Selective activation of 5-HT2C receptors stimulates GABA-ergic function in the rat substantia nigra pars reticulata: A combined in vivo electrophysiological and neurochemical study

Neuroscience 144 (2007) 1523–1535

SELECTIVE ACTIVATION OF 5-HT2C RECEPTORS STIMULATES GABA-ERGIC FUNCTION IN THE RAT SUBSTANTIA NIGRA PARS RETICULATA: A COMBINED IN VIVO ELECTROPHYSIOLOGICAL AND NEUROCHEMICAL STUDY R. W. INVERNIZZI,a1 M. PIERUCCI,b1 E. CALCAGNO,a1 G. DI GIOVANNI,c V. DI MATTEO,b A. BENIGNOc AND E. ESPOSITOb*

into the SNr only partially blocked RO 60-0175-induced GABA release. It is concluded that selective activation of 5-HT2C receptors stimulates GABA-ergic function in the SNr, and the clinical relevance of these data is discussed. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Istituto di Ricerche Farmacologiche “Mario Negri,” via Eritera 62, 20157 Milan, Italy

b

Istituto di Ricerche Farmacologiche “Mario Negri,” Consorzio “Mario Negri” Sud, 66030 Santa Maria Imbaro, Chieti, Italy

Key words: substantia nigra pars reticulata, basal ganglia, GABA, 5-HT2C receptors, electrophysiology, microdialysis.

c

Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana “G. Pagano,” Corso Tükory 129, 90134 Università di Palermo, Palermo, Italy

The basal ganglia are the largest subcortical nuclei of vertebrate brain including human forebrain, and they are placed in a key position to influence motor behavior, emotions, and cognition (Graybiel and Canales, 2001). Among the various neurotransmitters affecting basal ganglia function, 5-HT plays an important role (Soubrié et al., 1984; Blackburn, 2004; Di Giovanni et al., 2006). Thus, there is extensive evidence that 5-HT-containing neurons originating from midbrain raphe nuclei innervate all the basal ganglia circuitry, sending projections to the corpus striatum, the globus pallidus (GP), the subthalamic nucleus (STN) and the substantia nigra (SN) (Soubrié et al., 1984; Mori et al., 1987; Moukhles et al., 1997; Blackburn, 2004). The serotonin innervation of the SN is particularly dense, and there is indeed evidence that the substantia nigra pars reticulata (SNr) receives the largest 5-HT innervation from the dorsal raphe nucleus of all brain regions (Fibiger and Miller, 1977; Corvaja et al., 1993). It has been calculated that the density of 5-HT-immunoreactive varicosities in the SNr is in the order of 9⫻106/mm3, of which about 74% form synaptic specializations with GABA-ergic projection neurons (Moukhles et al., 1997). High to moderate density of 5-HT1B, 5-HT2A, 5-HT2C receptors has been found in several regions of the basal ganglia, including the striatum, the GP, the entopeduncular nucleus (EP) and the SN (Pazos and Palacios, 1985; Barnes and Sharp, 1999). Moreover moderate levels of mRNA for both 5-HT2A and 5-HT2C receptors have been detected in various areas of the basal ganglia, including the SNr (Pompeiano et al., 1994; Abramowski et al., 1995; Eberle-Wang et al., 1997). Interestingly, it was found that 5-HT2C receptor mRNA is expressed by GABA-ergic neurons but not by dopamine (DA)-containing neurons in the SNr (Eberle-Wang et al., 1997). Consistent with these findings, we showed that m-chlorophenylpiperazine (mCPP) excites SNr neurons by activating 5-HT2C receptors in vivo (Di Giovanni et al., 2001). This effect was evident both after systemic administration and local microiontophoretic application of mCPP. Inasmuch as combined electrophysiological and immuno-

Abstract—In vivo electrophysiology and microdialysis were used to investigate the physiological role of 5-HT2C receptors in the control of substantia nigra pars reticulata (SNr) function. Extracellular single-unit recordings were performed from putative GABA-containing neurons in the SNr of anesthetized rats, and local GABA release was studied by in vivo microdialysis in the SNr of awake freely-moving rats. Systemic administration of the selective 5-HT2C receptor agonist (S)-2-(chloro-5-fluoro-indol-1-yl)-1-methylethylamine 1:1 C4H4O4 (RO 60-0175) caused a dose-dependent excitation of about 30% of the SNr neurons recorded. However, the remaining neurons were either inhibited or unaffected by systemic RO 60-0175, in similar proportion. Local application of RO 60-0175 by microiontophoresis caused excitation in the majority of SNr neurons tested (48%), whereas a group of neurons was inhibited (16%) or unaffected (36%). Both the excitatory and the inhibitory effects of systemic and microiontophoretic RO 60-0175 were completely prevented by pretreatment with SB 243213 [5-methyl-1-({2-[(2-methyl-3pyridyl)oxy]-5-pyridyl}carbamoyl)-6-trifluoromethylindoline], a selective and potent 5-HT2C receptor antagonist. Consistent with these electrophysiological data, both systemic and intranigral administration of RO 60-0175 and m-chlorophenylpiperazine (mCPP), a non-selective 5-HT2C agonist, markedly increased extracellular GABA levels in the SNr. The stimulatory effect of systemic and local RO 60-0175 on GABA release was completely prevented by systemic administration of SB 243213, whereas local application of SB 243213 1

These authors contributed equally to the paper. *Corresponding author. Tel: ⫹39-872-570274; fax: ⫹39-872-570416. E-mail address: [email protected] (E. Esposito). Abbreviations: aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; AUC, area under the curve; ␤-cyclodx, 0.9% NaCl containing 8% hydroxypropyl-␤-cyclodextrin by weight and 25 mM citric acid; DA, dopamine; GP, globus pallidus; HFS, high frequency stimulation; ISIHs, interspike interval histograms; mCPP, m-chlorophenylpiperazine; OPA, o-phthalaldehyde; PD, Parkinson’s disease; PLSD, protected least significant difference; RO 60-0175, (S)-2-(chloro5-fluoro-indol-1-yl)-1-methylethylamine 1:1 C4H4O4; SB 243213, 5-methyl-1-({2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl}carbamoyl)-6trifluoromethylindoline; SN, substantia nigra; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus; TTX, tetrodotoxin; 5-HT, 5-hydroxytryptamine (serotonin).

0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.11.004

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cytochemical studies have shown that most non-DA neurons in the SNr are GABA-ergic (Hajós and Greenfield, 1994; Richards et al., 1997), it is possible to conclude that selective activation of 5-HT2C receptors stimulates GABAergic function in the SNr. It is therefore of interest to study the effects of selective 5-HT2C receptors ligands on the electrical activity of non-DA SNr neurons, and on GABA release. Based on the above considerations, in this study in vivo single-cell recordings and microdialysis techniques were used to investigate the effects of the 5-HT2C receptor agonist (S)-2-(chloro-5-fluoro-indol-1-yl)-1-methylethylamine 1:1 C4H4O4 (RO 60-0175) on the electrical activity of non-DA neurons (presumably GABA-ergic), and on GABA release in the SNr. Extracellular single-unit recordings were performed from non-DA neurons in the SNr of anesthetized rats, and local GABA release was studied by in vivo microdialysis in the SNr of awake freely-moving rats. Moreover, the effect of pretreatment with the selective 5-HT2C receptor antagonist [5-methyl-1-({2-[(2-methyl-3pyridyl)oxy]-5-pyridyl}carbamoyl)-6-trifluoromethylindoline] (SB 243213) on RO 60-0175-induced changes in the firing rate of putative GABA-ergic neurons and on GABA release was also evaluated.

EXPERIMENTAL PROCEDURES Animals Male Sprague–Dawley rats (CD-COBS, Charles River, Milano, Italy or Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy) weighing 280 –350 g were used in neurochemical and cell recording studies. Rats were housed under constant room temperature (21⫾1 °C) and relative humidity (60⫾5%) with a 12-h light/dark cycle (light on at 7:00 AM). Food and water were freely available throughout the study. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L. n. 116, G.U., suppl. 40, 18 Febbraio 1992) and international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication N. 85–23, 1985 and Guidelines for the Use of Animals in Biomedical Research, Thromb Hemost 58:1078 –1084, 1987; EEC Council Directive 86/609, OJ L 358,1, Dec. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication N. 85–23, 1985 and Guidelines for the Use of Animals in Biomedical Research, Thromb Hemost 58:1078 –1084, 1987). All efforts were made to minimize the number of animals used and their suffering.

Single-cell recording procedures Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted on a stereotaxic instrument (SR-6, Narishige, Japan). Supplemental doses of anesthetic were administered via a lateral tail vein cannula. Throughout the experiment, the animal’s body temperature was maintained at 36 –37 °C by a thermostatically regulated heating pad. The coordinates, relatively to the interaural line, for placement of the recording electrode in the SNr were anterior 2.7–3.4 mm, lateral 1.8 –2.2 mm, and ventral 7– 8 mm (Paxinos and Watson, 1986). Extracellular recordings were performed using either single or multi-barrel micropipettes. The single micropipettes, measuring 2–3 ␮m at the tip, were filled with 2% Pontamine Sky Blue dye in 2 M NaCl (in vitro resistance 4 –7 M⍀). Three to four-barrel homemade micropipettes were pulled to an optimal wide tip angle and mechanically bevelled under microscopic control to a final tip diameter of 4 – 6 ␮m. The central barrel,

filled with 2% Pontamine Sky Blue dye in 2 M NaCl, was used for recording (in vitro resistance 4 –7 M⍀) while one of the side barrels, filled with 2 M NaCl, was used for continuous automatic current balancing. The remaining barrels contained one of the following solutions: RO 60-0175 (20 mM, pH 4), and SB 243213 (20 mM). Non-dopaminergic (presumably GABA-ergic) neurons in the SNr were identified on the basis of established electrophysiological characteristics: short-duration (0.5– 0.7 ms), biphasic (positive/negative) or triphasic (positive/negative/positive) action potentials of large amplitude (1.0 –1.5 mV), baseline firing rates between 10 and 40 spikes/s, and location immediately ventral to nigral DA neurons (Grace and Bunney, 1979, 1985; Grace et al., 1980; Waszczak et al., 1980; Zhang et al., 1993). Electrical signals of spike activity were passed through a high impedance amplifier (bandpass filter setting: 0.1–3 kHz) whose output was led into an analog oscilloscope, audio monitor and window discriminator. Unit activity was then converted to an integrated histogram by a rate-averaging computer and displayed as spikes per 10-s intervals. After each experiment, the recording site was marked by the ejection of Pontamine Sky Blue dye from the electrode using a ⫺20 ␮A current for 10 min. Brains were removed and placed in 10% buffered formalin for 2 days before histological examination. Frozen sections were cut at 40-␮m intervals and stained with Neutral Red. Microscopic examination of the sections was carried out to verify that the electrode tip had been placed in the SNr.

Microdialysis procedure and neurochemical assays Rats were anesthetized with 3 ml/kg Equithesin i.p. and placed on a stereotaxic apparatus (model 900, David Kopf, Tujunga, CA, USA). Dialysis probes of the concentric type, 2 mm long, were made of a copolymer of acrylonitrile-sodium methallyl sulfonate (AN 69, Hospal SpA, Italy; 0.31 mm outer diameter, with 44,000 Da M.W. cutoff). Probes were lowered slowly into the rat SNr (at a 15° angle to the dorsal–ventral plane) at the following stereotaxic coordinates for the probe tip: AP⫽⫺5.6, L⫽⫾1.5 and V⫽⫺8.7 from the bregma with the incisor bar set at ⫺3.3 mm according to the Paxinos and Watson (1986) atlas. Twenty hours after probe implantation, each rat was placed in a Perspex cage and the inlet cannula connected by polythene tubing (Portex Ltd., Hythe, UK) to a 2.5 ml syringe, mounted on a CMA/100 microinjection pump (CMA Microdialysis, Stockholm, Sweden) containing artificial cerebrospinal fluid (aCSF, composition in mM: NaCl 145; KCl 3.0; CaCl2 1.26; MgCl2 1.0, buffered at pH 7.4 with 2 mM sodium phosphate buffer). The probes were perfused with aCSF at a constant flow-rate of 1 ␮l/min. After 1 h and 30 min washout, perfusate was collected every 20 min and assayed by HPLC with fluorometric detection for the measurement of GABA, essentially as previously described (Donzanti and Yamamoto, 1988). GABA was measured after derivatization with o-phthalaldehyde-based (OPA; Sigma-Aldrich, Milan, Italy) reagent. Stock derivatizing reagent was prepared by dissolving 27 mg OPA in 1 ml methanol, followed by 5 ␮l ␤-mercaptoethanol and 9 ml 0.1 M sodium tetraborate buffer (pH 9.3) prepared by dissolving 0.62 g boric acid in about 80 ml ultrapure water (MilliQ, Millipore, Milan, Italy). The pH was adjusted to 9.3 with 2–3 ml 5 M NaOH and the final volume brought to 100 ml with water. Stock reagent solution was maintained at room temperature in a darkened bottle for one week. Derivatizing reagent was prepared by diluting stock solution 1:4 with 0.1 M borate buffer, 24 h before use. On the next day, derivatizing reagent was filtered using a 0.2 ␮m cellulose acetate filter and 2 ␮l of derivatizing reagent were added to 15 ␮l of dialysate, thoroughly mixed and immediately injected into the HPLC. GABA was separated through a 4.6⫻80 mm C18 reversephase column (HR-80, ESA, Chelmsford, MA, USA). New Guard RP-18 guard column (3.2⫻15 mm; Perkin-Elmer, Shelton, CT, USA) was used to protect the analytical column. The mobile phase was as follows: 0.05 M Na2HPO4, 35% methanol, pH 6.25 with

R. W. Invernizzi et al. / Neuroscience 144 (2007) 1523–1535 85% phosphoric acid, pumped at 1.2 ml/min with a LC10-ADvp HPLC pump (Shimadzu, Milan, Italy). A solution consisting of methanol:water (80:20), was used to wash out late eluting peaks. This was connected to HPLC pumps through a three-way valve (Biggs et al., 1995; Piepponen and Skujins, 2001). Immediately after the GABA peak was recorded (retention time 10.5 min), the valve was manually switched to methanol:water solution for 2 min (washout step). The whole run (from injection to injection) took less than 15 min. GABA was measured by a fluorescence detector (F-1080; Merck-Hitachi, Düsseldorf, Germany). Excitation and emission wavelengths were 335 and 450 nm. Assay was calibrated daily by injecting 0.4 pmol/20 ␮l GABA, made up freshly in aCSF. Detection limit was 0.025 pmol on column (signal-tonoise ratio⫽2). Unless differently specified, all chemicals were analytical grade or better and were purchased from Merck (Bracco, Milan, Italy) or Carlo Erba Reagents (Milan, Italy). At the end of each experiment, rats were anesthetized with chloral hydrate (400 mg/kg i.p.) and killed by decapitation. The brains were removed and frozen on dry ice. Correct probe placement (⫾0.4 mm AP and ⫾0.1 mm L of given coordinates) was verified by visual inspection of the probe track on 40-␮m coronal sections from the SNr of each rat. Only rats with correct probe placement were considered in the results.

Drug administration protocols Electrophysiological experiments. Both RO 60-0175, a selective 5-HT2C agonist (Martin et al., 1998), and SB 243213, a potent and selective 5-HT2C antagonist (Bromidge et al., 2000), were given i.v. (via a lateral tail vein), and the effect on the activity of non-DA SNr neurons was recorded. SB 243213, dissolved in 0.9% NaCl containing 8% hydroxypropyl-␤-cyclodextrin by weight and 25 mM citric acid (␤-cyclodx), was administered as a single bolus of 50, 100 and 300 ␮g/kg, i.v., while control rats were injected with an equal volume of vehicle (100 ␮l, i.v.). RO 60-0175 (5, 5, 10, 20, 40, 80, 160, 320 ␮g/kg, i.v.) was dissolved in 0.9% NaCl and administered in exponentially increasing doses every 2 min, reaching a cumulative dose of 640 ␮g/kg. In a group of rats, SB 243213 (300 ␮g/kg, i.v.) was given 5 min before the administration of cumulative doses of RO 60-0175 to assess the specificity of this latter drug as a selective 5-HT2C agonist in our experimental conditions. Only one cell per animal was studied. In a last series of experiments, the effect of local administration of both RO 60-0175 and SB 243213 was evaluated using multibarrel micropipettes. RO 60-0175 (dissolved in 0.9% NaCl to a final concentration of 20 mM, pH 4) was applied by microiontophoresis using ejection currents of 20, 40 and 60 nA, while a retention current of ⫺10 nA was used to prevent leakage between ejection periods. SB 243213 (dissolved in ␤-cyclodx) was administered locally, by micropressure ejection at 12 p.s.i. (20 mM) 1–2 min before RO 60-0175, in order to assess the specificity of RO 60-0175 as a selective 5-HT2C agonist in our experimental conditions. It is important to point out that the microiontophoresis of SB 243213 was impossible to perform due its low solubility in water. Microdialysis experiments. Once the extracellular concentration of GABA was stable (at least three consecutive sample differing by less than 20% from the mean basal value), mCPP (1 and 3 mg/kg), RO 60-0175 (0.1, 0.3 and 1 mg/kg) or SB 243213 (0.3 mg/kg) was injected s.c. mCPP and RO 60-0175 were dissolved in saline (pH about 7) whereas SB 243213 was dissolved in ␤-cyclodx. SB 243213 solution was buffered to pH 7 with 100 ␮l of 1 M NaOH for 2 ml of solution. Control rats were injected with 2 ml/kg respective vehicles. For local perfusion through the probe, 1 mM solutions were prepared dissolving mCPP and RO 60-0175 in aCSF and SB 243213 in its vehicle as described above. These solutions were further diluted with aCSF to the final concentrations. The pH of the final solutions was about 7.4. Control rats

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were infused with normal aCSF or ␤-cyclodx diluted 1:1000 in aCSF (control groups for rats given SB 243213).

Data analysis and statistics Electrophysiological experiments. Data were acquired and stored on computer using an integrated software package for electrophysiology (RISI, Symbolic Logic, Dallas, TX, USA); offline analysis of recorded neurons was made by HiQ 4.1 (National Instruments, Austin, TX, USA), using an algorithm for spike train analysis of both baseline and post-injection periods, made by one of the authors (M.P.); statistical analyses were done using the StatView 5.0 for Windows based computers (SAS Institute Inc., Cary, NC, USA). Rate histograms were constructed integrating discharge frequencies over 10-s epochs; interspike interval histograms (ISIHs) and autocorrelograms were constructed using bin width of 1 ms. Mean firing rate was used to construct doseresponse or current-response curves, comparing post-injection periods to basal firing rate (percentage change); units showing a mean firing rate change in the range of ⫾15% were considered as responsive. Several ISIH parameters were quantitatively evaluated [mean, median, mode, skewness, kurtosis, coefficient of variation (CV), asymmetric index (AI)] in order to describe discharge patterns and quantify changes in firing patterns after drugs administration (Fedrowitz et al., 2003). Drug-induced modifications in mean, median and mode of ISIHs were evaluated as percentage changes, compared with basal values, while the other parameters were considered as absolute values. In addition, pattern analysis was implemented with the Poisson “surprise” algorithm described by Legéndy and Saleman (1985) to evaluate the degree of burstiness of recorded units; the number of bursts, percentage of spikes in burst and the number of spikes within burst were calculated for each considered segment of spike trains. Absolute changes in these bursting indexes [i.e. the difference (⌬) between post-injection and baseline periods] were used as a measure of drug-induced modifications in bursting activity. ISIHs and autocorrelograms computed from baseline period of at least 1000 intervals, together with ISIH parameters and visual analysis of raster plots, were used to classify single SNr neurons according to their firing patterns of activity (Murer et al., 1997). Data obtained from mean firing rate were subjected to a two-way ANOVA (analysis of variance) for repeated measures; ISIHs parameters and bursting indexes of the highest dose or current were compared between groups by one-way ANOVA for repeated measures; when significant effects were found, post hoc comparisons were made with Fisher’s PLSD (protected least significant difference) test. In microiontophoresis experiments, the effects of the highest current of RO 60-0175 on ISIH parameters and bursting indexes were compared with their basal values by either one-sample t-test or paired t-test. Microdialysis experiments. Dialysate GABA levels, not corrected for in vitro recovery, were expressed as pmol/20 ␮l. Data were analyzed by ANOVA for repeated measures with time as within factor and treatments as between factors. Post hoc comparisons were made with Tukey-Kramer’s test. Interaction between 5-methyl-1-({2[(2-methyl-3-pyridyl)oxy]-5-pyridyl}carbamoyl)-6-trifluoromethylindoline (SB 243213) and RO 60-0175 was further analyzed by comparing the area under the curve (AUC) calculated on the percentages of basal values with the Student’s t-test. Statistical analyses were done on raw data using the StatView 5.0 for Apple Macintosh computer (SAS Institute Inc., Cary, NC, USA).

Drugs 2-Hydroxypropyl-␤-cyclodextrin and mCPP were from Sigma (St. Louis, MO, USA); RO 60-0175 [Hoffmann-La Roche, Basel, Switzer-

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Fig. 1. Spontaneous patterns of activity of single units recorded extracellularly in the rat SNr. Each panel represents one single unit, characterized by its ISIH (upper panel), autocorrelogram (middle panel), and the digital raster plot (lower panel) of a representative segment of signal from which they were computed. (A) Regular unit, showing a narrow symmetric ISIH and several peaks in the autocorrelogram. (B) Slightly irregular unit, characterized by a larger symmetric ISIH and by only two peaks in the autocorrelogram. (C) Irregular unit with asymmetric ISIH and flat autocorrelogram. (D) Bursty unit, showing strongly asymmetric and widely distributed ISIH, and a flat autocorrelogram with a single long lasting initial peak.

land] was kindly donated by Dr. Eva-Maria Gutknech, F. Hoffmann-La Roche Ltd.; SB 243213 was a gift from Dr. Martyn Wood, GlaxoSmithKline, Harlow, UK. All drugs dosages refer to the weight of the salt.

RESULTS Characteristics of the spontaneous activity of extracellularly recorded SNr neurons A total number of 129 neurons were recorded from SNr showing both biphasic and triphasic spike waveforms with durations of about 1.5 ms, resembling SNr GABAergic neurons already described in scientific literature.

Recorded neurons showed different basal discharge patterns: on the basis of their ISIHs, autocorrelograms and raster plots they were classified as regular (N⫽16; 12.4% of total), slightly irregular (N⫽27; 20.9% of total), irregular (N⫽80; 62.0% of total) and bursty (N⫽6; 4.7% of total) units. Fig. 1 shows representative ISIHs, autocorrelograms and raster plots for each recognized pattern of activity. The degree of burstiness for each unit was also detected (see Experimental Procedures). Statistical analysis did not reveal any significant difference in the mean basal firing rates among the four different recognized patterns (F(3,125)⫽0.36; P⫽0.78) (Table 1), although mean values showed a slight tendency for

Table 1. Basal electrophysiological characteristics of total SNr neurons recorded Characteristic

Regular

Slightly irregular

Irregular

Bursty

Number of neurons Mean firing (Hz) Mean ISI (ms) Median ISI (ms) Mode ISI (ms) Skew ISI Kurtosis ISI AI ISI CV ISI Number of bursts % Spike in burst Number of spikes in bursts

16 22.66⫾1.67 48.77⫾4.42 47.09⫾4.21 45.38⫾4.29° 1.11⫾0.14°° 7.38⫾0.65 0.93⫾0.01** 0.29⫾0.01** 2.31⫾1.6** 0.12⫾0.06** 5.81⫾4.24**

27 20.51⫾1.39 54.43⫾3.58 51.15⫾3.36 47.19⫾3.05°°* 1.61⫾0.26°° 13.73⫾5.76 0.87⫾0.02** 0.36⫾0.02** 3.22⫾0.88** 0.72⫾0.22** 23.48⫾7.01**

80 21.11⫾0.91 54.58⫾2.39 48.37⫾2.01 38.3⫾1.87 1.68⫾0.13°° 11.05⫾2.43 0.7⫾0.02°° 0.52⫾0.02°° 18.88⫾2.52°° 5.27⫾0.74°° 151.75⫾20.37°°

6 19.45⫾2.5 55.79⫾6.96 40.17⫾4.61 26.33⫾4.73 2.97⫾0.56 22.38⫾8.26 0.48⫾0.08 0.87⫾0.16 87.67⫾17.82 28.4⫾7.42 863.67⫾215.47

Data represent mean value of firing rates, ISIH parameters and bursting activity indexes for the four recognized discharge pattern types. Comparisons between different firing patterns for each parameter mean value were made by one-way ANOVA followed by Fisher’s PLSD. * P⬍0.05 bursty vs. irregular. ** P⬍0.01 vs. irregular, bursty. ° P⬍0.05 vs. bursty. °° P⬍0.01 vs. bursty.

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Fig. 2. Effect of systemic administration of RO 60-0175 and SB 243213 on the firing rate of SNr neurons. (A) Representative rate histograms showing the effects elicited by i.v. administration of cumulative doses of RO 60-0175 (5, 10, 20, 40, 80, 160, 320, 640 ␮g/kg, at arrows) or saline (100 ␮l, at each arrow) which represented the control group. The last trace shows the effect of the same cumulative doses of RO 60-0175 after pretreatment (5 min before) with the selective 5-HT2C antagonist SB 243213 (300 ␮g/kg, i.v.). (B) Cumulative dose-response curves showing the mean percentage changes (⫾S.E.M.) in firing rate of SNr neurons elicited by administration of RO 60-0175 alone and after pretreatment with SB 243213. Statistical analysis evidenced a significant difference between both excitatory (N⫽5) and inhibitory (N⫽5) effects compared with the control group (N⫽5), that were abolished by pretreatment with SB 243213 (300 ␮g/kg, i.v.). ° P⬍0.05, °° P⬍0.01 vehicle⫹RO 60-0175 vs. vehicle⫹saline; * P⬍0.05, ** P⬍0.01 vehicle⫹RO 60-0175 vs. SB 243213⫹RO 60-0175 (Fisher’s PLSD).

regular neurons to fire at higher rates then irregular and bursty ones. Furthermore, chi-square test did not evidence any association between the described discharge patterns and the observed spike waveforms or the depth (relative to DA-ergic neurons) of extracellularly recorded SNr neurons. Effect of systemic administration of SB 243213 on the firing rate and pattern of SNr neurons In order to evaluate the effect of the systemic administration of the selective 5-HT2C antagonist SB 243213 on the activity of SNr neurons, the doses of 50 ␮g/kg (N⫽5), 100 ␮g/kg (N⫽6) and 300 ␮g/kg (N⫽7) were injected i.v. as a single bolus, and compared with the effect of its vehicle (␤-cyclodx, 100 ␮l i.v.; N⫽6). SB 243213 was inactive on the basal electrophysiological activity of SNr neurons (not shown).

Effect of systemic administration of cumulative doses of RO 60-0175 on the firing rate and pattern of SNr recorded neurons In order to evaluate the effect of the systemic administration of the selective 5-HT2C agonist RO 60-0175, a group of 16 rats was treated with cumulative doses of this drug ranging from 5 to 640 ␮g/kg (i.v.), while a control group of six rats was injected with the same volumes of saline. Thus, systemic administration of RO 60-0175 caused either an increase (N⫽5), a decrease (N⫽5) or had no effect (N⫽6) on the basal activity of SNr neurons (Fig. 2). The increase reached a peak of 22.6⫾7.9% (above baseline) at the cumulative dose of 160 ␮g/kg, while the decrease reached its maximum of 28.2⫾6.5% (below baseline) at the cumulative dose of 640 ␮g/kg. Statistical analysis of dose-response curves revealed a significant effect of RO 60-0175 for both the increasing and decreasing effects

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(treatments by dose F(28,189)⫽5.02; P⬍0.01), as compared with the control group injected with saline (100 ␮l i.v.; N⫽5). Systemic administration of RO 60-0175 did not cause any significant change in the firing pattern of SNr neurons (not shown). Furthermore, no significant associations were found among waveform, basal firing rate and pattern, location of SNr neurons and the effects elicited by systemic administration of RO 60-0175 (not shown). SB 243213 blocked the effects elicited by systemic administration of cumulative doses of RO 60-0175 on the firing rate of SNr neurons The potent and selective 5-HT2C receptor antagonist SB 243213 (300 ␮g/kg, i.v.) was given 5 min before administration of cumulative doses of RO 60-0175 (5– 640 ␮g/kg, i.v.) to assess the specificity of this latter drug as a selective 5-HT2C agonist in our experimental conditions. The dose of 300 ␮g/kg of SB 243213 was chosen on the basis of preliminary experiments showing that it did not modify the basal firing rate of SNr neurons. Nevertheless, pretreatment with SB 243213 prevented both excitatory and inhibitory (treatment by dose F(28,189)⫽5.02; P⬍0.01) effects elicited by RO 60-0175 on the firing rate of SNr neurons (N⫽10; Fig. 2). Effect of microiontophoretic application of RO 60-0175 on the firing rate and pattern of SNr neurons Fig. 3 shows the effect of microiontophoretically applied RO 60-0175 on the firing rate of SNr neurons (N⫽53). Local application of RO 60-0175 (20 – 60 nA for 1 min) caused an increase in the basal activity of the majority of SNr neurons (N⫽25) tested. Moreover, microiontophoretically applied RO 60-0175 elicited a decrease in the basal firing rate of few SNr neurons (N⫽9), whereas it did not cause any significant effect in the remaining SNr neurons recorded (N⫽19). When the microiontophoretic current was applied for a period of 3 min, a biphasic effect (i.e. excitation followed by inhibition) was usually observed (N⫽4) (Fig. 3). As can be seen in Fig. 3, both the excitatory and the inhibitory effects of RO 60-0175 were related to the amount of applied current. The mean increase in firing rate in the group of neurons which was excited by RO 60-0175 was statistically significant (dose F(2,48)⫽29.84; P⬍0.01), reaching a peak of 33.8⫾5.0% (above baseline) at the ejection current of 60 nA. The inhibitory effect elicited by RO 60-0175 was also statistically significant (dose F(2,16)⫽17.37; P⬍0.01), and reached a peak of 37.5⫾ 6.5% (below baseline) at the ejection current of 60 nA (Fig. 3). In order to evaluate the effect of locally applied RO 60-0175 on discharge pattern of SNr neurons, mean values of ISIHs parameters and bursting indexes elicited by the highest ejection current (60 nA) were statistically analyzed. Thus, microiontophoretically applied RO 60-0175 did not cause any significant change in the firing pattern of SNr neurons (not shown). Furthermore, no significant associations were found among waveform, firing rate and pattern, location of SNr neurons and the effects elicited by local ejection of RO 60-0175 (not shown).

Local application of SB 243213 by micropressure ejection prevented the effects elicited by microiontophoretically applied RO 60-0175 on the firing rate and pattern of SNr neurons The selective 5-HT2C antagonist SB 243213 was applied by micropressure ejection (12 p.s.i.) 1–2 min before microiontophoretic administration of RO 60-0175 (20 – 60 nA) (N⫽6) in order to verify the receptor selectivity of the locally administered 5-HT2C agonist. Local application of SB 243213 counteracted both excitatory and inhibitory (treatment by current F(5,74)⫽18.19; P⬍0.01) effects exerted by microiontophoresis of RO 60-0175 on the firing rate of SNr neurons (Fig. 3). These data show that SB 243213 applied locally in the SNr by microejection pressure was capable of antagonizing the effects of local administration of RO 60-0175, indicating an involvement of 5-HT2C receptor subtype in the effects elicited by this drug on SNr neurons. Basal extracellular GABA: effect of tetrodotoxin (TTX) Extracellular GABA levels were stable about 1 h 30 min after the beginning of probe perfusion. Mean basal GABA concentrations in the SNr, obtained by pooling basal value of different experimental groups, were 0.150⫾0.004 pmol/20 ␮l (N⫽142). No significant differences were found among basal GABA levels in the different groups (F(24,117)⫽1.5, P⫽0.07). Fig. 4 shows that 1 ␮M TTX reduced extracellular GABA by about 60% of basal levels (treatment F(6,36)⫽5.8, P⬍0.01). The effect was significant from 40 to 120 min. Effect of systemic administration of mCPP and RO 60-0175 Extracellular GABA levels were not significantly modified by the injection of saline over the 2 h observation period. S.c. administration of 3 mg/kg mCPP significantly increased extracellular GABA (treatment F(2,15)⫽7.3, P⬍ 0.01, Fig. 5A). The effect reached about 140% at 20 min, was back to basal levels at 40 min and rose again at 60-80 min (100% above baseline levels). One milligram per kilogram mCPP had no significant effect. The effects of systemic administration of RO 60-0175 (0.1, 0.3 and 1 mg/kg, s.c.) on extracellular GABA levels are shown in Fig. 5B. RO 60-0175, at 1 mg/kg, significantly increased extracellular GABA (treatment F(3,17)⫽17.8, P⬍0.01). GABA rose by about 100% at 20 min, returned to basal levels at 40 min and reached the maximum increase 60-80 min after injection (200% above baseline levels). Lower doses of RO 60-0175 (0.1 and 0.3 mg/kg) had no significant effects. Effect of intranigral mCPP, RO 60-0175 and SB 243213 on extracellular GABA in the SNr Fig. 6 shows that mCPP and RO 60-0175 infused through the probe into the SNr significantly raised extracellular GABA levels in this nucleus (treatment by time interaction for mCPP and RO 60-0175 was, respectively, F(12,78)⫽6.3,

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Fig. 3. Effect of local application of RO 60-0175 and SB 243213 on the firing rate of SNr neurons. (A) Typical rate histograms showing excitatory (N⫽25; first trace), inhibitory (N⫽9; second trace), biphasic (N⫽4; third trace) or null (N⫽19; fourth trace) effect elicited by microiontophoretically applied RO 60-0175. Local co-application of SB 243213 counteracted both the excitatory and inhibitory effects elicited by microiontophoretically applied RO 60-0175. (A) Representative rate histogram showing that local administration of SB 242313 by micropressure ejection (12 p.s.i.; ejection time indicated by largest horizontal bar), 1–2 min before, prevented both excitatory (fifth trace) and inhibitory (sixth trace) effects elicited by locally applied RO 60-0175 on the firing rate of SNr neurons. Numbers above each bar indicate the ejecting currents in nA. (B) Current-response curves showing the mean percentage changes (⫾S.E.M.) in firing rate, elicited by local administration of RO 60-0175 alone or SB 243213⫹RO 60-0175 (N⫽6) on SNr neurons. Statistical analysis evidenced a significant effect of RO 60-0175 for both stimulatory and inhibitory effects. ** P⬍0.01 vs. 20 nA (Fisher’s PLSD). Micropressure ejection of SB 243213 significantly counteracted the stimulatory and inhibitory effects exerted by locally applied RO 60-0175 on the firing rate of SNr neurons. °° P⬍0.01 vs. SB 243213⫹RO 60-0175 (Fisher’s PLSD).

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(F(7,84)⫽4.6, P⬍0.01). This was probably due to the opposite effect of SB 243213 on RO 60-0175-induced rise of extracellular GABA at 20 and 100 min. Thus, further analysis comparing the AUCs calculated in the period 80 –140 min showed that SB 243213 antagonized RO 60-0175induced rise of GABA (mean AUC⫾S.E.M., RO 60-0175 alone, 9980⫾450; SB 243213⫹RO 60-0175, 6581⫾665; P⬍0.01, Student’s t-test) whereas it enhanced the rise of GABA observed at 20 min (P⬍0.05, Student’s t-test) (Fig. 5D).

DISCUSSION

Fig. 4. Effect of 1 ␮M TTX on the extracellular levels of GABA in the SNr (N⫽7). Data represent mean⫾S.E.M. Horizontal bar indicates the duration of TTX infusion; * P⬍0.05 vs. basal values (Tukey-Kramer’s test).

P⬍0.01, F(2,72)⫽4.8, P⬍0.01). The effect was significant at the concentrations of 100 ␮M mCPP (80% above baseline levels) and 1 ␮M RO 60-0175 (140% above baseline levels). Although the infusion was continued for 2 h, the increase of GABA was short-lasting being significant 20 min after the beginning of the infusion of both drugs. Lower concentrations of mCPP (10 ␮M) and RO 60-0175 (0.1 ␮M) had no effects. Fig. 7A shows that SB 243213 (0.3 mg/kg, s.c.) or vehicle had no significant effect on extracellular GABA (treatment F(1,10)⫽3.1, P⫽0.09). Infusion of 1 ␮M SB 243213 through the probe did not affect extracellular GABA (treatment F(1,9)⫽1.1, P⫽0.33) (Fig. 7B). Blockade of 5-HT2C receptor antagonized the effect of RO 60-0175 on extracellular GABA: differences between systemic and intranigral SB 243213 RO 60-0175 (1 mg/kg, s.c.) raised by about twofold extracellular GABA in rats given ␤-cyclodx (Fig. 5C). The effect was significant (vs. baseline values) at 40, 100 and 120 min. Overall, ANOVA showed a significant interaction (treatments by time F(7,126)⫽5.5, P⬍0.01). SB 243213 (0.3 mg/kg) completely antagonized the rise of extracellular GABA induced by 1 mg/kg RO 60-0175 (RO 60-0175 alone vs. SB 243213⫹RO 60-175; F(1,10)⫽11.5, P⬍0.01). The effect of intra-SNr infusion of SB 243213 on RO 60-0175-induced rise of extracellular GABA is shown in Fig. 5D. The infusion of 1 ␮M SB 243213 into the SNr antagonized the effect of 1 mg/kg RO 60-0175 on extracellular GABA (treatment by time interaction F(7,140)⫽ 2.5, P⫽0.02). Comparison between RO 60-0175 alone and SB 243213⫹RO 60-0175 showed no significant differences between treatments (F(1,12)⫽0.4, P⬎0.05) but a significant interaction between treatment and time

The present study was undertaken with the aim to elucidate the role played by 5-HT2C receptors in the regulation of neuronal function in the rat SNr. Although the neurochemical identity of the neurons recorded in the SNr was not established, it is likely that the majority of them are GABA-ergic. Thus, combined electrophysiological and immunocytochemical experiments have demonstrated that most non-DA neurons in the SNr are indeed GABA-ergic (Hajós and Greenfield, 1994; Richards et al., 1997; Gonzalez-Hernandez and Rodriguez, 2000). One important feature of this study is the use of two different techniques to investigate the control exerted by 5-HT2C receptors on GABA-ergic transmission in the SNr: the in vivo electrophysiology that permits following of the changes in the firing rate of putative GABA-containing neurons and the microdialysis technique, which allows monitoring of extracellular GABA in awake freely-moving animals. Although the results are not directly comparable in terms of experimental conditions, two important aspects of the effect of 5-HT2C receptors on GABA-ergic neurotransmission (firing activity and neurotransmitter release) are assessed for the first time in the same study. These techniques combined with systemic and local administration of 5-HT2C receptor agonists and antagonists permit the investigation of the role of nigral and extranigral 5-HT2C receptors in controlling nigral GABA-ergic transmission. However, it is important to point out that electrophysiological techniques allow the recording of the electrical activity of the somato-dendritic component of SNr neurons, whereas the source of GABA in the SNr might be of different origins: i.e. it might derive from the somato-dendritic release of GABA-containing neurons in the SNr, from their recurrent collaterals, from striato-nigral GABA-ergic axons or from GP (Albin et al., 1989; Alexander and Crutcher, 1990; Mink, 1996; Celada et al., 1999; Maurice et al., 2003; Windels et al., 2005). Another important point to consider is that electrophysiological experiments are carried out in chloral-hydrate-anesthetized animals, whereas microdialysis experiments are conducted on unanesthetized rats. Unfortunately, this issue is presently unsolvable inasmuch as the methodology of electrophysiology in awake unanesthetized animals is not available in our laboratory. The fact that the selective 5-HT2C antagonist SB 243213 did not cause any change in the basal activity of SNr neurons suggests that 5-HT2C receptors do not exert any tonic control upon these neurons. Systemic adminis-

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Fig. 5. (A) Extracellular levels of GABA in the SNr of rats given 1 (N⫽7), 3 mg/kg (N⫽5) mCPP or saline (N⫽6) or (B) 0.1 (N⫽4), 0.3 (N⫽4) and 1 mg/kg (N⫽7) RO 60-0175 or saline (N⫽6). Arrow indicates the injection of mCPP, RO 60-0175 or saline. (C) The 5-HT2C receptor antagonist SB 243213 prevents the stimulatory effect of RO 60-0175 on extracellular GABA in the SNr. Rats were injected s.c. with 0.3 mg/kg SB 243213 or vehicle (first arrow) 20 min before 1 mg/kg RO 60-0175 or saline (N⫽5 or 6 per group). (D) SB 243213 (1 ␮M) was infused through the probe 20 min before s.c. RO 60-0175 (1 mg/kg) or saline (2 ml/kg) and infusion maintained for 140 min (N⫽5 or 7 per group). Data represent mean⫾S.E.M. * P⬍0.05 vs. saline; ° P⬍0.05 vs. vehicle⫹saline (Tukey-Kramer’s test).

tration of the selective 5-HT2C receptor agonist RO 600175 caused a dose-dependent excitation of about 1/3 of the SNr neurons recorded. However, the remaining 2/3 of neurons were either inhibited or unaffected by systemic RO 60-0175. Both the excitatory and the inhibitory effects

of RO 60-0175 were completely prevented by pretreatment with SB 243213, thus indicating that both effects were mediated by 5-HT2C receptors. Since RO 60-0175 was administered systemically, it is impossible to establish whether the effects of this 5-HT2C receptor agonist were

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inhibition) was evoked, probably as a consequence of local GABA release elicited by sustained stimulation of GABAcontaining neurons in the SNr. Nevertheless, a small group of SNr neurons recorded (nine of 53) were inhibited by microiontophoretically applied RO 60-0175. Both the excitatory and the inhibitory effects elicited by local application of RO 60-0175 were completely prevented by micropressure ejection of SB 243213 directly into the SNr. These latter data indicate that activation of 5-HT2C receptors expressed by neurons in the SNr is mostly associated with an excitatory response, although an inhibitory response

Fig. 6. (A) Extracellular levels of GABA in the SNr of rats given 10 ␮M (N⫽6), 100 ␮M (N⫽5) mCPP or aCSF (N⫽5) or (B) 0.1 ␮M (N⫽5), 1 ␮M (N⫽5) RO 60-0175 or aCSF (N⫽5) through the probe. Data represent mean⫾S.E.M. Horizontal bar indicates the duration of mCPP or RO 60-0175 infusion. * P⬍0.05 vs. aCSF (Tukey-Kramer’s test).

mediated directly by stimulation of the receptors present on GABA-containing neurons in the SNr or if they resulted from an indirect effect on other neurons (e.g. pallidal, striatal) impinging on SNr. It is, however, conceivable that the prevalent effect of 5-HT2C receptor activation in the SNr is stimulatory, inasmuch as the direct application of RO 60-0175 in the SNr by microiontophoresis caused excitation in about half of the neurons tested (25 of 53). When the microiontophoretic current was applied for a period of 3 min, a biphasic effect (i.e. excitation followed by

Fig. 7. (A) Extracellular levels of GABA in the SNr of rats given 0.3 mg/kg SB 243213 (N⫽5) or vehicle (N⫽6). Arrow indicates the injection of SB 243213 or vehicle. (B) Extracellular levels of GABA in the SNr of rats given 1 ␮M SB 243213 (N⫽7) or vehicle (N⫽4) through the probe. Horizontal bar indicates the duration of SB 243213 infusion. Data represent mean⫾S.E.M.

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might also be elicited in a minority of neurons. Apparently, these different responses (i.e. excitation versus inhibition) are not dependent on the different basal electrophysiological characteristics of SNr neurons, and it is presently impossible to establish if they are mediated by diverse subpopulations of these neurons. However, since the majority of SNr neurons are excited by local application of RO 60-0175, it is conceivable that the primary effect of 5-HT2C receptor stimulation would be excitatory. Thus, it is unlikely that RO 60-0175-induced reduction in basal firing rate of SNr neurons is mediated by inhibitory 5-HT2C receptors. It is therefore conceivable that RO 60-0175-induced inhibition of SNr neurons might derive from the stimulation of inhibitory neurons (e.g. GABA-ergic) expressing 5-HT2C receptors and impinging on the SNr cell recorded. Moreover, it is important to note that a subpopulation of SNr neurons was not affected by RO 60-0175, thus confirming neuroanatomical data showing that about half of SNr neurons do not express 5-HT2C receptors (Eberle-Wang et al., 1997). The present findings confirm and extend previous in vitro and in vivo data showing that 5-HT exerts a direct excitatory effect on GABA-ergic neurons in the SNr by acting on 5-HT2C receptors (Rick et al., 1995; Stanford and Lacey, 1996; Di Giovanni et al., 2001). These electrophysiological data are consistent with the present findings showing that the 5-HT2C receptor agonist RO 60-0175, administered s.c. at the dose of 1 mg/kg, markedly increased extracellular GABA levels in the SNr. Moreover, a similar pattern of increase in extracellular GABA levels was produced by administration of mCPP, an unselective 5-HT2C receptor agonist which was previously found to increase the basal firing rate of non-DA neurons in the SNr by specifically acting on 5-HT2C receptors (Di Giovanni et al., 2001). The effects of RO 60-0175 and mCPP on GABA release could be partly due to the firing-dependent release of the neurotransmitter from GABA-ergic neurons of the SNr. Consistently with this hypothesis, basal extracellular levels of GABA in the SNr are reduced by 60-80% in rats infused with 1 ␮M TTX (Biggs et al., 1995; Osborne et al., 1991; present study) suggesting that GABA release in the SNr is to a large extent dependent on neuronal activity. It is interesting to note that both mCPP and RO 600175 caused a biphasic increase in GABA release, with an early peak at 20 min, a return to almost basal level at 40 min, and a second peak 80 min after systemic administration of the drugs. It is tempting to speculate that the multiple sources of GABA released in the SNr are at the basis of the biphasic effect elicited by both mCPP and RO 600175 on nigral GABA outflow. Thus, it is conceivable that the initial increase in GABA release induced by both 5-HT2C agonists would trigger some compensatory inhibitory mechanism which would bring GABA output almost to the basal levels. It is likely that the second wave of GABA release would occur when this compensatory mechanism subsides and/or it is overwhelmed by the stimulatory action of mCPP and RO 60-0175 on 5-HT2C receptors in the SNr. The fact that systemic SB 243213 completely antagonized

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RO 60-0175-induced rise of extracellular GABA at 20 and 80 min strongly support the role of 5-HT2C receptors in these effects. It was also found that the infusion of RO 60-0175 and mCPP through the probe significantly increased extracellular GABA levels in the SNr. This suggests that the effects of RO 60-0175 and mCPP on extracellular GABA are at least partly related to a direct action of the drugs on 5-HT2C receptors of the SNr. In line with this interpretation, Eberle-Wang et al. (1997) found that 5-HT2C receptors mRNA and glutamic acid decarboxylase (GAD) mRNA were expressed by the same neurons in the SNr. Thus, it is conceivable that RO 60-0175 by stimulating 5-HT2C receptors of the SNr increases the activity of GABA-ergic neurons and the release of the neurotransmitter. The increase of extracellular GABA levels in response to systemic RO 60-0175 lasted longer (80 min) than after intranigral infusion (20 min). Although this may reflect a rapid elimination of the drug after intranigral infusion, an action of systemic RO 60-0175 on nigral and extranigral 5-HT2C receptors may also account for this finding. Thus, both mRNA and binding sites for 5-HT2C receptors have been found in various regions of the rat brain including several basal ganglia structures such as striatum, entopeduncular nucleus, the GP, and the STN (Mengod et al., 1990; Pompeiano et al., 1994; Ward and Dorsa, 1996; Eberle-Wang et al., 1997; Di Giovanni et al., 2006) and electrical stimulation of the caudate nucleus, the GP, the internal capsule, and the STN increased tritiated or endogenous GABA release in the SNr (Biggs et al., 1995; Gauchy et al., 1980; Kondo and Iwatsubo, 1978; Windels et al., 2000). It is therefore likely that GABA-containing afferents to SNr contribute to GABA release induced by systemic RO 60-0175 and mCPP. Interestingly, the local administration of 1 ␮M SB 243213 directly into the SNr through the microdialysis probe blocked only the increase of extracellular GABA at 80 min. This suggests that the control exerted by 5-HT2C receptors on extracellular GABA in the SNr likely involves both intra- and extra-nigral components. The mechanism by which RO 60-0175-induced rise of extracellular GABA at 20 min was enhanced by intra-nigral SB 243213 was not addressed in the present study. Therefore, we can only speculate that this results from the opposite control exerted by nigral and extranigral 5-HT2C receptors on extracellular GABA. In line with this interpretation, 5-HT2C receptors may have opposite effects on DA-mediated functions such as cocaine-induced locomotor activity (McMahon et al., 2001).

CONCLUSION In conclusion, this study shows that selective activation of 5-HT2C receptors stimulates non-DA neurons in the SNr and causes a marked and sustained increase in GABA extracellular levels in the SNr. It is interesting to note that a similar increase in GABA release was found after high frequency stimulation (HFS) of the STN both in normal and 6-hydroxydopamine-lesioned rats (Windels et al., 2005), which represents an animal model of Parkinson’s disease (PD). Inasmuch as HFS of the STN increases GABA re-

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lease in the SNr and represents an effective treatment of PD, it is conceivable that a similar effect might be exerted by 5-HT2C receptor agonists which cause a similar increase in extracellular GABA. There is also evidence that 5-HT increases the firing rate of STN neurons by acting, in part, through 5-HT2C receptors (Xiang et al., 2005). It is therefore plausible that pharmacological stimulation of 5-HT2C receptors in the STN might have effects similar to HFS. Acknowledgments—This work was supported by Tourette’s Syndrome Association.

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(Accepted 3 November 2006) (Available online 8 December 2006)