Original Article

Neuropsychopharmacology (2004) 29, 319–326, advance online publication, 15 October 2003; doi:10.1038/sj.npp.1300329

In Vivo Evidence that 5-HT2C Receptor Antagonist but not Agonist Modulates Cocaine-Induced Dopamine Outflow in the Rat Nucleus Accumbens and Striatum

Sylvia Navailles1, Philippe De Deurwaerdère1, Grégory Porras1 and Umberto Spampinato1

1UMR CNRS, 5541-Université Victor Segalen Bordeaux 2, Bordeaux Cedex, France

Correspondence: Professor U Spampinato, UMR CNRS, 5541-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, Boîte Postale 31, 33076 Bordeaux Cedex, France. Tel: +33 557 57 15 44; Fax: +33 556 90 02 78; E-mail: Umberto.Spampinato@lnpb.u-bordeaux2.fr

Received 29 April 2003; Revised 10 July 2003; Accepted 10 September 2003; Published online 15 October 2003.

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Abstract

During recent years, much attention has been devoted at investigating the modulatory role of central 5-HT2C receptors on dopamine (DA) neuron activity, and it has been proposed that these receptors modulate selectively DA exocytosis associated with increased firing of DA neurons. In the present study, using in vivo microdialysis in the nucleus accumbens (NAc) and the striatum of halothane-anesthetized rats, we addressed this hypothesis by assessing the ability of 5-HT2C agents to modulate the increase in DA outflow induced by haloperidol and cocaine, of which the effects on DA outflow are associated or not with an increase in DA neuron firing, respectively. The intraperitoneal administration of cocaine (10–30 mg/kg) induced a dose-dependent increase in DA extracellular levels in the NAc and the striatum. The effect of 15 mg/kg cocaine was potentiated by the mixed 5-HT2C/2B antagonist SB 206553 (5 mg/kg i.p.) and the selective 5-HT2C antagonist SB 242084 (1 mg/kg i.p.) in both brain regions. The mixed 5-HT2C/2B agonist, Ro 60-0175 (1 mg/kg i.p.), failed to affect cocaine-induced DA outflow, but reduced significantly the increase in DA outflow induced by the subcutaneous administration of 0.1 mg/kg haloperidol. The obtained results provide evidence that 5-HT2C receptors exert similar effects in both the NAc and the striatum, and they modulate DA exocytosis also when its increase occurs independently from an increase in DA neuron impulse activity. Furthermore, they show that 5-HT2C agonists, at variance with 5-HT2C antagonists, exert a preferential control on the impulse-stimulated release of DA.

Keywords:

In vivo dopamine release, cocaine, haloperidol, striatum, accumbens, 5-HT2C receptors

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INTRODUCTION

During recent years, a growing body of evidence has highlighted the potential of central serotonin2C (5-HT2C) receptors for improved treatment of neuropsychiatric disorders related to dopamine (DA) neuron dysfunctions, such as Parkinson's disease, schizophrenia, or drug addiction (Wood et al, 2001; Jones and Blackburn 2002). In this context, much attention has been devoted at investigating their modulatory role on DA neuron activity. However, even though compelling evidences indicate that 5-HT2C receptors do affect basal DA neuron activity, their influence on DA function in case of altered DA transmission remains unclear.

5-HT2C receptors are expressed along the mesocorticolimbic and nigrostriatal DA pathways (Pazos et al, 1985; Eberle-Wang et al, 1997), and have been shown to exert phasic and tonic inhibitory controls on both basal DA neuronal firing and basal DA release in the nucleus accumbens (NAc), the striatum and the frontal cortex (Di Giovanni et al, 1999; Gobert et al, 2000). 5-HT2C receptors have been also shown to modulate stimulated DA release although in restricted conditions. Specifically, 5-HT2C antagonists potentiate the increase in DA release induced by drugs stimulating DA neuron firing rate, such as haloperidol, morphine, or phencyclidine (Hutson et al, 2000; Lucas et al, 2000; Porras et al, 2002b), but not that induced by drugs eliciting an impulse-independent outflow of DA such as amphetamine (Porras et al, 2002b). Furthermore, it has been shown that the nonselective 5-HT2C agonist DOI does not affect cocaine-induced increase in accumbal DA outflow (Willins and Meltzer, 1998). Considering that, at variance with morphine or haloperidol, DA exocytosis induced by cocaine is not triggered by an increase in DA neuron firing (Pitts and Marwah, 1988; White, 1990; Benwell et al, 1993), it has been proposed that 5-HT2C receptors selectively modulate the impulse-dependent release of DA in the NAc and the striatum (Lucas et al, 2000; Porras et al, 2002b), but only when DA release is associated with increased firing of DA neurons (Willins and Meltzer, 1998).

This picture however becomes less clear when looking at recent data reporting the effect of 5-HT2C agents on behavioral and neurochemical DA responses elicited by cocaine. Indeed, it has been shown that locomotor stimulant and reinforcing properties of cocaine, two behavioral responses classically related to increased mesolimbic DA transmission, are sensitive to both 5-HT2C agonists and antagonists (McCreary and Cunningham, 1999; Grottick et al, 2000; Fletcher et al, 2002). In addition, it has been reported that 5-HT2C receptor knockout mice exhibit an enhanced locomotor response to cocaine, an effect associated with an increase in DA outflow in the NAc, but not in the striatum (Rocha et al, 2002). In the absence of neurochemical studies assessing the effect of selective 5-HT2C ligands on cocaine-induced DA outflow, the data reported above challenge the hypothesis that 5-HT2C receptors regulate selectively DA exocytosis associated with increased DA neuron firing. Furthermore, in contrast with previous data (De Deurwaerdère and Spampinato, 2001; Porras et al, 2002b), the study by Rocha et al (2002) raises the possibility that 5-HT2C receptors may exert a selective control of mesoaccumbens DA pathway activity. This point deserves attention in light of the therapeutic potential of 5-HT2C agents in drug addiction (Grottick et al, 2000). Indeed, an independent modulation of the mesoaccumbens DA pathway would avoid the emergence of extrapyramidal side effects related to altered nigrostriatal DA transmission (De Deurwaerdère et al, 1998; Wood et al, 2001).

Thus, the present study was performed to examine the ability of systemically injected selective 5-HT2C receptor agonist and antagonists to modulate the increase in DA extracellular levels induced by cocaine, and to determine the extent to which this control operates selectively in the NAc with respect to the striatum. To have a further insight into the role of DA neuron firing activity, the influence of 5-HT2C agonism was also studied on the impulse-stimulated release of DA induced by haloperidol. Experiments were performed using in vivo microdialysis in halothane-anesthetized rats, an experimental procedure permitting simultaneous monitoring of DA outflow in the ipsilateral NAc and striatum (De Deurwaerdère et al, 1998).

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MATERIALS AND METHODS

Animals

Male Sprague–Dawley rats (IFFA CREDO, Lyon, France) weighing 330–380 g were used. Animals were kept at a constant room temperature (21plusminus2°C) and relative humidity (60%) with a 12 light/dark cycle (dark from 8 p.m.) and had free access to water and food. All animals 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: Ro 60-0175.HCl (S-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine hydrochloride) kindly donated by Dr P Weber (F Hoffmann-La Roche, Basel, Switzerland); SB 206553 (5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f] indole), and SB 242084 (6-chloro-5-methyl-1-[6-(2-methylpiridin-3-yloxy)pyridin-3-yl carbamoyl] indoline) generously provided by Dr M Wood (Psychiatry CEDD, GlaxoSmithKline, Harlow, UK); cocaine hydrochloride (Calaire Chimie, Calais, France); haloperidol (4-[4-(p-chlorophenyl)-4-hydroxypiperidino]-4'-fluorobutyrophenone) as the commercially available solution (Haldol 5 mg/ml, Janssen Pharmaceutica, Beerse, Belgium). All others chemicals and reagents were the purest commercially available (VWR, Strasbourg, France; Sigma, Illkirch, France).

Microdialysis

Surgery and perfusion procedures were performed as previously described (Porras et al, 2002b), with minor modifications. Briefly, rats were anesthetized with a mixture of halothane and nitrous oxide-oxygen (2%; 2 : 1 v/v). After tracheotomy for artificial ventilation, the animals were placed in a stereotaxic frame, and their rectal temperature was monitored and maintained at 37.3°Cplusminus0.1 with a heating pad. Two microdialysis probes, 2 and 4 mm long, (CMA/11, 240 mum outer diameter, Cuprophan; Carnegie Medicin, Phymep, Paris, France) were implanted simultaneously using a dual probe holder (Carnegie Medicin, Phymep) in the right NAc and striatum (coordinates from interaural point: anteroposterior [AP]=11, lateral [L]=1.3, ventral [V]=2 and AP=9.8, L=3.3, V=2.7, respectively) according to the atlas of Paxinos and Watson (1986). Probes were perfused at a constant flow rate of 2 mul/min by means of a microperfusion pump (CMA 111, Carnegie Medicin, Phymep) with artificial cerebrospinal fluid (aCSF) containing (in mM): 154.1 Cl-, 147 Na+, 2.7 K+, 1 Mg2+, and 1.2 Ca2+, adjusted to pH 7.4 with 2 mM sodium phosphate buffer. Dialysates (30 mul) were collected on ice every 15 min. The in vitro recovery of the probes was about 10% for DA. At the end of each experiment, the brain was removed and fixed in NaCl (0.9%)/paraformaldehyde solution (10%). The location of the probes was determined histologically on serial coronal sections (60 mum) stained with cresyl violet, and only data obtained from rats with correctly implanted probes were included in the results.

Chromatographic Analysis

Dialysate samples were immediately analyzed by reverse-phase high-performance liquid chromatography (HPLC) coupled with electrochemical detection, as previously described (Bonhomme et al, 1995). The mobile phase (containing (in mM) 70 NaH2PO4, 0.1 Na2EDTA, 0.7 triethylamine, and 0.1 octylsulfonic acid plus 10% methanol, adjusted to pH 4.8 with orthophosphoric acid) was delivered at 1 ml/min flow rate (system LC-10AD-VP, Shimadzu, France) through a Hypersyl column (C18; 4.6 times 150 mm, particle size 5 mum; Touzard & Matignon, Paris, France). Detection of DA was carried out with a coulometric detector (Coulochem II, ESA, Paris, France) coupled to a dual-electrode analytical cell (model 5014, ESA). The potential of the electrodes was set at -175 and +175 mV. Output signals were recorded on a computer (system class VP-4, Shimadzu, France). Under these conditions, the sensitivity for DA was 0.5 rhog/30 mul with a signal/noise ratio of 3 : 1.

Pharmacological Treatments

Pharmacological treatments were performed after the stabilization of DA levels in the perfusate. A stable baseline, defined as three consecutive samples in which DA contents varied by less than 10% in both structures, was generally obtained 135 min after the beginning of the perfusion (stabilization period).

Cocaine, diluted in NaCl 0.9%, was administered intraperitoneally at 10, 15, or 30 mg/kg in a volume of 1 ml/kg. Each 5-HT2C compound was injected intraperitoneally in a volume of 2 ml/kg, 15 min before cocaine. The 5-HT2C/2B receptor agonist Ro 60-0175 was dissolved in a physiological saline (NaCl 0.9%) and administered at 1 mg/kg. The selective 5-HT2C receptor antagonist SB 242084, dissolved in a mixture of physiological saline (NaCl 0.9%) containing hydroxypropyl-beta-cyclodextrin (8% by weight) plus citric acid (25 mM), was administered at 1 mg/kg. The 5-HT2C/2B receptor antagonist SB 206553, diluted in a 99 : 1 v/v mixture of apyrogenic water and lactic acid, was administered at 5 mg/kg. In another set of experiments, Ro 60-0175 was injected 15 min before the DA receptor antagonist haloperidol, administered subcutaneously at 0.01 and 0.1 mg/kg in a volume of 1 ml/kg.

The doses of the different compounds used were chosen on the basis of previous studies to keep both selectivity and efficiency toward the targeted sites (Kennett et al, 1996, 1997; Gobert et al, 2000; Grottick et al, 2000; Lucas et al, 2000; Porras et al, 2002a, 2002b). All drug doses were calculated as the free base. In each experimental group, animals received either drugs or their appropriate vehicle.

Statistical Analysis

DA contents in each sample were expressed as the percentage of the average baseline level calculated from the three fractions preceding any treatment. Data correspond to the meanplusminusSEM values of the percentage obtained in each experimental group. Drug overall effect was calculated as the average of DA content from dialysates collected after drug treatment administration (time 15–180 and 15–120 for cocaine and haloperidol experiments, respectively).

A one-way ANOVA (using group as the main factor) was used to determine for each experiment statistical differences between groups with time as repeated measures for the samples corresponding to the treatment effect. When significant (p<0.05), the ANOVA was followed by the Fisher's protected least-significance difference test (PLSD) to allow adequate multiple comparisons between groups. For each experiment, statistical differences in baseline DA levels among groups were assessed by a one-way ANOVA (using group as the main factor).

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RESULTS

Basal Extracellular DA Concentrations in Dialysates from the Nac and the Striatum

All measurements were performed 150 min after the beginning of perfusion, by which time a steady state was achieved. Absolute basal levels of DA in dialysate simultaneously collected from the NAc and the striatum did not differ across the different experimental groups (see legend to figures for statistics) throughout the course of the study and were (meanplusminusSEM, without adjusting for probe recovery) 7.1plusminus1.1 pg/30 mul and 17.2plusminus1.6 pg/30 mul for the NAc and the striatum, respectively (n=129 animals).

Dose–Response Effect of Cocaine on DA Extracellular Levels

The effect of the intraperitoneal administration of 10, 15, and 30 mg/kg of cocaine on DA extracellular levels in the NAc and the striatum is shown in Figure 1. Cocaine induced a dose-dependent increase in DA outflow in both the NAc (one-way ANOVA F3,22=71, p<0.001) and the striatum (one-way ANOVA F3,22=46, p<0.001). The overall effect elicited by 10, 15, and 30 mg/kg of cocaine reached respectively, 189plusminus19%, 448plusminus46%, and 1290plusminus135% of baseline in the NAc and 160plusminus7%, 304plusminus36%, and 793plusminus93% of baseline in the striatum. In the context of our statistical analysis, only the effect elicited by the higher doses of cocaine reached statistical significance (p<0.001, Fisher's PLSD test for 15 and 30 mg/kg in both brain regions).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Time-course effect of cocaine (10, 15, and 30 mg/kg i.p.) on DA extracellular levels in the NAc and the striatum. Cocaine was injected at time zero. Data, obtained from four to eight animals per group, are presented as the meanplusminusSEM percentages of the baseline calculated from three samples preceding the injection of cocaine. Absolute basal levels of DA did not differ among groups in the NAc (one-way ANOVA F3,22=1.7, p>0.05, NS) and the striatum (one-way ANOVA F3,22=1.2, p>0.05, NS). ***p<0.001 vs the vehicle+vehicle group (Fisher's PLSD test).

Full figure and legend (73K)

Effect of SB 206553 and SB 242084 on Cocaine-Induced Increase in DA Extracellular Levels

Figure 2 illustrates the effect of the 5-HT2C/2B antagonist SB 206553 (5 mg/kg i.p.; upper panels) and the selective 5-HT2C antagonist SB 242084 (1 mg/kg i.p.; lower panels) on the increase in DA extracellular levels induced by 15 mg/kg of cocaine in the NAc and the striatum. The increase in accumbal and striatal DA outflow induced by cocaine (p<0.001, Fisher's PLSD test after a one-way ANOVA; F3,23=37.5, p<0.001 for the NAc; and F3,23=30.9, p<0.001 for the striatum) was significantly potentiated by SB 206553 (p<0.05 and <0.001 vs cocaine-treated rats, Fisher's PLSD test, for the NAc and the striatum respectively), and reached its maximum 30 min after cocaine administration (Figure 2, upper panels).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Time-course effect of the 5-HT2C/2B antagonist SB 206553 (5 mg/kg i.p.; upper panels) and the selective 5-HT2C antagonist SB 242084 (1 mg/kg i.p.; lower panels) on basal and cocaine-induced increase in DA extracellular levels in the NAc and the striatum. The 5-HT2C compounds were injected (vertical arrows) 15 min before the intraperitoneal administration of 15 mg/kg cocaine (time zero). Data, obtained from six to eight animals per group, are presented as the meanplusminusSEM percentages of the baseline calculated from the three samples preceding the first drug administration. Absolute basal levels of DA did not differ among groups in the NAc (one-way ANOVA F3,23=1.2, p>0.05, NS; F3,23=2.4, p>0.05, NS, for SB206553/cocaine and SB242084/cocaine experiments, respectively) and the striatum (one-way ANOVA F3,23=0.5, p>0.05, NS; F3,23=1.6, p>0.05, NS for SB206553/cocaine and SB242084/cocaine experiments, respectively). ***p<0.001 vs the vehicle+vehicle group; +p<0.05, ++p<0.01, +++p<0.001 vs the vehicle+cocaine group (Fisher's PLSD test).

Full figure and legend (124K)

Similarly, the increase in DA outflow induced by 10 mg/kg of cocaine (p<0.001, Fisher's PLSD test after a one-way ANOVA; F3,16=142, p<0.001 for the NAc; and F3,16=197, p<0.001 for the striatum) was significantly potentiated by SB 206553 (p<0.001 vs cocaine-treated rats, Fisher's PLSD test, for both the NAc and the striatum). Indeed, in the presence of SB 206553, DA extracellular levels stimulated by cocaine reached an overall increase of 364plusminus38% and of 276plusminus17% in the NAc and the striatum respectively. In both regions, the effect peaked 30 min after cocaine administration (937plusminus240 and 954plusminus174% of basal values for the NAc and the striatum respectively), and thereafter decreased progressively to about 286% (NAc) and 200% (striatum) of baseline at the end of the experiment (data not shown).

According to previous reports (Di Giovanni et al, 1999; Gobert et al, 2000), SB 206553 elicited a rapid increase in DA efflux per se in both brain regions, reaching approximately 45% above basal values 30–45 min after its injection. However, this effect, because of its small magnitude, did not reach statistical significance in the context of our statistical analysis (Fisher's PLSD test).

As shown in the lower panels of Figure 2, the enhancement of DA extracellular levels induced by 15 mg/kg of cocaine (p<0.001, Fisher's PLSD test after one-way ANOVA; F3,23=23.1, p<0.001 for the NAc; and F3,23=22.9, p<0.001 for the striatum) was significantly increased by SB 242084 pretreatment (p<0.01 vs cocaine-treated rats, Fisher's PLSD test, in both the NAc and the striatum). The time-course effect of this interaction was slightly different in both brain regions, the potentiation occurring later in the striatum.

Finally, in both the NAc and the striatum, SB 242084 elicited a progressive and long-lasting increase in DA efflux per se that reached almost 30% above control values at the end of the experimental period. However, as in the case of SB 206553, this effect did not reach statistical significance in the context of our statistical analysis (Fisher's PLSD test).

Effect of Ro 60-0175 on Cocaine-Induced Increase in DA Extracellular Levels

Figure 3 reports the effect of the 5-HT2C/2B agonist Ro 60-0175 (1 mg/kg i.p.) on the enhancement of DA extracellular levels induced by 15 mg/kg of cocaine in the NAc and the striatum. Cocaine-stimulated DA outflow (p<0.001, Fisher's PLSD test after one-way ANOVA; F3,25=17.2, p<0.001 for the NAc; and F3,25=12.7, p<0.001 for the striatum) was not affected by Ro 60-0175 in both brain regions (NS, Fisher's PLSD test). Moreover, Ro 60-0175 by itself did not modify basal DA efflux in both the NAc and the striatum (NS, Fisher's PLSD test).

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Time-course effect of the 5-HT2C/2B agonist Ro 60-0175 (1 mg/kg i.p.) on basal and cocaine-induced increase in DA extracellular levels in the NAc and the striatum. Ro 60-0175 was injected (vertical arrow) 15 min before the intraperitoneal administration of 15 mg/kg cocaine (time zero). Data, obtained from six to eight animals per group, are presented as the meanplusminusSEM percentages of the baseline calculated from the three samples preceding the first drug administration. Absolute basal levels of DA did not differ among groups in the NAc (one-way ANOVA F3,25=2.8, p>0.05, NS) and the striatum (one-way ANOVA F3,25=0.7, p>0.05, NS). ***p<0.001 vs the vehicle+vehicle group (Fisher's PLSD test).

Full figure and legend (74K)

Effect of Ro 60-0175 on Haloperidol-Induced Increase in DA Extracellular Levels

Figure 4 reports the effect of the 5-HT2C/2B agonist Ro 60-0175 (1 mg/kg i.p.) on the increase in DA outflow elicited by the subcutaneous administration of 0.1 mg/kg haloperidol. In the NAc, DA extracellular levels enhanced by haloperidol reached a plateau (199plusminus14% of basal values) 75 min after its injection (p<0.001, Fisher's PLSD test after a one-way ANOVA, F3,16=27.1, p<0.001) whereas, in the striatum, they increased progressively to reach 203plusminus5% of basal values at the end of the experimental period (p<0.001, Fisher's PLSD test after a one-way ANOVA, F3,16=227, p<0.001). The administration of Ro 60-0175 reduced the enhancement of DA outflow induced by haloperidol in the NAc and the striatum (p<0.01 vs haloperidol-treated rats, Fisher's PLSD test). At variance with the NAc, the inhibitory effect of Ro 60-0175 in the striatum appeared during the last 45 min of the experimental period.

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Time-course effect of the 5-HT2C/2B agonist Ro 60-0175 (1 mg/kg i.p.) on basal and haloperidol-induced increase in DA extracellular levels in the NAc and the striatum. Ro 60-0175 was injected (vertical arrow) 15 min before the intraperitoneal administration of 0.1 mg/kg haloperidol (time zero). Data, obtained from five to seven animals per group, are presented as the meanplusminusSEM percentages of the baseline calculated from the three samples preceding the first drug administration. Absolute basal levels of DA did not differ among groups in the NAc (one-way ANOVA F3,16=2.3, p>0.05, NS) and the striatum (one-way ANOVA F3,16=0.6, p>0.05, NS). ***p<0.001 vs the vehicle+vehicle group; ++p<0.01 vs the vehicle+haloperidol group (Fisher's PLSD test).

Full figure and legend (74K)

In another set of experiments, we have studied the effect of the same regimen of Ro 60-0175 on DA outflow stimulated by the subcutaneous administration of 0.01 mg/kg haloperidol. DA dialysate content reached its maximum 30 and 60 min after haloperidol injection in the NAc (135plusminus15%, p<0.001, Fisher's PLSD test after a one-way ANOVA, F3,19=11.6, p<0.001) and the striatum (160plusminus5%, p<0.001, Fisher's PLSD test after a one-way ANOVA, F3,19=16.4, p<0.001), respectively (data not shown). Ro 60-0175 did not affect haloperidol-stimulated DA outflow in both brain regions (NS, Fisher's PLSD). Finally, in each experiment, Ro 60-0175 failed to affect basal DA outflow per se in the NAc and the striatum (NS, Fisher's PLSD test).

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DISCUSSION

In this study, we provide evidence that 5-HT2C antagonists potentiate the enhancement of DA outflow induced by cocaine in the rat NAc and striatum, and that 5-HT2C agonist, while ineffective on cocaine-induced DA outflow, reduces haloperidol-stimulated DA release in both brain regions. When considering the cellular mechanisms underlying the increase in DA outflow induced by cocaine and haloperidol, these results show that 5-HT2C receptors are able to modulate DA exocytosis also when it is not triggered by an increase in DA neuron impulse activity. Furthermore, they indicate that 5-HT2C agonists, at variance with 5-HT2C antagonists, exert a preferential inhibitory control on impulse-stimulated DA release.

As previously reported (Di Chiara and Imperato, 1988; Porras et al, 2002a), cocaine elicits a significant and dose-dependent increase in DA extracellular levels in both the NAc and the striatum. The systemic administration of the 5-HT2B/2C antagonist SB 206553 potentiates the increase in DA outflow induced by cocaine (10 and 15 mg/kg) in both brain regions. As discussed elsewhere (Di Giovanni et al, 1999; Porras et al, 2002b), it is unlikely that the 5-HT2B-antagonist component of SB 206553 participates in the observed effects. Indeed, we have shown that the effect of cocaine on accumbal and striatal DA outflow is also potentiated by the selective 5-HT2C antagonist, SB 242084. However, the time course of the effects elicited by SB 206553 and SB 242084 on both basal and cocaine-stimulated DA outflow is different, the effect of SB 242084 being delayed. Distinct responses to these 5-HT2C antagonists have been already reported (De Deurwaerdère and Spampinato, 2001), and attributed to both pharmacokinetic and pharmacodynamic interactions (Gobert et al, 2000). Thus, our findings, in line with a recent study reporting a facilitation of cocaine-induced DA outflow in the NAc of mice lacking 5-HT2C receptors (Rocha et al, 2002), indicate that endogenous 5-HT inhibits cocaine-stimulated DA outflow via 5-HT2C receptor stimulation.

The finding that 5-HT2C antagonists modulate cocaine-induced increase in DA outflow dampens the proposal that 5-HT2C receptors affect selectively DA exocytosis originating from an increase in the impulse flow of DA neurons (Willins and Meltzer, 1998; Porras et al, 2002b). Indeed, the elevation of DA extracellular levels induced by cocaine is not triggered by an increase in DA neuron firing: it is consequent to DA transporter blockade, and is associated with a decrease in DA neuron impulse activity induced by the stimulatory action of DA itself on somatodendritic D2 autoreceptors (Pitts and Marwah, 1988; White, 1990; Benwell et al, 1993). Considering that 5-HT2C receptor blockade is known to disinhibit DA neuron firing (Di Giovanni et al, 1999; Gobert et al, 2000), it is tempting to suggest that the enhancement of cocaine-stimulated DA outflow elicited by 5-HT2C antagonists could result from their opposite action on DA neuron firing (ie blockade of 5-HT2C receptors could reverse the inhibitory effect of cocaine on DA neuron firing). Favoring this hypothesis, previous studies have shown that the increase in DA outflow induced by DA reuptake blockers other than cocaine is sensitive to increased DA neuron impulse activity (Westerink et al, 1989). Also, blockade of 5-HT2 receptors has been shown to counteract the ability of 5-HT to enhance the autoinhibitory control exerted by DA on D2 receptors (Brodie and Bunney, 1996). Thus, our results, together with the above considerations, indicate that 5-HT2C receptor antagonists may modulate DA exocytosis also when it is not associated with an increase in DA neuron firing. In addition to its ability to block DA transporter, cocaine is also a potent inhibitor of the 5-HT transporter, thereby increasing 5-HT extracellular levels and reducing 5-HT neuron firing (Cunningham and Lakoski, 1990; Teneud et al, 1996). It is possible that the raise in 5-HT extracellular concentration strengthens the inhibitory tone of 5-HT2C receptors on DA neuron activity. Alternatively, blockade of the tonic inhibitory control of 5-HT2C receptors may unmask excitatory and phasic influences on DA outflow exerted by endogenous 5-HT via other 5-HT receptors, as already shown in case of concomitant increase in DA and 5-HT transmission (Kankaanpää et al, 2002; Bubar et al, 2003; Porras et al, 2003). Additional experiments are warranted to evaluate this possibility.

At variance with 5-HT2C antagonists, stimulation of 5-HT2C receptors by Ro 60-0175 affects neither basal nor cocaine-stimulated DA outflow, whatever the brain area considered. A similar result has been reported in a previous study showing that the nonselective 5-HT2 agonist DOI fails to alter the effect of 10 mg/kg cocaine in the NAc of freely moving rats (Willins and Meltzer, 1998). It is unlikely that the lack of effectiveness of Ro 60-0175 is due to inappropriate dose and route administration, or to the 5-HT2C receptor occupancy by cocaine-induced endogenous 5-HT outflow. Indeed, behavioral data have shown that a similar regimen dramatically reduced, via a 5-HT2C receptor-dependent mechanism, locomotor hyperactivity and self-administration induced by nicotine, ethanol, and cocaine (Grottick et al, 2000, 2001; Tomkins et al, 2002). More likely, the failure of Ro 60-0175 to affect cocaine-induced DA outflow is related to the cellular mechanisms underlying their interaction on DA neurons. To further address this point, we have studied the effect of Ro 60-0175 on DA outflow induced by haloperidol, a drug that, at variance with cocaine, increases DA release as a consequence of increased DA neuron firing induced by DA-D2 autoreceptor blockade (Mereu et al, 1984; Di Chiara and Imperato, 1988). Interestingly, we found that, in both the NAc and the striatum, 1 mg/kg Ro 60-0175 has no influence on basal and 0.01 mg/kg haloperidol-stimulated DA release, but inhibits the increase in DA release induced by 0.1 mg/kg haloperidol. These findings are in line with recent studies in the rat NAc and prefrontal cortex showing that 5-HT2C agonists exert a preferential effect on impulse-stimulated release of DA (Willins and Meltzer, 1998; Pozzi et al, 2002), probably consequent to their property to inhibit DA neuron firing (Gobert et al, 2000). In addition, the fact that the effect of 0.01 mg/kg haloperidol is insensitive to Ro 60-0175 suggests that, as already shown for 5-HT2C antagonism (Lucas et al, 2000), the degree of DA neuron activation is a critical factor for the expression of this interaction. Indeed, we have already shown that the facilitatory effect of SB 206553 on haloperidol-stimulated striatal DA release is no longer observed when haloperidol is administered at doses inducing a maximal increase in DA neuron firing rate (Lucas et al, 2000). By the above-discussed data, it is conceivable that the inhibitory effect of cocaine on DA neuron firing precludes the action of 5-HT2C agonists on cocaine-induced DA outflow. Altogether, these data suggest that DA neuron impulse activity is the main cellular mechanism targeted by 5-HT2C agents to control DA exocytosis (Willins and Meltzer, 1998; Porras et al, 2002b). Electrophysiological studies are warranted to confirm this hypothesis.

The simultaneous monitoring of accumbal and striatal DA outflow allow us to show that, as already reported for other drugs increasing DA release (Porras et al, 2002b), 5-HT2C agents exert similar influence on basal and cocaine-stimulated DA outflow (10 and 15 mg/kg) in both the NAc and the striatum. However, a recent study in 5-HT2C receptor knockout mice has reported that cocaine-stimulated DA outflow is selectively potentiated in the NAc with respect to the striatum (Rocha et al, 2002). It is possible that, given the different experimental context (5-HT2C receptor gene inactivation vs acute antagonist administration), adaptative responses subsequent to chronic deletion of 5-HT2C receptors in mice may have altered differentially the responsiveness of nigrostriatal and mesoaccumbal DA neurons to cocaine. For instance, previous studies in rats have shown that chronic, but not acute, pharmacological blockade of 5-HT2C receptors leads to a selective inhibition of the mesoaccumbal DA neuron firing with respect to the nigrostriatal one (Blackburn et al, 2002).

Finally, the obtained results raise the issue of the role of DA neurons in the effects of 5-HT2C ligands on DA-dependent behavioral responses induced by cocaine. Indeed, while 1 mg/kg Ro 60-0175 does not modulate cocaine-stimulated accumbal DA outflow in both anesthetized (this study) and freely moving rats (unpublished observation), it potently reduces the hyperlocomotive, discriminative stimulus, and reinforcing properties of cocaine (Grottick et al, 2000). Furthermore, although 5-HT2C antagonists increase both behavioral (McCreary and Cunningham, 1999; Fletcher et al, 2002) and neurochemical (present study) effects of cocaine, in some cases their potentiative effects do not follow the same time course. For instance, the facilitatory effect of SB 242084 on locomotor hyperactivity (Fletcher et al, 2002) occurs earlier than that observed on DA outflow (present study). Similarly, Hutson et al (2000) have reported different time-course effects of SB 242084 on phencyclidine-induced accumbal DA release and locomotor activity. Furthermore, it has been shown that SB 206553, without effect on amphetamine-stimulated DA outflow (Porras et al, 2002b), potentiates amphetamine-induced locomotor hyperactivity (Bankson and Cunningham, 2002). These data together point out that, as already shown for 5-HT1A receptors (Müller et al, 2002), 5-HT2C receptors may inhibit cocaine-induced DA-dependent behaviors independently from an action on DA outflow itself, thereby controlling DA transmission by acting downstream from DA neurons. This conclusion is supported by anatomical findings showing that 5-HT2C receptors are mainly expressed by non-DA neurons in various brain structures innervated by DA neurons (Eberle-Wang et al, 1997). Consistent with previous studies reporting that 5-HT2C receptors modulate cellular activity in various brain areas involved in the control of DA-dependent behaviors (Fox et al, 1998; De Deurwaerdère and Chesselet, 2000; Di Giovanni et al, 2001; Filip and Cunningham, 2002), it has been recently shown that stimulation of frontocortical 5-HT2C receptors reduces cocaine-induced locomotor hyperactivity (Filip and Cunningham, 2003). Moreover, from a molecular point of view, recent data have reported that 5-HT2 receptors regulate the phosphorylation of the DA and cyclic 3'–5' adenosine monophosphate-regulated phosphoprotein (DARPP-32), a protein located in dopaminoceptive neurons and involved in the mediation of the reinforcing effects of cocaine by processes acting independently from changes of DA outflow (Svenningsson et al, 2002; Zachariou et al, 2002).

In conclusion, the present study provides evidence that 5-HT2C receptors are able to modulate DA exocytosis in both the NAc and the striatum, also when it occurs independently from an increase in DA neuron impulse activity. Furthermore, the obtained results, in agreement with previous findings (Pozzi et al, 2002), indicate that the phasic inhibitory control exerted by 5-HT2C agonists occurs preferentially on impulse-stimulated release of DA. When considering the ability of 5-HT2C agonists to inhibit DA-dependent behaviors induced by cocaine (Grottick et al, 2000), our findings also indicate that 5-HT2C receptor stimulation can modulate altered DA transmission independently from an action on DA neuron activity itself. These findings add further insight into the mechanisms underlying the 5-HT2C receptors/DA neuron interaction and its involvement in the DA effects of cocaine, thus providing a neurochemical basis for a better understanding of the therapeutic potential of 5-HT2C agonists for treating cocaine abuse and dependence (Grottick et al, 2000; Rocha et al, 2002).

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Acknowledgments

This work was supported by grants from Centre National de la Recherche Scientifique and Bordeaux 2 University. S Navailles was a fellowship recipient from the Ministère de la Recherche et de l'Enseignement Supérieur during the course of this study. We are grateful to Dr M Wood (Psychiatry CEDD, GlaxoSmithKline, Harlow, UK) for the generous gift of SB 206553 and SB 242084 and Dr P Weber (F Hoffmann-La Roche, Basel, Switzerland) for the gift of Ro 60-0175. We thank D Moison for the technical assistance.

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