C, sections were incubated for 90 minutes at 20°C with biotinylated goat antirabbit antibody (1:200 dilution, DakoCytomation, Glostrup, Denmark). C-Fos immunoreactivity was visualized by the biotinstreptavidin technique (ABC kit, Dako S.A., Trappes, France) using 3,3′-diaminobenzidine chromogen. The density of c-Fos positive cells was estimated using a 25 × microscope objective (Leitz Aristoplan, Wetzlar, Germany) on two sections per animal. For each animal, c-Fos positive nuclei were counted unilaterally (right side) using a 0.4 × 0.4 mm grid within the NAc shell and core subregions (see Fig. 4a) with the observer blinded to treatment. Laser microdissection and pressure catapulting (LMPC) and immunoblotting—combined experiments On the test day, in a first experiment, rats were randomly assigned to four groups (n = 6 animals/group), and each group received a pretreatment with Ro 60-0175 or vehicle and a treatment with vehicle or cocaine. In a second experiment (n = 5/6 animals/group, four groups), each group received a pretreatment with SB 242084 or vehicle and a treatment with vehicle or Ro 60-0175; all animals received a cocaine injection. Treatments were administered according to the schedule reported in the ‘Pharmacological treatments’ section. In both experiments, 45 minutes after the last injection, animals were killed by a pentobarbital overdose (100 mg/kg). Brains were then quickly dissected and snap-frozen in isopentane (SigmaAldrich, Saint Louis, MO, USA) and stored at −80°C before to be subjected to LMPC. Brain sections obtained from LMPC were prepared using a procedure previously described (Maitre et al. 2011). Briefly, laser-assisted microdissection of the shell and core NAc subregions was performed using a P.A.L.M. MicroBeam microdissection system (P.A.L.M. Microlaser Technologies AG, Zeiss, Germany) on 60-μm-thick cresyl violet counterstained coronal sections. Protein extracts from rat NAc shell and core were prepared using the homogenizer Precellys 24 (Bertin Technologies, Artigues prés Bordeaux, France) as previously described (Maitre et al. 2011). Protein concentration was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA,, USA). For immunoblotting analysis, microdissectionisolated proteins suspended in RIPA/Laemmli buffer were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, © 2014 Society for the Study of Addiction

MA, USA). The proteins were then revealed as previously described (Maitre et al. 2011) with affinity-purified rabbit anti-DARPP-32 antibody (#2306; 1/1500 dilution, Cell Signaling Technology, Beverly, MA, USA), anti-phosphoDARPP-32 at threonine 34 (#2304 and #5393, 1/250 dilution, CST), and threonine 75 residues (#2301; 1/250 dilution, CST), respectively. The mouse monoclonal α-tubulin antibody N356 (Amersham Biosciences, Amersham, UK) at 1/100 000 dilution was used as a loading control. X-ray films (Kodak, Rochester, NY, USA) were quantified by densitometry (optical density) using a GS-800 scanner coupled with Quantity One software (Bio-Rad, Hercules, CA, USA). Statistics Statistical analysis was carried out by Statistica 8.0 for Windows (Statsoft, Maisons-Alfort, France). The ability of Ro 60-0175 (pretreatment) to modify the effects of cocaine or quinpirole (treatment) was analyzed by a multi-factorial ANOVA with pretreatment and treatment as the between-subject factors and time as the withinsubject factor in the case of microdialysis experiments (including values from time −15 to 120 min) and quinpirole effects on locomotion. As well, the interaction between SB 242084 (pretreatment) and Ro 60-0175 (treatment) in the presence of cocaine (DARPP-32 phosphorylation) was studied by a multi-factorial ANOVA with pretreatment and treatment as the betweensubject factors, the group vehicle/vehicle/cocaine being used as control. When ANOVA results were significant (P < 0.05), the Fisher’s protected least significant difference (PLSD) test was performed to allow adequate multiple comparisons between groups. Finally, statistical differences in basal DA (microdialysis experiments), DARPP-32 and α-tubulin (immunoblotting experiments) values among groups were assessed by a one-way ANOVA using group as a main factor.

RESULTS Effect of Ro 60-0175 on cocaine-induced hyperlocomotion Figure 1 illustrates the effect of Ro 60-0175 on the increase in locomotor activity induced by cocaine. Statistical analysis revealed a significant main effect of pretreatment (FRo (1,27) = 21.44, P < 0.001) and treatment (Fcoc (1,27) = 39.94, P < 0.001), as well as a significant pretreatment × treatment interaction (FRo × coc (1,27) = 5.43, P < 0.05). Post hoc analysis revealed that, as reported previously (Fletcher, Grottick & Higgins 2002), cocaine produced a significant increase in total locomotor counts recorded over the 120-minute test session, reaching about 239% of basal activity (P < 0.001 versus the Addiction Biology

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Figure 1 Effect of Ro 60-0175 on cocaine-induced hyperlocomotion. Ro 60-0175 (Ro, 1 mg/kg, i.p.) was injected 15 minutes prior to the administration of cocaine (coc, 15 mg/kg, i.p.). Histograms represent the mean ± SEM horizontal activity counts over a 2-hour test period (n = 7–8 animals/group). ***P < 0.001 versus the vehicle/vehicle (v/v) group and +++P < 0.001 versus the vehicle/cocaine (v/coc) group (Fisher’s PLSD test)

vehicle/vehicle group). Cocaine-induced hyperlocomotion was significantly reduced by about 47% by Ro 60-0175 pretreatment (P < 0.001 versus the vehicle/ cocaine group). Finally, as reported previously (Grottick et al. 2000), administration of Ro 60-0175 alone did not significantly alter basal locomotor activity (P > 0.05 versus the vehicle/vehicle group). Effect of Ro 60-0175 on cocaine-induced increase in accumbal DA outflow Figure 2 illustrates the effect of Ro 60-0175 on cocaineinduced increase in DA outflow in the shell (upper panel) and core (lower panel) subregions of the NAc. In the NAc shell, statistical analysis revealed a significant main effect of treatment (Fcoc (1,15) = 271.42, P < 0.001) and a significant treatment by time interaction (Fcoc × time (9,135) = 53.14, P < 0.001). As expected (Navailles et al. 2004; Cadoni & Di Chiara 2007), cocaine elicited an overall significant increase (approximately 295% of baseline) in DA outflow, which reached its significance 15 minutes after injection and remained significant during the entire experimental period (P < 0.001 versus cocaine-untreated groups). There was no significant main effect of pretreatment (FRo (1,15) = 0.10, NS), no significant pretreatment × treatment interaction (FRo × coc (1,15) = 1.88, NS), and no significant time interactions (FRo × time (9,135) = 0.72, NS; FRo × coc × time (9,135) = 0.99, NS). In the NAc core, statistical analysis revealed a significant main effect of treatment (Fcoc (1,18) = 202.66, P < 0.001), pretreatment (FRo (1,18) = 13.68, P < 0.01), and a significant pretreatment × treatment interaction (FRo × coc (1,18) = 13.72, P < 0.01). Moreover, these effects were dependent on the time (Fcoc × time (9,162) = © 2014 Society for the Study of Addiction

Figure 2 Time course effect of the administration of Ro 60-0175 on cocaine-induced increase in dopamine (DA) outflow in the (a) shell and (b) core subregions of the nucleus accumbens (NAc). Ro 60-0175 (Ro, 1 mg/kg, i.p.) was injected (vertical arrow) 15 minutes before cocaine (coc, 15 mg/kg, i.p., time zero) administration. Data are presented as the mean ± SEM percentages of the baseline calculated from the three samples preceding the first drug administration (n = 4–6 animals/group). Absolute basal levels of DA in dialysate collected from the NAc shell and core subregions did not differ across the different experimental groups (shell: ANOVA F(3,15) = 2.14, NS; core: F(3,18) = 0.76, NS) and were: 10.5 ± 0.5 pg/20 μl and 8.5 ± 0.7 pg/20 μl for the shell and the core, respectively (mean ± SEM, n = 10 animals chosen randomly from the cohort of 19 rats for the shell and 22 rats for the core NAc subregions). ***P < 0.001, overall effect versus the vehicle/vehicle (v/v) group; +++P < 0.001, overall effect versus the vehicle/cocaine (v/coc) group (Fisher’s PLSD test)

43.42, P < 0.001, FRo × time (9,162) = 6.98, P < 0.001, FRo × coc × time (9,162) = 6.66, P < 0.001). Post hoc analysis revealed that, as expected (Navailles et al. 2004; Cadoni & Di Chiara 2007), cocaine elicited an overall significant increase in DA outflow, reaching approximately 336% of baseline (P < 0.001 versus the vehicle/vehicle group). The effect of cocaine reached its significance 15 minutes after injection and remained significant during the entire experimental period (at least P < 0.01 versus vehicle/ Addiction Biology

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vehicle group). Cocaine-induced DA outflow was significantly increased (about 165%) by Ro 60-0175 (P < 0.001 versus the vehicle/cocaine group), the effect reaching its significance between 30 and 75 minutes after cocaine injection (at least P < 0.05 versus the vehicle/cocaine group). Finally, Ro 60-0175 per se had no effect on basal DA outflow (P > 0.05 versus the vehicle/ vehicle group). Effect of Ro 60-0175 on quinpirole-induced locomotor activity The effect of Ro 60-0175 on quinpirole-induced locomotor activity is shown in Fig. 3. As reported previously (Basso et al. 2005; Benaliouad et al. 2009), quinpirole produced time-dependent changes of spontaneous locomotion, encompassing first a reduction, and then a mod-

erate and sustained increase in locomotor activity (Fquinp × time (17,425) = 16.46, P < 0.001 versus quinpiroleuntreated groups, Fig. 3a). The effect of Ro 60-0175 pretreatment on quinpiroleinduced locomotion was studied on total horizontal activity counts recorded from 0 to 60 minutes and from 61 to 180 minutes after the last drug injection (see Fig. 3b). During the first 60 minutes post-injection at a time when spontaneous locomotion is maximal in vehicle-treated rats, statistical analysis revealed a significant main effect of treatment (Fquinp (1,25) = 25.50, P < 0.001). There was no significant main effect of pretreatment (FRo (1,25) = 0.06, NS) as well as no significant pretreatment × treatment interaction (FRo × quinp (1,25) = 0.24, NS). Analysis performed at a later time post-injection (61–180 minutes) revealed a significant main effect of pretreatment (FRo (1,25) = 4.36, P < 0.05), treatment (Fquinp (1,25) = 26.17, P < 0.001), and a significant pretreatment × treatment interaction (FRo × quinp (1,25) = 4.88, P < 0.05). Post hoc analysis revealed that quinpirole induced a significant increase in locomotor activity reaching about 572% of basal activity (P < 0.001 versus the vehicle/vehicle group). With no effect on basal locomotion (P > 0.05 versus the vehicle/vehicle group), Ro 60-0175 significantly diminished (overall reduction of approximately 48%) quinpirole-induced locomotor activity (P < 0.01 versus the vehicle/quinpirole group). Effect of Ro 60-0175 on cocaine-induced c-Fos expression in the NAc

Figure 3 Effect of the administration of Ro 60-0175 on quinpiroleinduced changes of spontaneous locomotor activity. Ro 60-0175 (Ro, 1 mg/kg, i.p.) was injected immediately before the subcutaneous administration of 0.5 mg/kg quinpirole (Q, time zero). (a) Time course effect; data represent the mean ± SEM horizontal activity counts in each 10-minute period of the 3-hour test; (b) histograms represent mean ± SEM horizontal activity counts averaged over 0–60 minutes and 61–180 minutes monitoring (n = 5–7 animals/group). ***P < 0.001, overall effect versus the vehicle/vehicle (v/v) group; ++ P < 0.01, overall effect versus the vehicle/quinpirole (v/Q) group (Fisher’s PLSD test) © 2014 Society for the Study of Addiction

Figure 4 reports the effect of Ro 60-0175 on cocaineinduced c-Fos expression in the shell and core subregions of the NAc. In the shell, statistical analysis revealed a significant main effect of treatment (Fcoc (1,22) = 91.35, P < 0.001), but not pretreatment (FRo (1,22) = 0.06, NS), and a significant pretreatment × treatment interaction (FRo × coc (1,22) = 25.29, P < 0.001). Post hoc analysis revealed that, as reported previously (Barrot et al. 1999), cocaine induced a significant increase in c-Fos expression, reaching approximately 426% of basal values (P < 0.001 versus the vehicle/vehicle group). The effect of cocaine was significantly reduced by about 25% by Ro 60-0175 (P < 0.01 versus the vehicle/cocaine group). Finally, in agreement with previous findings (Beyeler et al. 2010), administration of Ro 60-0175 alone significantly increased basal c-Fos expression by 217% (P < 0.001 versus the vehicle/vehicle group). In the core subregion, statistical analysis revealed a significant main effect of treatment (Fcoc (1,22) = 28.73, P < 0.001), but not pretreatment (FRo (1,22) = 0.02, NS), and a significant pretreatment × treatment interaction (FRo × coc (1,22) = 16.63, P < 0.001). Post hoc analysis revealed that, as reported previously (Barrot et al. 1999), cocaine Addiction Biology

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induced a significant increase in c-Fos expression, reaching approximately 316% of basal values (P < 0.001 versus the vehicle/vehicle group). The effect of cocaine was significantly reduced by about 30% by Ro 60-0175 (P < 0.01 versus the vehicle/cocaine group). In agreement with previous findings (Beyeler et al. 2010), administration of Ro 60-0175 alone significantly increased basal c-Fos expression by 190% (P < 0.05 versus the vehicle/vehicle group). Effect of Ro 60-0175 on cocaine-induced phosphorylation of DARPP-32

Figure 4 Effect of Ro 60-0175 on cocaine-induced increase in c-Fos expression in the shell and core subregions of the nucleus accumbens (NAc). Ro 60-0175 (Ro, 1 mg/kg, i.p.) was injected 15 min before the administration of cocaine (coc, 15 mg/kg, i.p.). (a) Schematic diagram taken from the Paxinos and Watson atlas (1986) showing anatomical localization of areas (solid boxes) explored for quantification of c-Fos positive cells in the shell and core subdivisions of the NAc. aca: anterior part of the anterior commissure. (b) Representative bright-field photomicrographs illustrating the immunohistochemical staining of c-Fos expression in the shell and core subregion of the NAc following pretreatment with Ro or vehicle (v) and treatment with v or coc. (c) Histograms representing the mean ± SEM number (n) of c-Fos positive cells in a 1 mm2 area of tissue (n = 5–7 animals/group). *P < 0.05, ***P < 0.001 versus the corresponding v/v groups; ++P < 0.01 versus the corresponding v/coc groups (Fisher’s PLSD test)

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Figure 5 illustrates the effect of Ro 60-0175 on cocaineinduced phosphorylation of DARPP-32 at threonine 34 and 75 residues in the NAc shell (Fig. 5b) and core (Fig. 5c) subregions. In the shell, statistical analysis revealed no significant effect of pretreatment, treatment, and pretreatment × treatment interaction for both threonine 34 (FRo (1,20) = 0.77, NS; Fcoc (1,20) = 0.04, NS; FRo × coc (1,20) = 0.20, NS) and threonine 75 (FRo (1,20) = 0.75, NS; Fcoc (1,20) = 0.90, NS; FRo × coc (1,20) = 0.04, NS) residues. In the core subregion, statistical analysis revealed a significant effect of pretreatment (FRo (1,20) = 10.06, P < 0.01) and treatment (Fcoc (1,20) = 6.28, P < 0.05), as well as a significant pretreatment × treatment interaction (FRo × coc (1,20) = 7.24, P < 0.05) on DARPP-32 phosphorylation at threonine 34. Post hoc analysis revealed that cocaine induced a significant increase in the phosphorylation of DARPP-32 at threonine 34, reaching approximately 192% of basal values (P < 0.01 versus the vehicle/vehicle groups). Without an effect by itself (P > 0.05 versus the vehicle/vehicle group), Ro 60-0175 reduced significantly the effect of cocaine by about 55% (P < 0.001 versus the vehicle/cocaine group). In the core subregion, statistical analysis also showed a significant main effect of treatment (Fcoc (1,20) = 8.28, P < 0.01), but not pretreatment (FRo (1,20) = 4.10, NS), and a significant pretreatment × treatment interaction (FRo × coc (1,20) = 4.52, P < 0.05) on DARPP-32 phosphorylation at threonine 75. Post hoc analysis revealed that cocaine induced a significant increase in DARPP-32 phosphorylation reaching approximately 195% of basal values (P < 0.01 versus the vehicle/vehicle groups). The effect of cocaine was significantly reduced by about 44% by Ro 60-0175 (P < 0.01 versus the vehicle/cocaine group), which per se had no effect on DARPP-32 phosphorylation (P > 0.05 versus the vehicle/vehicle group). Effect of SB 242084 on Ro 60-0175 inhibition of cocaine-induced phosphorylation of DARPP-32 Figure 6 illustrates the effect of SB 242084 on the suppressant effect of Ro 60-0175 on cocaine-induced phosphorylation of DARPP-32 at threonine 34 and 75 Addiction Biology

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Figure 5 Effect of Ro 60-0175 on cocaine-induced stimulation of DARPP-32 signaling pathway in the shell and core subregions of the nucleus accumbens (NAc). Ro 60-0175 (Ro, 1 mg/kg, i.p.) was injected 15 minutes prior to cocaine (coc, 15 mg/kg, i.p.). (a) Schematic diagram taken from the Paxinos and Watson atlas (1986) showing representative pictures of the laser microdissection and pressure catapulting (LMPC) treatment of NAc. Shell and core subregions before and after LMPC, and captured zones are shown. Scale bar = 300 μm, aca: anterior part of the anterior commissure, mfba: medial forebrain bundle.Western blot analysis and densitometric quantification of (b) NAc shell and (c) NAc core expressions of P-DARPP-32Thr34, P-DARPP-32Thr75, DARPP-32 and α -tubulin proteins. Histograms represent the mean ± SEM optical density (OD) (n = 6 animals/group). DARPP-32 and α-tubulin values did not differ across the different experimental groups in both the shell (ANOVA, DARPP-32: F(3,20) = 0.63, NS; α-tubulin: F(3,20) = 0.48, NS) and the core subregion (ANOVA, DARPP-32: F(3,20) = 0.35, NS; α-tubulin: F(3,20) = 0.58, NS). **P < 0.01 versus the corresponding vehicle/vehicle (v/v) groups; ++P < 0.01, +++P < 0.001 versus the corresponding v/coc groups (Fisher’s PLSD test) © 2014 Society for the Study of Addiction

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Figure 6 Effect of SB 242084 on Ro 60-0175 inhibition of cocaine-induced stimulation of DARPP-32 signaling pathway in the core subregion of the nucleus accumbens (NAc). Rats were injected with SB 242084 (SB, 0.5 mg/kg, i.p.) or its vehicle (v) and Ro 60-0175 (Ro, 1 mg/kg, i.p.) or its v respectively 30 and 15 minutes prior to cocaine (coc, 15 mg/kg, i.p.) administration. Western blot analysis and densitometric quantification of expressions of P-DARPP-32Thr34, P-DARPP-32Thr75, DARPP-32 and α-tubulin proteins are shown. Histograms represent the mean ± SEM optical density (OD) (n = 5–6 animals/group). DARPP-32 and α-tubulin values did not differ across the different experimental groups (ANOVA, DARPP-32: F(3,18) = 0.08, NS; α-tubulin: F(3,18) = 0.46, NS). *P < 0.05 versus the vehicle/vehicle/coc (v/v/coc) group; +++P < 0.001 versus the v/Ro/coc group (Fisher’s PLSD test)

residues in the core subregion of the NAc. Statistical analysis revealed a significant main effect of pretreatment (FSB (1,18) = 13.37, P < 0.01), but not treatment (FRo (1,18) = 0.23, NS), and a significant pretreatment × treatment interaction (FSB × Ro (1,18) = 6.88, P < 0.05) on DARPP-32 phosphorylation at threonine 34. Post hoc analysis revealed that Ro 60-0175 significantly reduced cocaine-induced phosphorylation of DARPP-32 (P < 0.05 versus the vehicle/vehicle/cocaine group). Without effect by itself (P > 0.05 versus the vehicle/ vehicle/cocaine group), SB 242084 completely blocked the suppressant effect of Ro 60-0175 (P < 0.001 versus the vehicle/Ro 60-0175/cocaine group). Statistical analysis failed to reveal a significant effect of pretreatment (FRo (1,18) = 4.00, NS), treatment (Fcoc (1,18) = 3.36, NS), and pretreatment × treatment interaction (FRo × coc (1,18) = 2.47, NS) on DARPP-32 phosphorylation at threonine 75 residue, although there was a trend for SB 242084 to block the suppressant effect of Ro 60-0175 on cocaine-induced DARPP-32 phosphorylation (see Fig. 6). DISCUSSION Assessing the effect of the 5-HT2CR agonist Ro 60-0175 on cocaine-induced neurochemical, behavioral and © 2014 Society for the Study of Addiction

molecular responses, the present study provides the first evidence that 5-HT2CRs modulate mesoaccumbal DA pathway activity at post-synaptic level, specifically by controlling DARPP-32 phosphorylation within the core subregion of the NAc. In keeping with the tight relationship between NAc DA activity and cocaine-induced DA-dependent behaviors (Dunnett & Robbins 1992; Di Chiara 2002), modulation of NAc core DA transmission could participate to the inhibitory effect of Ro 60-0175 on cocaine-induced hyperlocomotion. Likewise in previous neurochemical and behavioral investigations (Filip et al. 2012), in the present study, the functional role of central 5-HT2CRs in the effects of cocaine was assessed using the brain-penetrant and potent 5-HT2CR agonist Ro 60-0175, administered at a dose (1 mg/kg) known to selectively target central 5-HT2CRs and modulate cocaine-induced behavioral effects (Grottick et al. 2000; De Deurwaerdère et al. 2004; Fletcher et al. 2008; Filip et al. 2012). Noteworthy, the fact that this dose of Ro 60-0175 has no effect on basal and cocaine-induced DA outflow in the NAc (De Deurwaerdère et al. 2004; Navailles et al. 2004) provides a useful pharmacological tool to identify possible postsynaptic effects of Ro 60–0175 occurring independently of changes of DA outflow itself. Addiction Biology

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As previously reported (Grottick et al. 2000; Cadoni & Di Chiara 2007), we found that peripheral administration of 15 mg/kg cocaine elicited a significant increase in locomotor activity and DA outflow in both the shell and core subregions of the NAc. Furthermore, Ro 60-0175 pretreatment significantly reduced cocaine-induced hyperlocomotion but had no effect on cocaine-increased DA in the shell. These findings are in agreement with previous behavioral studies (Grottick et al. 2000) and confirm microdialysis investigations in halothaneanesthetized rats (Navailles et al. 2004). A recent study in squirrel monkeys reported that Ro 60-0175 has no effect on basal DA release, but attenuates cocaine-induced DA outflow in the NAc (Manvich, Kimmel & Leonard 2012). Nevertheless, the different experimental conditions used, such as differences in doses, route administration, probe implantation, in addition to the different 5-HT2CR distribution in the rodent and monkey brain (Mengod 2011), do not allow direct comparison with the present study. At variance with the shell, we found that cocaineevoked DA outflow in the core subregion of the NAc was significantly increased by Ro 60-0175. To our knowledge, this is the first report that Ro 60-0175 modulates cocaine-induced DA outflow in this NAc subregion. Despite Ro 60-0175 is known to possess affinity for the 5-HT2BR (Porter et al. 1999), the observed effect is likely due to the selective stimulation of the 5-HT2CR over the 5-HT2BR. Indeed, 5-HT2BRs have been shown to have no effect on both basal (Auclair et al. 2010) and cocaineevoked DA outflow (unpublished results) in the core subregion of the NAc. That 5-HT2CR stimulation can exert a facilitatory control on DA outflow is not so surprising, although 5-HT2CR agents are generally known to afford an overall inhibitory influence on DA release at terminals (Filip et al. 2012; Devroye et al. 2013). Indeed, excitatory effects of 5-HT2CRs on cocaine-induced DA-dependent behaviors and NAc shell DA release have been already reported in the literature after systemic (McCreary & Cunningham 1999) or local administration of 5-HT2C compounds (McMahon, Filip & Cunningham 2001; Filip & Cunningham 2002; Navailles et al. 2008; Leggio et al. 2009; Devroye et al. 2013). Indeed, behavioral investigations have first shown that the 5-HT2CR inverse agonist SB 206553 bidirectionally affects acute cocainestimulated activity in a dose-dependent manner (McCreary & Cunningham 1999). Furthermore, microinjection studies with 5-HT2CR agonist and antagonist have shown that NAc 5-HT2CRs exert an excitatory control on both cocaine-induced behaviors (McMahon et al. 2001; Filip & Cunningham 2002) and NAc DA release (Navailles et al. 2008). Finally, local injections of 5-HT2CR agonist or antagonist into the medial prefrontal cortex (mPFC) have demonstrated that mPFC 5-HT2CRs © 2014 Society for the Study of Addiction

facilitate accumbal DA outflow (Leggio et al. 2009), but inhibit behavioral responses induced by cocaine (Filip & Cunningham 2003). On the other hand, it is worth mentioning that 5-HT2CRs located into the ventral tegmental area (VTA) have been shown to exert an inhibitory control on DA-dependent behaviors and NAc DA release evoked by cocaine (McMahon et al. 2001; Fletcher et al. 2004). By these findings, 5-HT2CR control of the mesoaccumbens DA pathway is currently considered as a composite response involving a functional balance between excitatory and inhibitory inputs to DA neurons (Filip & Cunningham 2002, 2003; Navailles et al. 2006, 2008; Leggio et al. 2009) related to different receptor populations located within multiple brain areas (Clemett et al. 2000). Thus, the facilitatory effect of Ro 60-0175 on cocaine-induced DA in the NAc core reported in the present study could result from the stimulation of 5-HT2CRs locally expressed within this subregion, but further studies are needed to address this issue. Whatever the mechanism underlying the positive influence of Ro 60-0175 on cocaine-induced DA outflow, it is unlikely that this effect may account for its motor suppressant effects. Indeed, given the positive relationship between NAc DA release and locomotion (Dunnett & Robbins 1992), 5-HT2CR agonist-induced inhibition of cocaineevoked hyperlocomotion should have been paralleled by a reduction in accumbal DA outflow. Hence, our results suggest that, as already shown for the 5-HT1AR (Müller et al. 2007), 5-HT2CR stimulation could control cocaine hyperactivity by a mechanism which bypasses DA release itself. To assess a possible post-synaptic interaction, we studied the effect of Ro 60-0175 on quinpirole-induced changes of spontaneous locomotion. Noteworthy, quinpirole produces time-dependent effects on locomotion, encompassing first a reduction, and then a moderate and sustained increase in locomotor activity (Koeltzow et al. 2003; Basso et al. 2005). These opposite effects have been related to a direct stimulation of preand post-synaptic DA receptors, respectively (Basso et al. 2005; Benaliouad et al. 2009). Interestingly, we found that Ro 60-0175 did not modify the pre-synaptic action of quinpirole, but blocked significantly its late activating effect. Our findings altogether indicate that 5-HT2CR control of stimulated locomotor activity could involve an action downstream to DA neurons likely by controlling DA transmission in the NAc. In support to this proposal, we found that cocaine-induced increase in c-Fos immunoreactivity, a post-synaptic marker of neuronal activity (Barrot et al. 1999), was significantly reduced by Ro 60-0175 in both the shell and core subregions of the NAc. Furthermore, we found that Ro 60-0175 was able to inhibit cocaine-induced phosphorylation of DARPP-32 Addiction Biology

5-HT2CRs and cocaine

at threonine residues in the NAc core subregion. Furthermore, the suppressant effect of Ro 60-0175 was reversed by the selective 5-HT2CR antagonist SB 242084. Demonstrating that the observed effect of Ro 60-0175 is mediated by the 5-HT2CR, this result confirms and extends previous studies in various experimental conditions and/or models showing that Ro 60-0175 behaves as a 5-HT2CR agonist in vivo (Grottick et al. 2000; De Deurwaerdère et al. 2004; Di Matteo et al. 2004; Zaniewska et al. 2007; Fletcher et al. 2008; Quérée, Peters & Sharp 2009; Zaniewska, McCreary & Filip 2009). In keeping with its location in dopaminoceptive cells, DARPP-32 is an essential factor for DA neurotransmission (Svenningsson et al. 2002) and the NAc core is involved in the modulation of DA-mediated locomotion (Rouillon, Abraini & David 2008). It is therefore tempting to suggest that inhibition of cocaineinduced DARPP-32 phosphorylation could participate in the motor suppressant effects of Ro 60-0175. Thus, the facilitatory effect of Ro 60-0175 on cocaine-induced core DA outflow could reflect a compensatory effect resulting from Ro 60-0175 blockade of DA transmission. As in the case of neuroleptic-induced blockade of DA transmission, indirect blockade of DA transmission by Ro 60-0175 could trigger a long-loop GABAergic feedback to the VTA resulting in a disinhibition of DA neurons and increased DA release in the NAc core (Di Chiara & Imperato 1988). In addition to the NAc core, other brain regions such as the striatum, the VTA and the mPFC are known to participate in the control of cocaine-induced hyperactivity (McMahon et al. 2001; Filip & Cunningham 2003; Fletcher et al. 2004; Burton et al. 2013; Devroye et al. 2013). Specifically, intra-mPFC injection of a 5-HT2CR agonist has been shown to decrease cocaine-induced hyperlocomotion independently of changes of accumbal DA release (Filip & Cunningham 2003; Leggio et al. 2009). Furthermore, we found that Ro 60-0175 was able to reduce cocaine-induced c-Fos immunoreactivity in the mPFC (unpublished results). Hence, it is possible that Ro 60-0175 inhibition of cocaine-induced DARPP-32 phosphorylation in the core may involve indirect regulations through polysynaptic corticosubcortical pathways afferent to the NAc (Leggio et al. 2009). Additional studies are needed to unravel the mechanisms and circuits underlying this interaction. Noteworthy, this is the first report demonstrating that 5-HT2CRs modulate DARPP-32 phosphorylation in the NAc, thereby extending previous findings showing that 5-HT2 receptors are capable of controlling DARPP-32 signaling in the mouse striatum (Svenningsson et al. 2002). Furthermore, to our knowledge, none of the previous studies assessing cocaine-induced DARPP-32 © 2014 Society for the Study of Addiction

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phosphorylation in mice or rats investigated possible effects within the NAc subregions (Svenningsson et al. 2005; Chen, Chen & Chiang 2009). At variance with the core, we found that cocaine and/or Ro 60-0175 administration had no effect on DARPP-32 phosphorylation in the shell. Showing that NAc subregions respond in a different manner to pharmacological stimuli (Auclair et al. 2010; present study), this result is in line with previous studies reporting a differential effect of nicotine or food consumption on DARPP-32 phosphorylation in the shell and core subregions of the rat NAc (Abdolahi et al. 2010; Danielli et al. 2010). Interestingly, these latter studies also indicate that regulation of DARPP-32 phosphorylation at threonine 34 and 75 residues occurs in a time-dependent manner. Thus, further investigations are needed to explore a possible time-dependent effect of cocaine and/or Ro 60-0175 on DARPP-32 phosphorylation, thereby establishing the role of the shell subregion in this interaction. Nonetheless, it is noteworthy that the dichotomous effects of Ro 60-0175 on c-Fos and DARPP-32 phosphorylation in the NAc shell suggest that other neurotransmitter systems could also participate in this interaction. From a functional point of view, this issue deserves interest given that DA activity in the shell subregion of the NAc is thought to play a key role in mediating the behavioral responses of drugs of abuse (Di Chiara & Bassareo 2007; Pierce & Vanderschuren 2010), and that Ro 60-0175 is known to inhibit cocaineinduced DA behaviors such as discriminative stimulus and reinforcing properties (Filip et al. 2012). Nonetheless, it is important to remind that the ability of Ro 60-0175 to modulate these behavioral responses could also involve its action on DA transmission at the level of the core, given that this NAc subregion is known to play an important role in the motivation in drug seeking and the incentive value to drugs of abuse (Di Chiara 2002). In conclusion, our study provides the first evidence that 5-HT2CRs are capable of controlling cocaine-induced changes of DA transmission specifically in the core subregion of the NAc, independently of pre-synaptic DA release. In keeping with the tight relationship between locomotor activity and NAc DA function (Dunnett & Robbins 1992; Di Chiara 2002), this regulatory control could participate in the motor suppressant effects of Ro 60-0175. On their whole, the obtained results afford additional knowledge into the prominent role of the 5-HT2CR into the regulatory neurochemistry of mesoaccumbens DA functions, and provide new information allowing a better understanding of the mechanisms underlying the 5-HT2CR-dependent control of cocaineinduced responses (Filip et al. 2012; Devroye et al. 2013). Addiction Biology

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Acknowledgements This work was supported by grants from the Institut National de la Recherche et de la Santé (INSERM) and Bordeaux 2 University. The authors wish to thank Dr G.M. Leggio for assistance in behavioral experiments and Mrs M. Neny, A. Le Roux and V. Roullot-Lacarrière for technical assistance. Conflict of Interest None. Authors Contribution AC and US were responsible for the study concept and design. AC, CD, MM, DNA and JMR contributed to the acquisition of animal data. AC and CD performed the microdialysis and behavioral experiments. AC, MM, DNA and JMR performed the molecular experiments. AC, CD and US assisted with data analysis and interpretation of findings, and drafted the manuscript. AC, CD, US, JMR, DNA and PVP provided critical revision of the manuscript for important intellectual content. All authors critically reviewed content and approved final version for publication. References Abdolahi A, Acosta G, Breslin FJ, Hemby SE, Lynch WJ (2010) Incubation of nicotine seeking is associated with enhanced protein kinase A-regulated signaling of dopamine- and cAMP-regulated phosphoprotein of 32 kDa in the insular cortex. Eur J Neurosci 31:733–741. Auclair AL, Cathala A, Sarrazin F, Depoortère R, Piazza PV, Newman-Tancredi A, Spampinato U (2010) The central serotonin 2B receptor: a new pharmacological target to modulate the mesoaccumbens dopaminergic pathway activity. J Neurochem 114:1323–1332. Barrot M, Marinelli M, Abrous DN, Rougé-Pont F, Le Moal M, Piazza PV (1999) Functional heterogeneity in dopamine release and in the expression of Fos-like proteins within the rat striatal complex. Eur J Neurosci 11:1155–1566. Basso AM, Gallagher KB, Bratcher NA, Brioni JD, Moreland RB, Hsieh GC, Drescher K, Fox GB, Decker MW, Rueter LE (2005) Antidepressant-like effect of D(2/3) receptor-, but not D(4) receptor-activation in the rat forced swim test. Neuropsychopharmacology 30:1257–1268. Benaliouad F, Kapur S, Natesan S, Rompré PP (2009) Effects of the dopamine stabilizer, OSU-6162, on brain stimulation reward and on quinpirole-induced changes in reward and locomotion. Eur Neuropsychopharmacol 19:416–430. Beyeler A, Kadiri N, Navailles S, Boujema MB, Gonon F, Moine CL, Gross C, De Deurwaerdère P (2010) Stimulation of serotonin2C receptors elicits abnormal oral movements by acting on pathways other than the sensorimotor one in the rat basal ganglia. Neuroscience 169:158–170. Burton CL, Rizos Z, Diwan M, Nobrega JN, Fletcher PJ (2013) Antagonizing 5-HT2A receptors with M100907 and stimulating 5-HT2C receptors with Ro60-0175 blocks cocaine© 2014 Society for the Study of Addiction

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