modulation of dopamine release by striatal 5-ht2c receptors

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Modulation of Dopamine Release by Striatal 5-HT2C Receptors KATHERINE D. ALEX, 1,4 GREGORY J. YAVANIAN, 3 HEWLET G. MCFARLANE, 5 CHARLES P. PLUTO, 6 AND ELIZABETH A. PEHEK 1,2,4 * 1 Department of Neuroscience, Case Western Reserve University, Cleveland, Ohio 44106 2 Department of Psychiatry, Case Western Reserve University, Cleveland, Ohio 44106 3 Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106 4 Louis Stokes VA Medical Center, W151, Cleveland, Ohio 44106 5 Department of Psychology, Kenyon College, Gambier, Ohio 43022 6 Department of Neuroscience, Medical College of Ohio, Toledo, Ohio 43614 KEY WORDS striatum; in vivo microdialysis; SB 206553; mCPP; serotonin; prefrontal cortex ABSTRACT Previous work has demonstrated that dopamine (DA) transmission is regulated by serotonin-2C (5-HT2C) receptors but the site(s) in the brain where these receptors are localized is not known. The present work utilized in vivo microdialysis to investigate the modulation of DA release by 5-HT2C receptors localized in the nerve terminal regions of the mesocortical and nigrostriatal DA pathways. Microdialysis probes implanted in the striatum or the prefrontal cortex (PFC) measured dialysate DA concentrations, while the selective 5-HT2B/2C inverse agonist SB 206553 was given locally by reverse dialysis into these terminal regions. Additionally, the effects of the 5-HT2C agonist mCPP on striatal DA were measured. Local administration of SB 206553 (0.1-100 M) into the striatum increased DA efflux in a concentration-dependent manner. Systemic administration of mCPP (1.0 mg/kg i.p.) decreased striatal DA and attenuated the SB 206553-induced increase. In contrast, infusion of SB 206553 (0.1– 500 M) by reverse dialysis into the PFC had no significant effect on basal DA efflux in this region. Additionally, high concentrations of SB 206553 had no effect on high potassium (K )-stimulated DA release in the PFC. These data contribute to a body of evidence indicating that 5-HT2C receptors inhibit nigrostriatal dopaminergic transmis- sion. In addition, the results suggest that the nigrostriatal system is regulated by 5-HT2C receptors localized in the dorsal striatum. Elucidating the mechanisms by which serotonin (5-HT) modulates striatal and prefrontocortical DA concentrations may lead to improvements in the treatment of diverse syndromes such as schizophrenia, Parkinson’s disease, anxiety, drug abuse, and/or depression. Synapse 55:242–251, 2005. Published 2005 Wiley-Liss, Inc. INTRODUCTION The nigrostriatal dopamine (DA) pathway is one of four major dopaminergic systems in the brain. The cell bodies of neurons in this pathway reside in the sub- stantia nigra pars compacta (SNpc) and project to the dorsal striatum (caudate-putamen). Degeneration of these neurons results in the subsequent motor deficits of Parkinson’s disease. While attention has focused on the motor functions of the striatum, recent evidence indicates that this structure may be important for nor- mal cognitive behavior and perception. For example, Laurelle et al. (1999) provide evidence for dopaminer- gic hyperactivity in the striatum of schizophrenic pa- tients and suggest that it underlies the positive symp- toms of the disease. In contrast, the mesocortical DA pathway, which originates in the ventral tegmental area (VTA) and terminates in the prefrontal cortex (PFC), has long been associated with the regulation of complex cognitive processes. In this case, hypoactivity of mesocortical DA has been linked to the negative symptoms and impaired cognition associated with schizophrenia (Weinberger et al., 1987). Contract grant sponsor: National Institutes of Health; Contract grant number: MH52220; Contract grant sponsor: Department of Veterans Affairs, Merit Re- view Award (to E.A.P.). *Correspondence to: Elizabeth A. Pehek, Louis Stokes Cleveland VA Medical Center W151, 10701 East Blvd., Cleveland OH 44106. Received 13 February 2004; Accepted 30 November 2004 DOI 10.1002/syn.20109 Published online in Wiley InterScience (www.interscience.wiley.com). SYNAPSE 55:242–251 (2005) Published 2005 WILEY-LISS, INC. This article is a US government work and, as such, is in the public domain in the United States of America.

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Modulation of Dopamine Release byStriatal 5-HT2C Receptors

KATHERINE D. ALEX,1,4 GREGORY J. YAVANIAN,3 HEWLET G. MCFARLANE,5CHARLES P. PLUTO,6 AND ELIZABETH A. PEHEK1,2,4*

1Department of Neuroscience, Case Western Reserve University, Cleveland, Ohio 441062Department of Psychiatry, Case Western Reserve University, Cleveland, Ohio 44106

3Department of Biology, Case Western Reserve University, Cleveland, Ohio 441064Louis Stokes VA Medical Center, W151, Cleveland, Ohio 44106

5Department of Psychology, Kenyon College, Gambier, Ohio 430226Department of Neuroscience, Medical College of Ohio, Toledo, Ohio 43614

KEY WORDS striatum; in vivo microdialysis; SB 206553; mCPP; serotonin;prefrontal cortex

ABSTRACT Previous work has demonstrated that dopamine (DA) transmission isregulated by serotonin-2C (5-HT2C) receptors but the site(s) in the brain where thesereceptors are localized is not known. The present work utilized in vivo microdialysis toinvestigate the modulation of DA release by 5-HT2C receptors localized in the nerveterminal regions of the mesocortical and nigrostriatal DA pathways. Microdialysisprobes implanted in the striatum or the prefrontal cortex (PFC) measured dialysate DAconcentrations, while the selective 5-HT2B/2C inverse agonist SB 206553 was givenlocally by reverse dialysis into these terminal regions. Additionally, the effects of the5-HT2C agonist mCPP on striatal DA were measured. Local administration of SB206553 (0.1-100 �M) into the striatum increased DA efflux in a concentration-dependentmanner. Systemic administration of mCPP (1.0 mg/kg i.p.) decreased striatal DA andattenuated the SB 206553-induced increase. In contrast, infusion of SB 206553 (0.1–500 �M) by reverse dialysis into the PFC had no significant effect on basal DA efflux inthis region. Additionally, high concentrations of SB 206553 had no effect on highpotassium (K�)-stimulated DA release in the PFC. These data contribute to a body ofevidence indicating that 5-HT2C receptors inhibit nigrostriatal dopaminergic transmis-sion. In addition, the results suggest that the nigrostriatal system is regulated by5-HT2C receptors localized in the dorsal striatum. Elucidating the mechanisms bywhich serotonin (5-HT) modulates striatal and prefrontocortical DA concentrations maylead to improvements in the treatment of diverse syndromes such as schizophrenia,Parkinson’s disease, anxiety, drug abuse, and/or depression. Synapse 55:242–251,2005. Published 2005 Wiley-Liss, Inc.†

INTRODUCTION

The nigrostriatal dopamine (DA) pathway is one offour major dopaminergic systems in the brain. The cellbodies of neurons in this pathway reside in the sub-stantia nigra pars compacta (SNpc) and project to thedorsal striatum (caudate-putamen). Degeneration ofthese neurons results in the subsequent motor deficitsof Parkinson’s disease. While attention has focused onthe motor functions of the striatum, recent evidenceindicates that this structure may be important for nor-mal cognitive behavior and perception. For example,Laurelle et al. (1999) provide evidence for dopaminer-gic hyperactivity in the striatum of schizophrenic pa-tients and suggest that it underlies the positive symp-toms of the disease. In contrast, the mesocortical DA

pathway, which originates in the ventral tegmentalarea (VTA) and terminates in the prefrontal cortex(PFC), has long been associated with the regulation ofcomplex cognitive processes. In this case, hypoactivityof mesocortical DA has been linked to the negativesymptoms and impaired cognition associated withschizophrenia (Weinberger et al., 1987).

Contract grant sponsor: National Institutes of Health; Contract grant number:MH52220; Contract grant sponsor: Department of Veterans Affairs, Merit Re-view Award (to E.A.P.).

*Correspondence to: Elizabeth A. Pehek, Louis Stokes Cleveland VA MedicalCenter W151, 10701 East Blvd., Cleveland OH 44106.

Received 13 February 2004; Accepted 30 November 2004

DOI 10.1002/syn.20109

Published online in Wiley InterScience (www.interscience.wiley.com).

SYNAPSE 55:242–251 (2005)

Published 2005 WILEY-LISS, INC. †This article is a US governmentwork and, as such, is in the public domain in the United States of America.

An accumulating body of evidence has demonstratedimportant neurochemical differences in the regulationof DA release between the nigrostriatal and mesocor-tical pathways (see Wolf et al., 1987, for review). Oneclass of drugs that differentially modulate DA releasein the PFC and the striatum are the atypical antipsy-chotic agents (Kuroki et al., 1999; Moghaddam andBunney, 1990; Pehek and Yamamoto, 1994). Whilethese drugs bind to many different receptor subtypes,evidence indicates that their ability to alter DA releasemay result, in part, from their actions as antagonistsand/or inverse agonists at serotonin (5-HT) receptors(Pehek et al., 2001; Kuroki et al., 1999).

The cell bodies and the terminal regions of the nigro-striatal and mesocortical pathways are innervated by5-HT neurons originating in the raphe nucleus (Azmi-tia and Segal 1978; Herve et al., 1987). In vivo micro-dialysis studies have shown that serotonergic ligandsalter DA release (e.g., Bowers et al., 2000; Galloway etal., 1993; Pehek, 1996). In particular, systemic admin-istration of inverse agonists at the 5-HT2C receptorsubtype increases DA efflux in both the striatum (DeDeurwaerdere and Spampinato, 1999; Di Giovanni etal., 1999; Porras et al., 2002) and PFC (Gobert et al.,2000; Millan et al., 1998; Pozzi et al., 2002). Receptorbinding, in situ hybridization, and immunohistochem-ical studies have demonstrated that 5-HT2C receptorsare expressed in specific brain regions (Abramowski etal., 1995; Clemett et al., 2000; Eberle-Wang et al.,1997; Molineaux et al., 1989; Pasqualetti et al., 1999;Pompeiano et al., 1994). These areas include thosecontaining mesocortical and nigrostriatal cell bodies:the VTA (Eberle-Wang et al., 1997; Molineaux et al.,1989; Pompeiano et al., 1994), and the SNpc(Abramowski et al., 1995; Eberle-Wang et al., 1997;Molineaux et al., 1989; Pasqualetti et al., 1999; Pom-peiano et al., 1994), respectively. 5-HT2C receptors arealso present in the terminal regions: the striatum(Abramowski et al., 1995; Clemett et al., 2000; Eberle-Wang et al., 1997; Pasqualetti et al., 1999; Pompeianoet al., 1994) and the PFC (Pasqualetti et al., 1999;Pompeiano et al., 1994). Thus, 5-HT2C receptors couldmodulate DA transmission at the level of the cell bodiesor nerve terminals.

Previous microdialysis studies demonstrating thatsystemically administered 5-HT2C inverse agonistsand antagonists increase DA release (see above) in-dicate that 5-HT2C receptors inhibit dopaminergicneurons comprising the nigrostriatal and mesocorti-cal pathways. However, because systemic injectionsblock all 5-HT2C receptors, the anatomical localiza-tion of the relevant receptor population(s) remainsunknown. To address this, the present experimentsused SB 206553 to examine the specific contributionsof 5-HT2C receptors in DA nerve terminal areas. SB206553 has been characterized as an inverse agonistat these receptors (Berg et al., 1999). The affinity of

this ligand is at least 100-fold greater for 5-HT2B(pKI 8.9) and 5-HT2C (pKI 7.9) receptors than for anyother receptor (Kennett et al., 1996). SB 206553 wasinfused, by reverse dialysis, into either the striatumor PFC and changes in dialysate DA in these regionswere measured. Additionally, the 5-HT2C agonistmCPP was administered systemically, alone and incombination with intrastriatal SB 206553, while stri-atal DA was quantified. Lastly, the ability of SB206553 to alter high potassium (K�)-stimulated DArelease in the PFC was tested. It was hypothesizedthat local administration of the inverse agonistwould increase extracellular DA in the striatum andPFC and potentiate high K�-stimulated DA releasein the PFC. It was also predicted that administrationof mCPP would decrease striatal DA and attenuatethe SB 206553-induced increase, providing evidencethat the effects are 5-HT2C receptor-mediated.These results would indicate that 5-HT2C receptorsin the terminal regions mediate the increases in ex-tracellular DA observed in response to the systemicadministration of 5-HT2C ligands.

MATERIALS AND METHODSAnimals and surgery

The animals used in this study were male Sprague-Dawley rats (Harlan, Indianapolis, IN, or Zivic-Miller,Zelienople, PA) weighing from 200–400 g at the time ofsurgery. Rats were housed in pairs in a temperature-controlled room with free access to food and water on a12/12-h light/dark schedule. Prior to surgery, rats wereanesthetized with a ketamine (131.25 mg/kg) and xy-lazine (11.25 mg/kg) cocktail that was injected i.m.Subjects were then mounted in a stereotaxic frame.Measurements from bregma were subsequently used todrill a hole through the skull above the anterior stria-tum (AP 1.2, ML 3.0) or the PFC (AP 3.0, ML 0.7)according to the atlas of Paxinos and Watson (1998).Then 21-gauge stainless steel guide cannulae werechronically implanted to sit directly on the brain sur-face without disturbing the brain matter. The guidecannulae were secured in position with cyanoacrylategel at the skull/cannulae junctions and with three skullscrews covered with cranio-plastic cement. Each ratwas placed on a heating pad during the initial recoveryto maintain a body temperature of 37°C. After awak-ening from anesthesia, the animals were placed in in-dividual cages where they remained for the 3–7-day period between surgery and microdialysis exper-iments. All animal use procedures were in strict accor-dance with the NIH Guide for the Care and Use ofLaboratory Animals and were approved by the localanimal care committee.

Microdialysis

Microdialysis data were collected using probes of aconcentric flow design (for details, see Yamamoto and

5-HT2C REGULATION OF DOPAMINE RELEASE 243

Pehek, 1990). PFC probes were constructed to dialyzethe dorsally located anterior cingulate cortex, the pre-limbic cortex, and the ventrally located infralimbic sub-region. Striatal probes dialyzed the lateral aspects ofthe anterior striatum. The active dialyzing surface ofthe membrane (Spectra/Por Hollow, MW cutoff �13,000, diameter � 200 �m) was 5 mm for the PFC and3 mm for the striatum. At 18–24 h before the start ofthe experiments, the probes were slowly insertedthrough the guide cannulae into the brains of awakerats and secured in place with cyanoacrylate gel. Theanimals were then placed in clear Plexiglas test cham-bers and tethered to counterbalance arms that allowedrelatively free movement. Food and water were avail-able ad libitum in these test chambers until the start ofthe experiment.

A micro-infusion pump (Pump 22, Harvard Appara-tus, South Natick, MA) and liquid swivels (Instech,Plymouth Meeting, PA) were used to perfuse artificialcerebrospinal fluid (aCSF) through the probes at aconstant rate. Unless otherwise noted, the aCSF usedwas modified from commercially available Dulbecco’sphosphate-buffered saline (137 mM NaCL, 2.7 mMKCl, 0.5 mM MgCl2, 1.5 mM KH2PO4, 8.1 mMNA2HPO4, pH: 7.4) by the addition of CaCl2 (1.2 mM)and glucose (10 mM) and was pumped at a rate of1 �L/min. Dialysate samples were collected every30 min until basal DA concentrations were stable(�180 min). At this time, drug was administered eitherby a systemic injection or by manually switching tub-ing connections to allow drug diluted in aCSF to pumpthrough the probes. These tubing switches were per-formed rapidly to maintain constant flow rates andcollection volumes. Sample collections continued every30 min for another 3–7 h (see Procedures for individualexperiments). Each rat was used for only one microdi-alysis experiment. At the conclusion of each experi-ment probe placements were verified histologically.Only data from animals whose probe placements wereverified to be in the desired brain region were includedin the study.

Drugs

The free base form of SB 206553, N-3-Pyridinyl-3,5-dihydro-5-methylbenzo(1,2-b:4,5-b�)dipyrrole-1(2H)car-boxamide, (kindly donated by SmithKline BeechamPharmaceuticals, Harlow, UK) was used in all experi-ments. For reverse dialysis into the striatum, SB206553 was dissolved in 5 �L glacial acetic acid (GAA)and 1 mL deionized water to create a 10-mM stocksolution. This solution was then diluted with aCSFto the appropriate micromolar concentration. (0.1–500 �M). The pH of the final solutions was 7.4. Forsystemic mCPP administration, mCPP (m-chlorophe-nylpiperazine) hydrochloride (Tocris, Bristol, UK) wasdissolved in deionized water to 1 mg/ml and injected ata volume of 1 ml/kg body weight (i.p.). This dose refers

to the salt and was chosen based on previous studies(e.g., Di Giovanni et al., 2000). The pH of the solutionwas �5.5. Vehicle consisted of deionized water ad-justed to the same pH as the drug solution (1 ml/kg i.p.).

Chromatography

High-performance liquid chromatography (HPLC)and electrochemical detection were used to isolateand measure DA in each dialysate sample. Twenty-microliter dialysis samples were injected onto anUltracarb column (Phenomenex, Torrance, CA; 3 �Mparticle size, ODS 20, 100 � 2 mm). The mobilephase consisted of 32 mM citric acid, 54 mM sodiumacetate, 0.074 mM EDTA, 0.215 mM octylsulfonicacid, and 3% methanol (vol/vol), pH 4.2. When thismobile phase did not provide a clear separation of DAfrom its metabolite DOPAC and the 5-HT metabolite5-hydroxyindoleacetic acid, the pH of the mobilephase and the concentration of octylsulfonic acidwere adjusted in order to achieve optimal separation.Either aBAS LC-4C or an Antec Intro electrochemi-cal detector with a glassy carbon electrode, main-tained at a potential of �0.60 V relative to an Ag/AgCl reference electrode, was employed. The limit ofdetection for DA was 0.1 pg/20 �L.

ProceduresEffects of intrastriatal SB 206553 on striatal DA

This experiment tested the concentration-depen-dence of reverse dialysis with SB 206553 into the stri-atum. There were three groups: low concentrations,higher concentrations, and control (aCSF withoutdrug). For the first group, a 0.1 �M concentration wasinfused for 120 min followed by perfusion with a1.0 �M concentration for 60 min. Drug-free aCSF wasthen infused for 60 min before the animals were sacri-ficed. The second group was similar but 10 �M wasinfused for 120 min followed by 100 �M for 60 min.These groups were compared to the control group.

Effects of mCPP on the 10 �M intrastriatalSB 206553 DA increase

This experiment tested the ability of the 5-HT2Cagonist mCPP to block the effects of SB 206553. Therewere four groups tested in this experiment: mCPP �drug-free aCSF, VehiclemCPP � drug-free aCSF,mCPP � 10 �M SB 206553, and VehiclemCPP � 10 �MSB 206553. mCPP (1 mg/kg) or vehicle was given sys-temically after baselines stabilized. One hour after themCPP or vehicle injection, SB 206553 or drug-freeaCSF was infused into the brain through the microdi-alysis probe for 90 min. This was followed by perfusionwith drug-free aCSF for an additional 90 min. The flowrate for this experiment was 1.5 �L/min.

244 K.D. ALEX ET AL.

Effects of mCPP on the 100 �M intrastriatalSB 206553 DA increase

This experiment tested the ability of mCPP to blockthe effects of a higher concentration of SB 206553. Twonew groups were added: VehiclemCPP � 100 �M SB206553, and mCPP (1 mg/kg) � 100 �M SB 206553 inplace of the 10 �M SB 206553 groups. Other than thechange in the concentration of SB 206553, the experi-mental procedure was identical to the preceding exper-iment.

Effects of 0.1, 1.0, and 10 �M intracorticalSB 206553 on PFC DA

This experiment tested the ability of lower concen-trations of SB 206553, infused intracortically, to affectDA release in the PFC. Three concentrations ofSB206553 were infused into the PFC in increasingorder (0.1, 1.0, and 10 �M) for 120 min each. Drug-freeaCSF was then infused through the probe for 60 minbefore the termination of the experiment.

Effects of 100 and 500 �M intracorticalSB 206553 on PFC DA

This experiment tested the ability of higher concen-trations of SB 206553, infused intracortically, to affectDA release in the PFC. For this experiment and theexperiment that follows, the basal perfusion mediumused was a Krebs-Ringer buffer (137 mM NaCl, 3 mMKCl, 1.2 mM MgSO4, 0.4 mM KH2PO4, 1.2 mM CaCl2,and 10 mM glucose; pH: 7.4). This buffer was used sothat the K� concentration of the medium could bealtered as needed. The 10-mM stock solution of SB206553 was made as detailed in the Drugs section andwas diluted with this Krebs-Ringer buffer to 100 or500 �M (pH 7.4). Either the 100 or 500 �M solutionwas infused into the PFC for 60 min. Drug-free aCSFwas then infused through the probe for 3.5 h before thetermination of the experiment.

Effects of intracortical SB 206553 on highK�-stimulated DA release in the PFC

This experiment tested the ability of 100 and 500 �MSB 206553, infused intracortically, to alter high K�-stimulated DA release in the PFC. The high K� bufferwas made by increasing the KCl to 80 mM and decreas-ing the NaCl to 60 mM in order to maintain the osmo-larity of the solution. One group of rats was perfusedwith this high K� buffer for 30 min after baselinesamples were collected with the normal Krebs-Ringerbuffer. Two additional groups of rats received pretreat-ments with SB 206553 (100 or 500 �M) beginning30 min before the initiation of high K� buffer perfu-sion. The SB 206553 perfusion lasted 60 min, andtherefore was terminated at the conclusion of the30-min high K� buffer perfusion period. Normal drug-

free Krebs-Ringer buffer was then infused for 3.5 hbefore the experiment was terminated.

Data analysis

Data were expressed as a percentage of the averageof the last three predrug baseline samples. Statisticalanalyses were performed using repeated measuresANOVAs. For two-way ANOVAs, time was the re-peated measures factor and drug condition was theindependent factor. For one-way ANOVAs, time wasthe repeated factor. Post-hoc comparisons utilizedDunnett’s test for comparing treatment means with acontrol value.

RESULTSEffects of intrastriatal SB 206553 on striatal DA

Infusions of 0.1 and 1.0 �M SB 206553 signifi-cantly increased dialysate DA in the striatum rela-tive to drug-free aCSF controls (two-way ANOVA:significant time by drug interaction F(8,84) � 3.860,P � 0.001; see Fig. 1A). Post-hoc tests demonstrateda significant increase in DA following both the0.1 �M and 1.0 �M concentrations (Fig. 1A). Duringthe 0.1 �M infusion, dialysate DA reached a maxi-mum of 168% of baseline 120 min after the start ofdrug infusion. Once the concentration was increasedto 1.0 �M, DA continued to rise to a maximum of175% of baseline. Switching to nondrug aCSF re-sulted in a decrease in dialysate DA.

Infusions of 10 and 100 �M SB 206553 significantlyincreased dialysate DA in the striatum relative todrug-free aCSF controls (two-way ANOVA: significanttime by drug interaction F(8,88) � 6.640, P � 0.001; seeFig. 1B). Post-hoc tests demonstrated a significant in-crease in DA following either the 10 �M or 100 �Mconcentrations (Fig. 1B). During the 10 �M infusion,dialysate DA reached a maximum of 142% of baseline60 min after the start of drug infusion and was main-tained at elevated levels until the concentration wasincreased to 100 �M. Following this change in concen-tration DA continued to rise to a maximum of 212% ofbaseline. Switching to nondrug aCSF resulted in adecrease in dialysate DA that approached basal con-centrations after 1 h. The basal level of dialysate DAaveraged from the three separate intrastriatal groupsin Figure 1 was 6.38 � 1.05 pg / 20 �l (n � 19).

Effects of mCPP on the 10 �M intrastriatalSB 206553 DA increase

There were four groups tested in this experiment:mCPP � drug-free aCSF, VehiclemCPP � drug-freeaCSF, mCPP � 10 �M SB 206553, and VehiclemCPP �10 �M SB 206553. mCPP was administered systemi-cally (1 mg/kg i.p.), while SB 206553 was administeredby reverse dialysis into the striatum. A two-wayANOVA comparing all four drug treatments revealed a

5-HT2C REGULATION OF DOPAMINE RELEASE 245

significant interaction between drug treatment andtime (F(24,176) � 1.829, P � 0.014; see Fig. 2A). Treat-ment with mCPP resulted in a small but significantdecrease in striatal DA (one-way ANOVA: F(8,40) �2.398, P � 0.032; see Fig. 2A,B). For the VehiclemCPP �drug-free aCSF group there was no significant changein striatal DA. Similar to the first experiment, infusionof 10 �M SB 206553 significantly increased dialysateDA in the striatum (one-way ANOVA: F(8,56) � 2.316,P � 0.032; see Fig. 2A). The maximal increase was143% of baseline. Pretreatment with systemic mCPPattenuated this increase in striatal DA (one-wayANOVA on the mCPP � 10 �M SB 206553 groupshowed no significant effect of time: F(8,32) � 1.252,P � 0.302; see Fig. 2A).

Effects of mCPP on the 100 �M intrastriatalSB 206553 DA increase

Two new groups were added: VehiclemCPP � 100 �MSB 206553, and mCPP (1 mg/kg) � 100 �M SB 206553.These groups were compared to the mCPP � drug-freeaCSF and VehiclemCPP � drug-free aCSF groups fromthe preceding experiment. A two-way ANOVA on allfour treatments groups showed a significant time bydrug interaction (F(24,144) � 11.197, P � 0.001) asshown in Figure 2B. A comparison of Figure 2A,B il-lustrates the concentration-dependence of the SB206553-induced increase in striatal DA. The infusion of100 �M SB 206553 significantly increased dialysateDA to a maximum of 345% (one-way ANOVA: F(8,32) �13.939, P � 0.001; see Fig. 2B). Pretreatment with

Fig. 1. A: Effects of local administration of 0.1 and 1.0 �M SB206553 on dialysate DA concentrations in the striatum. For thedrug group, 0.1 �M SB 206553 was perfused intrastriatally for120 min followed by 60 min of 1.0 �M. Drug-free aCSF was thendelivered for the final 60 min of the experiment. The control groupreceived drug-free aCSF for the duration of the experiment. Dataare the means � SEM of seven animals for each group. *P � 0.05relative to predrug baseline. B: Effects of local administration of 10and 100 �M SB 206553 on dialysate DA concentrations in thestriatum. For the drug group, 10 �M SB 206553 was perfusedintrastriatally for 120 min followed by 60 min of 100 �M. Drug-freeaCSF was then delivered for the final 60 min of the experiment.The control group received drug-free aCSF for the duration of theexperiment. Data are the means � SEM of 5–7 animals. *P � 0.05relative to predrug baseline.

Fig. 2. A: Effects of systemic administration of mCPP (1 mg/kgi.p.) on the 10 �M SB 206553-induced increase in dialysate DA inthe striatum. mCPP or vehicle were injected at time zero as indi-cated by the arrow. SB 206553 or drug-free aCSF (control) wasperfused intrastriatally 1 h later for 90 min as indicated by the bar.Data are means � SEM of 5– 8 animals per group. *P � 0.05relative to predrug baseline. B: Effects of systemic administrationof mCPP (1 mg/kg i.p.) on the 100 �M SB 206553-induced increasein dialysate DA in the striatum. mCPP or vehicle were injected attime zero as indicated by the arrow. SB 206553 or drug-free aCSF(control) was perfused 1 h later for 90 min as indicated by the bar.Data are means � SEM of 4 – 8 animals per group. *P � 0.05relative to predrug baseline.

246 K.D. ALEX ET AL.

mCPP (1 mg/kg) slightly attenuated this increase instriatal DA. However, this attenuation was not signif-icant, as infusions of 100 �M SB 206553 still increaseddialysate DA following mCPP administration (one-wayANOVA: F(8,24) � 9.869, P � 0.001; see Fig 2B). Theaverage basal level of dialysate DA for the six groups ofrats that comprise Figure 2A,B was 3.98 � 0.29 pg/20 �l (n � 35).

Effects of 0.1, 1.0, and 10 �M intracorticalSB 206553 on PFC DA

Figure 3 shows the dialysate DA concentrations afterinfusion of three increasing concentrations of SB206553 (0.1, 1.0, and 10 �M). No significant increasefrom basal DA levels was seen in response to anyconcentration of SB 206553 (one-way ANOVA:F(14,70) � 0.922, P � 0.540; see Fig. 3). The basal levelof dialysate DA for the intracortical group was 0.52 �0.06 pg/20 �l (n � 6).

Effects of 100 and 500 �M intracorticalSB 206553 on PFC DA

There also was no significant increase in PFC DAin response to higher concentrations (100 and500 �M) of SB 206553, as shown in Figure 4A. Theaverage basal level of dialysate DA for the twogroups of rats that comprise Figure 4A was 0.75 �0.08 pg/20 �l (n � 14).

Effects of intracortical SB 206553 on highK�-stimulated DA release in the PFC

PFC DA was increased to a maximum of 292% ofbaseline in response to perfusion with high K� buffer(one-way ANOVA shows significant effect of timeF(9,81) � 8.839, P � 0.001; see Fig. 4B). Pretreatmentwith 100 or 500 �M SB 206553 had no significant effecton high K�-stimulated DA release in the PFC, as illus-

trated in Figure 4B (one-way ANOVAs continue toshow significant effects of time F(9,45) � 3.197, P �0.005 and F(9,54) � 3.616, P � 0.001 respectively). Theaverage basal level of dialysate DA for the four groupsof rats that comprise Figure 4B was 0.62 � 0.05 pg/20 �l (n � 31).

DISCUSSION

These data demonstrate that local administration ofthe 5-HT2C inverse agonist SB 206553 into the stria-tum increases striatal DA in a concentration-depen-dent manner. This increase was attenuated by thesystemic administration of the 5-HT2C agonist mCPP.These results are the first to demonstrate that 5-HT2Creceptors localized in the striatum may normally serveto inhibit DA release in the nigrostriatal pathway.They indicate that the effects of systemically adminis-

Fig. 3. Effects of local administration of 0.1, 1.0, and 10 �M SB206553 on dialysate DA concentrations in the PFC; 0.1 �M SB 206553was perfused intracortically for 120 min followed by 120 min of 1.0 �Mand then 120 min of 10 �M. Drug-free aCSF was then delivered forthe final 60 min of the experiment. Data are the means � SEM of sixanimals.

Fig. 4. A: Effects of local administration of 100 and 500 �M SB206553 on dialysate DA concentrations in the PFC; 100 or 500 �M SB206553 was perfused intracortically for 60 min followed by 210 min ofdrug-free aCSF. Data are the means � SEM of seven animals for eachgroup. B: Effects of local administration of 100 and 500 �M SB 206553on high (80 mM) K�-induced increases in dialysate DA concentrationsin the PFC. High K� solution was infused for 30 min as indicated bythe solid bar. SB 206553 was infused for 60 min as indicated by thedashed bar. Data are the means � SEM of 3–6 animals per group.*P � 0.05 relative to predrug baseline.

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tered SB 206553 may be mediated, at least in part, byactions on striatal 5-HT2C receptors.

Previous work has shown that systemic administra-tion of SB 206553 also increases DA release in the PFC(Gobert et al., 2000; Millan et al., 1998). However, inthe present work intracortical administration of thisligand in concentrations ranging from 0.1–500 �M didnot alter basal or high K�-stimulated DA release.These results indicate that the effects of systemic SB206553 are not due to actions on 5-HT2C receptorslocalized in the PFC.

The present increase in striatal DA after local ad-ministration of SB 206553 is consistent with and ex-tends previous work employing the systemic adminis-tration of 5-HT2C ligands. A recent electrophysiologicalstudy demonstrated that treatment with SB 206553increases the firing rate of DA neurons in the SNpc (DiGiovanni et al., 1999). In addition, dialysis studieshave shown that systemic administration of SB 206553results in increased striatal DA concentrations (DeDeurwaerdere and Spampinato, 1999; Di Giovanni etal., 1999; Gobert et al., 2000), whereas injections of the5-HT2C agonist Ro 60-0175 decreases dialysate DA inthis region (Gobert et al., 2000). Several studies usinga variety of approaches from receptor binding to immu-nocytochemistry and in situ hybridization have deter-mined that 5-HT2C receptors are present in the stria-tum (Abramowski et al., 1995; Clemet et al., 2000;Eberle-Wang et al., 1997; Pasqualetti et al., 1999; Pom-peiano et al., 1994). The present experiments demon-strate that these receptors regulate DA release. Intra-striatal administration of SB 206553 increased DAefflux significantly at concentrations from 0.1–100 �M(Figs. 1, 2). This result is inconsistent with one studythat found a 30% reduction in striatal DA in responseto 1 �M intrastriatal SB 206553 (Lucas and Spampi-nato, 2000). The discrepancy between these resultsmay reflect differences in experimental procedures. Inparticular, differences in probe placements could beresponsible. In this study probes were implanted in theanterolateral striatum, whereas the previous studyemployed striatal placements that were both more pos-terior and more medial. Other previous work illus-trates the heterogeneity of the dopaminergic system inthe striatum (Yamamoto and Pehek, 1990). In addition,5-HT2C mRNA has been shown to vary between stria-tal subregions (Eberle-Wang et al., 1997). The presentfinding that local administration of SB 206553 into thestriatum resulted in a concentration-dependent in-crease in striatal DA suggests that the nigrostriatal DAsystem is regulated by 5-HT2C receptors in the antero-lateral striatum. The question of whether or not theseneurons are also regulated by 5-HT2C receptors at thelevel of the cell bodies remains unanswered. InfusingSB 206553 into the SNpc by reverse dialysis couldresolve this matter.

It is worth noting that SB 206553 binds to the5-HT2B receptor with approximately the same affinityas for the 5-HT2C receptor (Kennett et al., 1996). Whilethis is a limitation of the present data, evidence sug-gests that 5-HT2B receptors are located primarily inthe stomach fundus (Duxon et al., 1997). The limitednumber of these receptors in the brain has been shownto be restricted to the cerebellum, lateral septum, dor-sal hypothalamus, and medial amygdala (Duxon et al.,1997). Therefore, in the brain regions examined in thisstudy the effects of SB 206553 should not be due toactions at 5-HT2B receptors. In addition, a recentstudy has shown that the effects on striatal DA ofsystemic administration of the inverse agonist SB206553 can be reversed by pretreatment with the se-lective 5-HT2C antagonist SB 242084 (De Deurwaer-dere et al., 2004).

In order to provide additional evidence for 5-HT2Cregulation of nigrostriatal neurons, one aim of thepresent research was to examine the effects of systemicadministration of the 5-HT2C agonist mCPP on DArelease in the striatum, alone and in combination withSB 206553. mCPP is a widely used agonist with highaffinity for the 5-HT2C receptor (pKI � 6.9) (Bonhauset al., 1997) and moderate affinities for 5-HT1A,5-HT1B, and 5-HT2A receptors and the 5-HT trans-porter (Bonhaus et al., 1997; Matsumoto et al., 1992;Owens et al., 1997). Despite this limitation, mCPP hasbeen shown to act in a 5-HT2C-specific manner inmany studies. Systemically administered mCPP hasbeen shown to decrease dialysate DA in the nucleusaccumbens and decrease the firing rate of DA cell bod-ies in both the VTA and the SNpc (Di Giovanni et al.,2000). These effects were attenuated by the selective5-HT2C antagonist SB 242084, indicating that theywere due to actions of mCPP at 5-HT2C receptors (DiGiovanni et al., 2000). The data illustrated in Figure2A,B are consistent with and extend this previouswork. Systemic administration of mCPP alone resultedin a small but significant decrease in striatal DA, pro-viding further evidence that stimulation of 5-HT2Creceptors inhibits dopaminergic activity. Additionally,pretreatment with mCPP attenuated the 10 �M SB206553-induced increases in striatal DA. These resultssuggest that the two drugs, when given in combination,compete for 5-HT2C receptors and support the hypoth-esis that stimulation of these receptors inhibits thenigrostriatal DA system.

Several recent studies have shown that treatmentwith SB 206553 (Gobert et al., 2000) or SB 242084(Gobert et al., 2000; Millan et al., 1998; Pozzi et al.,2002) increases PFC DA efflux, while administration ofthe selective 5-HT2C agonist Ro 60-0175 decreases DAlevels in this region (Gobert et al., 2000; Millan et al.,1998). Anatomical studies have demonstrated the pres-ence of 5-HT2C receptors in the PFC (Pasqualetti et al.,1999; Pompeiano et al., 1994). However, in the present

248 K.D. ALEX ET AL.

study intracortical infusions of SB 206553 over a wideconcentration range (0.1–500 �M) did not alter basal orhigh K�-stimulated DA efflux (Figures 3, 4). Thus,unlike the nigrostriatal system, these results do notsupport the hypothesis that cortical 5-HT2C receptorsregulate the release of DA from the mesocortical sys-tem. They are also consistent with recent work demon-strating that intracortical infusions of the agonist Ro60-0175 (Pozzi et al., 2002), or the antagonist SB242084 (F. Artigas, pers. commun.), did not affect PFCDA. However, infusions of the 5-HT2C antagonist RS102221 into the PFC potentiated the hyperlocomotioninduced by a systemic injection of cocaine (Filip andCunningham, 2003). Thus, further work must be per-formed in order to more fully assess the role of cortical5-HT2C receptors in the regulation of mesocortical DAfunction. There is evidence that 5-HT2C receptors lo-calized in the VTA regulate mesocortical DA release(Pozzi et al., 2002).

The cellular localization of 5-HT2C receptors in thestriatum has yet to be determined. Thus, the circuitryunderlying the regulation of DA release by these recep-tors remains unknown. Previous work indicates that5-HT2C receptors are not localized presynaptically onnigrostriatal DA terminals, for lesions of DA neuronsdo not alter 5-HT2 receptor binding in the striatum orother DA-rich brain areas (Leysen et al., 1982). Rele-vant anatomical evidence does demonstrate that5-HT2C receptors are localized on GABAergic, but notdopaminergic, neurons in the midbrain SNpc and VTA(Eberle-Wang et al., 1997). Thus, it is possible that5-HT2C receptors are localized to GABAergic interneu-rons or striatonigral projection neurons in the stri-atum.

The present findings in the striatum are consistentwith behavioral data examining the effects of 5-HT2Cligands on DA-mediated behaviors. Administration of5-HT2C agonists decreases locomotion (Kennett et al.,2000) and attenuates cocaine-induced hyperactivity(Filip and Cunningham, 2003; Grottick et al., 2000),suggesting an inhibition of dopaminergic function.Treatment with 5-HT2C antagonists potentiates co-caine-induced hyperlocomotion (Filip and Cunning-ham, 2003) and blocks the ability of 5-HT2C agonists toattenuate this hyperlocomotion (Grottick et al., 2000).Additionally, 5-HT2C knockout mice show increasedhyperlocomotion in response to cocaine (Rocha et al.,2002). In support of these behavioral data, one recentstudy demonstrates that both SB 206553 and the5-HT2C antagonist SB 242084 potentiate the cocaine-induced increase in dialysate DA in both the striatumand the nucleus accumbens (Navailles et al., 2004).

In addition to alterations in responses to cocaine,5-HT2C knockout mice show increased obesity (Heisleret al., 1998) and susceptibility to seizures (Applegateand Tecott, 1998), suggesting that 5-HT2C receptorsplay a role in the control of feeding behavior and in

modulating neuronal network excitability. 5-HT2C re-ceptors have also been implicated in anxiety. Both5-HT2C antagonists and inverse agonists possess anx-iolytic-like properties in animal models of anxiety(Kennett et al., 1996, 1997). 5-HT2C receptor down-regulation following chronic exercise (Broocks et al.,1999) and desensitization in response to SSRI-treat-ment (Questad et al., 1997) have been proposed as themechanisms by which these two treatments are anxi-olytic. Thus, 5-HT2C receptors have been implicated inthe regulation of a wide range of behaviors/syndromes.

Studies suggest that serotonergic ligands may havetherapeutic potential. Recent work suggests that acombination of the DA-releaser phentermine (Phen)and the 5-HT-releaser fenfluramine (Fen) may be aneffective treatment for drug abuse (Brauer et al., 1996;Rea et al., 1998). Phen has been shown to have a profileof effects similar to d-amphetamine, both in enhancingmood and a high potential for abuse (Brauer et al.,1996). Combining Phen with Fen, however, reduces thepotential for abuse (Brauer et al., 1996; Rea et al.,1998). Since 5-HT2C agonists can substitute for Fen indrug-discrimination studies, it is thought that thesereceptors mediate the effects of the drug (McCreary etal., 2003). Thus, Fen may decrease the abuse potentialof Phen by stimulating 5-HT2C receptors that reduceDA release. Additionally, 5-HT2C antagonists havebeen shown to be beneficial in animal models of Par-kinson’s disease (Fox et al., 1998; Fox and Brotchie,2000). This evidence is consistent with the presentfinding that administration of SB 206553 results inincreased DA release in the striatum. Thus, elucidat-ing the role of 5-HT2C receptors in the regulation of DAsystems may be critically important to the develop-ment of new therapeutic agents.

In summary, the present work indicates that thenigrostriatal DA system is under inhibitory control by5-HT2C receptors localized within the terminal region,the striatum. Knowledge of the anatomical localizationof 5-HT2C receptors regulating DA release will aid inthe elucidation of the circuitry underlying this regula-tion. In turn, knowledge of the underlying circuitrymay have implications for the understanding andtreatment of numerous syndromes such as schizophre-nia, depression, Parkinson’s disease, drug abuse, andanxiety.

ACKNOWLEDGMENT

The authors thank SmithKline and Beecham fortheir generous donation of SB 206553.

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