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m-Chlorophenylpiperazine excites non-dopaminergic neurons in the rat substantia nigra and ventral tegmental area by activating serotonin-2C receptors
111 Effect of mCPP on non-DA neurons in the SNr and VTA Neuroscience Vol. 103, No. 1, pp. 111±116, 2001
Pergamon
PII: S0306-4522(00)00561-3
q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00
www.elsevier.com/locate/neuroscience
m-CHLOROPHENYLPIPERAZINE EXCITES NON-DOPAMINERGIC NEURONS IN THE RAT SUBSTANTIA NIGRA AND VENTRAL TEGMENTAL AREA BY ACTIVATING SEROTONIN-2C RECEPTORS G. DI GIOVANNI, a V. DI MATTEO, a V. LA GRUTTA b and E. ESPOSITO a* a
Istituto di Ricerche Farmacologiche ªMario Negriº, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy b Istituto di Fisiologia Umana ªG. Paganoº, UniversitaÁ di Palermo, 90134 Palermo, Italy
AbstractÐIn vivo electrophysiological techniques were used to study the effect of m-chlorophenylpiperazine, a nonselective serotonin-2C receptor agonist, on the activity of non-dopaminergic neurons in the substantia nigra pars reticulata and the ventral tegmental area of anesthetized rats. Intravenous administration of m-chlorophenylpiperazine (5±320 mg/kg) caused a dose-dependent increase in the basal ®ring rate of a subpopulation of nigral neurons which do not respond to a footpinch stimulus [P(0) neurons], whereas it did not affect the activity of neurons which are responsive to the footpinch [P(1) neurons]. However, m-chlorophenylpiperazine (5±320 mg/kg) excited all non-dopaminergic neurons sampled in the ventral tegmental area. Moreover, microiontophoretic application of m-chlorophenylpiperazine (10±40 nA) caused an excitation of P(0) nigral and ventral tegmental area neurons. Pretreatment with the selective serotonin-2C receptor antagonist SB 242084 (200 mg/kg, i.v.) completely blocked the excitatory effect of i.v. m-chlorophenylpiperazine (5±320 mg/kg), both in the substantia nigra pars reticulata and in the ventral tegmental area. It is concluded that stimulation of serotonin-2C receptors by m-chlorophenylpiperazine activates non-dopaminergic (presumably GABA-containing) neurons in the substantia nigra pars reticulata and ventral tegmental area. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: GABAergic neurons, zona reticulata, ventral tegmental area, 5-HT2C receptors, interneurons, electrophysiology.
receptors, 1,11 its inhibitory effect on DA neurons in the VTA was completely prevented by the potent and selective 5-HT2C antagonist, SB 242084 {6-chloro-5-methyl-1-[2(2-methylpyridyl-3-oxy)-pyrid-5-yl-carbamoyl]indoline}.3 Therefore, it appears that mCPP reduces the mesolimbic DA function by acting on 5-HT2C receptors. 3 These data are consistent with the distribution of 5-HT2C receptors. 5,12,13 Moreover, it has recently been found that 5HT2C receptor mRNA is expressed by GABAergic neurons but not by DA neurons within the substantia nigra and VTA. 5 Therefore, it is conceivable that the effect of 5HT2C agonists on DA cell activity in the SNc and VTA would be mediated indirectly by GABAergic neurons. In this study, in vivo electrophysiological techniques were used to investigate the effects of the 5-HT2C receptor agonist mCPP on non-DA neurons. Extracellular single-unit recordings were performed from non-DA (presumably GABAergic) neurons in the SNr and VTA. Moreover, the effect of pretreatment with the selective 5-HT2C receptor antagonist SB 242084 on mCCP-induced changes in the ®ring rate of non-DA neurons was also evaluated.
Serotonin (5-HT)-containing neurons originating from the midbrain raphe nuclei innervate both the substantia nigra and the ventral tegmental area (VTA). Thus, neuroanatomical studies have shown a high density of 5-HT-immunoreactive ®bers in the substantia nigra pars compacta (SNc) and pars reticulata (SNr), and the VTA. 10,14,15 Serotonergic terminals make synaptic contacts with both dopaminergic (DA) and non-DA neurons in the SNc, SNr and VTA. 10,15 Several studies from our laboratory have shown that the serotonergic system controls the activity of DA neurons in the VTA by acting through 5HT2C receptors. 3,17,18 Thus, m-chlorophenylpiperazine (mCPP), a non-selective 5-HT2C receptor agonist, reduced the basal ®ring rate of dopamine-containing neurons in the VTA. 18 However, mCPP caused only a slight inhibition of DA neurons in the SNc (217.2 ^ 14.5%) compared to the VTA (242.6 ^ 12.8%). 3 Although mCPP is not an ideal ligand for 5-HT2C receptors, in that it also binds to 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2B *Corresponding author. Tel.: 139-872-570274; fax: 139-872578240. E-mail address: [email protected] (E. Esposito). Abbreviations: DA, dopaminergic; 5-HT, 5-hydroxytryptamine (serotonin); mCPP, m-chlorophenylpiperazine; P(1), nigral neurons that are excited by footpinch; P(0), nigral neurons that are not affected by footpinch; SB 242084, 6-chloro-5-methyl-1-[2-(2-methylpyridyl-3-oxy)-pyrid-5-yl-carbamoyl]indoline; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area.
EXPERIMENTAL PROCEDURES
Animals Male Sprague±Dawley rats (Consorzio Mario Negri Sud, Italy) weighing 300±350 g were used. Animals were kept at constant room temperature (21 ^ 28C) and relative humidity (60%) with a 111
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Fig. 1. Effect of mCPP on the ®ring rate of non-DA neurons in the SNr. (A) Representative rate histogram showing the typical excitatory effect produced by i.v. administration of mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on a P(0) neuron in the SNr. (B) Lack of effect of i.v. mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on a P(1) neuron in the SNr. (C) Cumulative dose± response curves showing the mean percentage changes (^S.E.M.) in ®ring rate of non-DA neurons after i.v. administration of mCPP, which signi®cantly enhanced the ®ring rate of P(0) neurons (n 10). The effect of mCPP on P(1) neurons (n 8) was not statistically signi®cant. *P , 0.05, **P , 0.01 compared to basal ®ring rate (one-way ANOVA, followed by Tukey's test).
12-h/12-h light±dark cycle (dark from 8.00 p.m.), and had free access to water and food. Procedures involving animals and their care were conducted in conformity with institutional guidelines that are in compliance with national (D.L. no. 116, G.U., Suppl. 40, 18 February 1992) and international laws and policies [EEC Council Directive 86/609, OJ L 358,1, 12 December 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication no. 85-23, 1985 and Guidelines for the Use of Animals in Biomedical Research (1987) Thromb. Haemost. 58, 1078±1084]. All efforts were made to minimize animal suffering and to reduce the number of animals used. 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±378C by a thermostatically regulated heating pad. The coordinates, relative to the interaural line, for placement of the recording electrode in the areas studied were, for the SNr: A 2.7±3.4 mm, L 1.8±2.2 mm, V 7±8 mm; for the VTA: A 2.7±3.4 mm, L 0.3±0.5 mm, V 7± 8 mm (to the level of exposed tissue). 16 Extracellular recordings
were performed by using either single- or ®ve-barreled micropipettes. The single micropipettes, measuring 1 mm at the tip, were ®lled with 2% Pontamine Sky Blue dye in 2 M NaCl (in vitro resistance 4±7 MV). Five-barreled micropipettes were pulled to an optimal wide tip angle and mechanically beveled under microscopic control to a ®nal tip diameter of 4±5 mm. The protruding center barrel, ®lled with 2% Pontamine Sky Blue dye in 2 M NaCl, was used for recording (in vitro resistance 4±7 MV), while one of the side barrels, ®lled with 2 M NaCl, was used for continuous automatic current balancing. The remaining barrels contained one of the following solutions: mCPP (30 mM, pH 4), GABA (1 mM, pH 4) and dopamine (100 mM, pH 4). These solutions were retained with a 210 nA current between ejection periods. Non-DA neurons in the SNr were identi®ed based on established electrophysiological characteristics: shortduration (0.5±0.7 ms), biphasic (positive/negative) action potentials of large amplitude (0.5±1.0 mV), baseline ®ring rates between 10 and 40 spikes/s, and location immediately ventral to nigral DA neurons. 6,8,22,23 SNr neurons can be distinguished by their excitatory response to footpinch. 6,8,23 Hemostats were used to administer a brief (,1 s) footpinch. SNr neurons were classi®ed as excited [P(1); brief increase in rate .10%] or not affected [P(0)] by footpinch. 6,8,23 Non-DA neurons in the VTA were also sampled. Although less well studied than SNr neurons,
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Effect of mCPP on non-DA neurons in the SNr and VTA
Fig. 2. Effect of mCPP on the ®ring rate of non-DA neurons in the VTA. (A) Representative rate histogram showing the typical excitatory effect produced by i.v. administration of mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on a non-DA neuron in the VTA. (B) Cumulative dose±response curves showing the mean percentage changes (^S.E.M.) in ®ring rate of non-DA neurons after i.v. administration of mCPP (n 11). *P , 0.05, **P , 0.01 compared to basal ®ring rate (one-way ANOVA, followed by Tukey's test).
they have similar characteristics. 21,23 Electrical signals of spike activity were passed through a high-impedance ampli®er (bandpass ®lter 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 interval. After each experiment, the recording site was marked by the ejection of Pontamine Sky Blue dye from the electrode using a 220 mA current for 10 min. Brains were removed and placed in 10% buffered formalin for two days before histological examination. Frozen sections were cut at 40-mm intervals and stained with Neutral Red. Microscopic examination of the sections was carried out to verify that the electrode tip was in the SNr or in the VTA. Drug administration protocol mCPP (5±320 mg/kg) was dissolved in 0.9% saline and administered i.v. (via a lateral tail vein) in exponentially increasing doses every 2 min, and the effect on the activity of non-DA neurons was recorded. SB 242084 was dissolved in 0.9% NaCl containing 8% hydroxypropyl-b-cyclodextrin by weight and 25 mM citric acid, and injected i.v. (200 mg/kg) 8±10 min prior to the administration of the ®rst dose of mCPP. Only one cell per animal was studied. All drug dosages refer to the weight of the salt. Statistical analysis Data acquisition and analysis were accomplished with an 83286-based PC and an integrated software package for electrophysiology (RISI, Symbolic Logic, Dallas, TX, USA). Dose± response curves were constructed by comparing the mean ®ring rate during 2 min, starting immediately after the injection of each dose, with the basal ®ring rate. The modi®cations in ®ring rate induced by microiontophoretic application of mCPP were calculated as percentages of drug-induced changes relative to the baseline. The data obtained were subjected to an ANOVA for repeated measures. When signi®cant effects were found, post hoc comparisons were made with Tukey's test. The interaction between SB
242084 and mCPP was analyzed by ANOVA (two-factor mixed design), followed by Tukey's test. In all cases, P , 0.05 was considered statistically signi®cant. Materials mCPP and hydroxypropyl-b-cyclodextrin were from Research Biochemicals Incorporated (RBI; Natick, MA, USA). SB 242084 was kindly provided by SmithKline Beecham Pharmaceuticals (Harlow, UK). RESULTS
Administration of mCPP (5±320 mg/kg, i.v.) caused a marked excitation of non-DA neurons in the P(0) group (n 10) of SNr neurons (Fig. 1A, C). A representative rate histogram is reported in Fig. 1A, which shows the typical excitatory action of mCPP. The effect of mCPP on P(0) neurons in the SNr was already evident at the cumulative dose of 20 mg/kg, which signi®cantly enhanced the basal ®ring rate by 31.8 ^ 19.9% (Fig. 1C). The maximum excitatory effect (59.0 ^ 15.0%) on this group of neurons was observed after administration of 320 mg/kg mCPP (Fig. 1C). The P(1) group (n 8) of SNr neurons was unaffected by the same dose range of mCPP (Fig. 1B, C). In contrast, all non-DA neurons tested in the VTA increased their ®ring rate following mCPP administration. The typical excitatory effect of mCPP on non-DA neurons in the VTA is shown in Fig. 2A. The effect of mCPP (5±320 mg/kg, i.v.) on non-DA cells in the VTA (n 11) was somewhat more marked than on P(0) neurons in the SNr (Fig. 2B). Thus, the dose of 320 mg/kg elicited a maximal excitation of 74.0 ^ 16.5% (Fig. 2B). Moreover, local application of mCPP by microiontophoresis caused a clear-cut excitation of
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Fig. 3. (A) Typical rate histogram showing the excitatory effect of microiontophoretically applied mCPP on non-DA neurons in the VTA. Numbers above each bar indicate the ejecting currents in nA. (B) Dose±response relationship for the effect of mCPP on non-DA neurons in the VTA (n 5). The dose (ejecting current in nA) is reported on the abscissa. Ordinate: mean percentage (^S.E.M.) of excitation of VTA non-DA neurons. *P , 0.05, **P , 0.01 compared to basal ®ring rate (one-way ANOVA, followed by Tukey's test).
non-DA VTA neurons (n 5; Fig. 3). As can be seen in Fig. 3B, the excitatory effect of mCPP was related to the amount of current applied, which ranged from 10 to 40 nA. The current±response curve shows that 40 nA mCPP caused a marked excitation (83.5 ^ 17.3%) of non-DA cells in the VTA (Fig. 3B). A similar excitatory effect was observed after microiontophoretic application of mCPP on P(0) non-DA neurons of the SNr (n 4; not shown). In one series of experiments, the effect of pretreatment with the potent and selective 5-HT2C receptor antagonist SB 242084 on mCCP-induced excitation of non-DA neurons was evaluated. Administration of SB 242084 (200 mg/kg, i.v.) 8±10 min before the injection of the ®rst dose of mCPP abolished the excitatory effect of this drug. Thus, Fig. 4A represents a typical rate histogram showing that pretreatment with SB 242084 prevents the stimulating effect of mCPP on this nonDA P(0) neuron in the SNr. Cumulative dose±response curves clearly show that SB 242084 abolishes the mCCPinduced excitation of P(0) non-DA neurons in the SNr (n 4; ANOVA, two-factor mixed design, F1,13 1.6; Fig. 4B). The maximal reduction was observed for the highest dose of mCPP injected (Tukey's test, P , 0.05 compared to the corresponding dose of mCPP alone; Fig. 4B). Administration of SB 242084 (200 mg/kg, i.v.) also antagonized the stimulatory effect of mCPP on non-DA neurons in the VTA (Fig. 5). As can be seen in Fig. 5, SB 242084 pretreatment completely blocked the effect of mCPP on non-DA neurons in the VTA (ANOVA,
Fig. 4. Pretreatment with SB 242084 blocks the excitatory effect of mCPP on non-DA neurons in the SNr. (A) Typical rate histogram showing that i.v. administration of SB 242084 (200 mg/kg, at arrow) prevents the effect of mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on this P(0) non-DA neuron. (B) Cumulative dose±response curves showing the mean percentage changes (^ S.E.M.) in ®ring rate of non-DA neurons after i.v. administration of mCPP alone (A; n 10) or SB 242084 1 mCPP (B; n 4). Pretreatment with SB 242084 abolished the stimulatory effect of mCPP on P(0) non-DA neurons. *P , 0.05, **P , 0.01 compared to basal ®ring rate of mCPP group (one-way ANOVA, followed by Tukey's test). 8P , 0.05 compared to the corresponding dose of mCPP alone (ANOVA, two-factor mixed design, followed by Tukey's test).
two-factor mixed design, F1,15 3.5; n 5). Also in this case, the maximal reduction was observed for the highest doses of mCPP injected (Tukey's test, P , 0.01 compared to the corresponding doses of mCPP alone). DISCUSSION
The present study shows that the non-selective 5-HT2C receptor agonist mCPP increases the basal ®ring rate of non-DA neurons in the SNr and VTA, recorded extracellularly in vivo. This effect is evident both after systemic administration and local microiontophoretic application of mCPP. Although mCPP is not a selective 5-HT2C receptor agonist, because it also acts on 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2B receptors, 1,11 its effects on non-DA neurons in the SNr and VTA are mediated by 5HT2C receptor, in as much as SB 242084, a potent and selective 5-HT2C antagonist, completely blocked the excitation elicited by mCPP. These ®ndings are consistent with in vitro electrophysiological studies showing that 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2B receptors are not involved in the excitatory effect exerted by 5-HT on non-DA neurons in the SNr. 20 Moreover, the inhibitory effect of mCPP on DA neurons in the VTA was completely prevented by SB 242084, a selective 5-HT2C
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Effect of mCPP on non-DA neurons in the SNr and VTA
Fig. 5. Pretreatment with SB 242084 blocks the excitatory effect of mCPP on non-DA neurons in the VTA. (A) Typical rate histogram showing that i.v. administration of SB 242084 (200 mg/kg, at arrow) prevents the effect of mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on this non-DA neuron. (B) Cumulative dose±response curves showing the mean percentage changes (^S.E.M.) in ®ring rate of non-DA neurons after i.v. administration of mCPP alone (A; n 11) or SB 242084 1 mCPP (B; n 5). Pretreatment with SB 242084 abolished the stimulatory effect of mCPP on non-DA neurons. *P , 0.05, **P , 0.01 compared to basal ®ring rate of mCPP group (one-way ANOVA, followed by Tukey's test). 88P , 0.01 compared to the corresponding dose of mCPP alone (ANOVA, two-factor mixed design, followed by Tukey's test).
receptor antagonist. 3 Combined electrophysiological and immunocytochemical studies have shown that most nonDA neurons in the SNr and VTA are indeed GABAergic. 9,19,21 Therefore, it is conceivable that the neurons recorded in this study are GABAergic. Thus, our data are consistent with the evidence that 5-HT2C receptor mRNA is expressed by GABA-containing neurons within the SNr and VTA. 5 Moreover, our ®ndings con®rm and extend previous data, obtained in vitro, showing that 5HT exerts a direct excitatory effect on GABAergic neurons in the SNr by acting on 5-HT2C receptors. 20 One interesting ®nding of our study was the differential effect exerted by mCPP on subpopulations of SNr
neurons. Thus, mCPP caused a marked excitation of the so-called P(0) non-DA neurons in the SNr, whereas it did not affect the P(1) neurons. These neurons are identi®ed on the basis of the presence [P(1)] or the absence [P(0)] of an excitatory response to a noxious stimulus (footpinch). 6±8,23 There is evidence that P(1) neurons in the SNr are GABAergic interneurons that exert a direct inhibitory in¯uence on DA neurons in the substantia nigra, whereas P(0) cells represent SNr projection neurons. 6,8,23 All non-DA neurons in the VTA were equally excited by mCPP. However, it is important to consider that the maximal excitation exerted by mCPP in the VTA was 74 ^ 16% compared to 59 ^ 15% on the P(0) subpopulation of non-DA neurons in the SNr. It is tempting to speculate that this differential response to mCPP might be the basis of the preferential inhibitory effect of 5-HT2C agonists on the mesolimbic versus the nigrostriatal dopaminergic function. Thus, there is evidence that 5-HT2C receptor agonists such as mCPP, MK 212 and RO 60-0175 markedly inhibit the spontaneous ®ring rate of DA neurons in the VTA, whereas they do not signi®cantly affect the activity of DA neurons in the SNc. 3,4 Therefore, it is conceivable that 5-HT2C receptor agonists might be useful in clinical conditions where it is necessary to reduce the mesolimbic dopaminergic activity without affecting the nigrostriatal function. Moreover, since projection neurons originating in the SNr are involved in the control of basal ganglia function, 2 activation of 5-HT2C receptors in the SNr might induce motor effects which are independent of the nigrostriatal DA system. CONCLUSIONS
This study shows that mCPP stimulates non-DA neurons in the SNr and VTA by activating 5-HT2C receptors. However, mCPP excites only one subpopulation of non-DA cells in the SNr, which are referred to as P(0) neurons, whereas in the VTA all neurons sampled are affected. This is the ®rst in vivo electrophysiological study showing that stimulation of 5-HT2C receptors enhances the activity of non-DA (presumably GABAergic) neurons in the SNr and VTA.
AcknowledgementÐThis work was partially supported by the Italian National Research Council (CNR, Rome, Italy), ShortTerm Mobility Programme 2000.
REFERENCES
1. Barnes N. M. and Sharp T. (1999) A review of central 5-HT receptors and their function. Neuropharmacology 38, 1083±1152. 2. Deniau J. M. and Chevalier G. (1992) The lamellar organization of the rat substantia nigra pars reticulata: distribution of projection neurons. Neuroscience 46, 361±377. 3. Di Giovanni G., Di Matteo V., Di Mascio M. and Esposito E. (2000) Preferential modulation of mesolimbic versus nigrostriatal dopaminergic function by serotonin2C/2B receptor agonists: a combined in vivo electrophysiological and microdialysis study. Synapse 35, 53±61. 4. Di Matteo V., Di Giovanni G., Di Mascio M. and Esposito E. (1999) SB 242084, a selective serotonin2C receptor antagonist, increases dopaminergic transmission in the mesolimbic system. Neuropharmacology 38, 1195±1205. 5. Eberle-Wang K., Mikeladze Z., Uryu K. and Chesselet M.-F. (1997) Pattern of expression of the serotonin2C receptor messenger RNA in the basal ganglia of adult rats. J. comp. Neurol. 384, 233±247. 6. Grace A. A. and Bunney B. S. (1979) Paradoxical excitation of nigral dopaminergic cells: indirect mediation through reticulata inhibitory neurons. Eur. J. Pharmac. 59, 211±218.
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7. Grace A. A. and Bunney B. S. (1985) Opposing effects of striatonigral feedback pathways on midbrain dopamine cell activity. Brain Res. 335, 271±284. 8. Grace A. A., Hommer D. W. and Bunney B. S. (1980) Peripheral and striatal in¯uences on nigral dopamine cells: mediation by reticulata neurons. Brain Res. Bull. 5, Suppl. 2, 105±109. 9. HajoÂs M. and Green®eld S. A. (1994) Synaptic connections between pars compacta and pars reticulata neurons: electrophysiological evidence for functional modules. Brain Res. 660, 216±224. 10. Herve D., Pickel V. M., Tong H. J. and Beaudet A. (1987) Serotonin axon terminals in the ventral tegmental area of the rat: ®ne structure and synaptic input to dopaminergic neurons. Brain Res. 435, 71±83. 11. Hoyer D. (1988) Functional correlates of serotonin 5-HT1 recognition sites. J. Recept. Res. 8, 59±81. 12. Mengod G., Nguyen H., Le H., Waeber C., LuÈbbert H. and Palacios J. M. (1990) The distribution and cellular localization of the serotonin 1C receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution. Neuroscience 15, 577±591. 13. Molineaux S. M., Jessell T. M., Axel R. and Julius D. (1989) 5-HT1C receptor is a prominent serotonin receptor subtype in the central nervous system. Proc. natn. Acad. Sci. USA 86, 6793±6797. 14. Mori S., Matsuura T., Takino T. and Sano Y. (1987) Light and electron microscopic immunohistochemical studies of serotonin nerve ®bers in the substantia nigra of the rat, cat and monkey. Anat. Embryol. 176, 13±18. 15. Moukhles H., Bosler O., Bolam J. P., ValleÂe A., Umbriaco D., Geffard M. and Doucet G. (1997) Quantitative and morphometric data indicate precise cellular interactions between serotonin and postsynaptic targets in rat substantia nigra. Neuroscience 76, 1159±1171. 16. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic, New York. 17. Prisco S. and Esposito E. (1995) Differential effects of acute and chronic ¯uoxetine administration on the spontaneous activity of dopaminergic neurones in the ventral tegmental area. Br. J. Pharmac. 116, 1923±1931. 18. Prisco S., Pagannone S. and Esposito E. (1994) Serotonin±dopamine interaction in the rat ventral tegmental area: an electrophysiological study in vivo. J. Pharmac. exp. Ther. 271, 83±90. 19. Richards C. D., Shiroyama T. and Kitai S. T. (1997) Electrophysiological and immunocytochemical characterization of GABA and dopamine neurons in the substantia nigra of the rat. Neuroscience 80, 545±557. 20. Rick C. E., Stamford I. M. and Lacey M. G. (1995) Excitation of rat substantia nigra pars reticulata neurons by 5-hydroxytryptamine in vitro: evidence for a direct action mediated by 5-hydroxytryptamine2C receptors. Neuroscience 69, 903±913. 21. Steffensen S. C., Svingos A. L., Pickel V. M. and Henriksen S. J. (1998) Electrophysiological characterization of GABAergic neurons in the ventral tegmental area. J. Neurosci. 18, 8003±8015. 22. Waszczak B. L., Eng N. and Walters J. R. (1980) Effects of muscimol and picrotoxin on single unit activity of substantia nigra neurons. Brain Res. 188, 185±197. 23. Zhang J., Chiodo L. A. and Freeman A. S. (1993) Effects of phencyclidine, MK-108 and 1,3-di(2-tolyl)guanidine on non-dopaminergic midbrain neurons. Eur. J. Pharmac. 230, 371±374. (Accepted 28 November 2000)
Pergamon
PII: S0306-4522(00)00561-3
q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00
www.elsevier.com/locate/neuroscience
m-CHLOROPHENYLPIPERAZINE EXCITES NON-DOPAMINERGIC NEURONS IN THE RAT SUBSTANTIA NIGRA AND VENTRAL TEGMENTAL AREA BY ACTIVATING SEROTONIN-2C RECEPTORS G. DI GIOVANNI, a V. DI MATTEO, a V. LA GRUTTA b and E. ESPOSITO a* a
Istituto di Ricerche Farmacologiche ªMario Negriº, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy b Istituto di Fisiologia Umana ªG. Paganoº, UniversitaÁ di Palermo, 90134 Palermo, Italy
AbstractÐIn vivo electrophysiological techniques were used to study the effect of m-chlorophenylpiperazine, a nonselective serotonin-2C receptor agonist, on the activity of non-dopaminergic neurons in the substantia nigra pars reticulata and the ventral tegmental area of anesthetized rats. Intravenous administration of m-chlorophenylpiperazine (5±320 mg/kg) caused a dose-dependent increase in the basal ®ring rate of a subpopulation of nigral neurons which do not respond to a footpinch stimulus [P(0) neurons], whereas it did not affect the activity of neurons which are responsive to the footpinch [P(1) neurons]. However, m-chlorophenylpiperazine (5±320 mg/kg) excited all non-dopaminergic neurons sampled in the ventral tegmental area. Moreover, microiontophoretic application of m-chlorophenylpiperazine (10±40 nA) caused an excitation of P(0) nigral and ventral tegmental area neurons. Pretreatment with the selective serotonin-2C receptor antagonist SB 242084 (200 mg/kg, i.v.) completely blocked the excitatory effect of i.v. m-chlorophenylpiperazine (5±320 mg/kg), both in the substantia nigra pars reticulata and in the ventral tegmental area. It is concluded that stimulation of serotonin-2C receptors by m-chlorophenylpiperazine activates non-dopaminergic (presumably GABA-containing) neurons in the substantia nigra pars reticulata and ventral tegmental area. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: GABAergic neurons, zona reticulata, ventral tegmental area, 5-HT2C receptors, interneurons, electrophysiology.
receptors, 1,11 its inhibitory effect on DA neurons in the VTA was completely prevented by the potent and selective 5-HT2C antagonist, SB 242084 {6-chloro-5-methyl-1-[2(2-methylpyridyl-3-oxy)-pyrid-5-yl-carbamoyl]indoline}.3 Therefore, it appears that mCPP reduces the mesolimbic DA function by acting on 5-HT2C receptors. 3 These data are consistent with the distribution of 5-HT2C receptors. 5,12,13 Moreover, it has recently been found that 5HT2C receptor mRNA is expressed by GABAergic neurons but not by DA neurons within the substantia nigra and VTA. 5 Therefore, it is conceivable that the effect of 5HT2C agonists on DA cell activity in the SNc and VTA would be mediated indirectly by GABAergic neurons. In this study, in vivo electrophysiological techniques were used to investigate the effects of the 5-HT2C receptor agonist mCPP on non-DA neurons. Extracellular single-unit recordings were performed from non-DA (presumably GABAergic) neurons in the SNr and VTA. Moreover, the effect of pretreatment with the selective 5-HT2C receptor antagonist SB 242084 on mCCP-induced changes in the ®ring rate of non-DA neurons was also evaluated.
Serotonin (5-HT)-containing neurons originating from the midbrain raphe nuclei innervate both the substantia nigra and the ventral tegmental area (VTA). Thus, neuroanatomical studies have shown a high density of 5-HT-immunoreactive ®bers in the substantia nigra pars compacta (SNc) and pars reticulata (SNr), and the VTA. 10,14,15 Serotonergic terminals make synaptic contacts with both dopaminergic (DA) and non-DA neurons in the SNc, SNr and VTA. 10,15 Several studies from our laboratory have shown that the serotonergic system controls the activity of DA neurons in the VTA by acting through 5HT2C receptors. 3,17,18 Thus, m-chlorophenylpiperazine (mCPP), a non-selective 5-HT2C receptor agonist, reduced the basal ®ring rate of dopamine-containing neurons in the VTA. 18 However, mCPP caused only a slight inhibition of DA neurons in the SNc (217.2 ^ 14.5%) compared to the VTA (242.6 ^ 12.8%). 3 Although mCPP is not an ideal ligand for 5-HT2C receptors, in that it also binds to 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2B *Corresponding author. Tel.: 139-872-570274; fax: 139-872578240. E-mail address: [email protected] (E. Esposito). Abbreviations: DA, dopaminergic; 5-HT, 5-hydroxytryptamine (serotonin); mCPP, m-chlorophenylpiperazine; P(1), nigral neurons that are excited by footpinch; P(0), nigral neurons that are not affected by footpinch; SB 242084, 6-chloro-5-methyl-1-[2-(2-methylpyridyl-3-oxy)-pyrid-5-yl-carbamoyl]indoline; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area.
EXPERIMENTAL PROCEDURES
Animals Male Sprague±Dawley rats (Consorzio Mario Negri Sud, Italy) weighing 300±350 g were used. Animals were kept at constant room temperature (21 ^ 28C) and relative humidity (60%) with a 111
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Fig. 1. Effect of mCPP on the ®ring rate of non-DA neurons in the SNr. (A) Representative rate histogram showing the typical excitatory effect produced by i.v. administration of mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on a P(0) neuron in the SNr. (B) Lack of effect of i.v. mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on a P(1) neuron in the SNr. (C) Cumulative dose± response curves showing the mean percentage changes (^S.E.M.) in ®ring rate of non-DA neurons after i.v. administration of mCPP, which signi®cantly enhanced the ®ring rate of P(0) neurons (n 10). The effect of mCPP on P(1) neurons (n 8) was not statistically signi®cant. *P , 0.05, **P , 0.01 compared to basal ®ring rate (one-way ANOVA, followed by Tukey's test).
12-h/12-h light±dark cycle (dark from 8.00 p.m.), and had free access to water and food. Procedures involving animals and their care were conducted in conformity with institutional guidelines that are in compliance with national (D.L. no. 116, G.U., Suppl. 40, 18 February 1992) and international laws and policies [EEC Council Directive 86/609, OJ L 358,1, 12 December 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication no. 85-23, 1985 and Guidelines for the Use of Animals in Biomedical Research (1987) Thromb. Haemost. 58, 1078±1084]. All efforts were made to minimize animal suffering and to reduce the number of animals used. 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±378C by a thermostatically regulated heating pad. The coordinates, relative to the interaural line, for placement of the recording electrode in the areas studied were, for the SNr: A 2.7±3.4 mm, L 1.8±2.2 mm, V 7±8 mm; for the VTA: A 2.7±3.4 mm, L 0.3±0.5 mm, V 7± 8 mm (to the level of exposed tissue). 16 Extracellular recordings
were performed by using either single- or ®ve-barreled micropipettes. The single micropipettes, measuring 1 mm at the tip, were ®lled with 2% Pontamine Sky Blue dye in 2 M NaCl (in vitro resistance 4±7 MV). Five-barreled micropipettes were pulled to an optimal wide tip angle and mechanically beveled under microscopic control to a ®nal tip diameter of 4±5 mm. The protruding center barrel, ®lled with 2% Pontamine Sky Blue dye in 2 M NaCl, was used for recording (in vitro resistance 4±7 MV), while one of the side barrels, ®lled with 2 M NaCl, was used for continuous automatic current balancing. The remaining barrels contained one of the following solutions: mCPP (30 mM, pH 4), GABA (1 mM, pH 4) and dopamine (100 mM, pH 4). These solutions were retained with a 210 nA current between ejection periods. Non-DA neurons in the SNr were identi®ed based on established electrophysiological characteristics: shortduration (0.5±0.7 ms), biphasic (positive/negative) action potentials of large amplitude (0.5±1.0 mV), baseline ®ring rates between 10 and 40 spikes/s, and location immediately ventral to nigral DA neurons. 6,8,22,23 SNr neurons can be distinguished by their excitatory response to footpinch. 6,8,23 Hemostats were used to administer a brief (,1 s) footpinch. SNr neurons were classi®ed as excited [P(1); brief increase in rate .10%] or not affected [P(0)] by footpinch. 6,8,23 Non-DA neurons in the VTA were also sampled. Although less well studied than SNr neurons,
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Effect of mCPP on non-DA neurons in the SNr and VTA
Fig. 2. Effect of mCPP on the ®ring rate of non-DA neurons in the VTA. (A) Representative rate histogram showing the typical excitatory effect produced by i.v. administration of mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on a non-DA neuron in the VTA. (B) Cumulative dose±response curves showing the mean percentage changes (^S.E.M.) in ®ring rate of non-DA neurons after i.v. administration of mCPP (n 11). *P , 0.05, **P , 0.01 compared to basal ®ring rate (one-way ANOVA, followed by Tukey's test).
they have similar characteristics. 21,23 Electrical signals of spike activity were passed through a high-impedance ampli®er (bandpass ®lter 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 interval. After each experiment, the recording site was marked by the ejection of Pontamine Sky Blue dye from the electrode using a 220 mA current for 10 min. Brains were removed and placed in 10% buffered formalin for two days before histological examination. Frozen sections were cut at 40-mm intervals and stained with Neutral Red. Microscopic examination of the sections was carried out to verify that the electrode tip was in the SNr or in the VTA. Drug administration protocol mCPP (5±320 mg/kg) was dissolved in 0.9% saline and administered i.v. (via a lateral tail vein) in exponentially increasing doses every 2 min, and the effect on the activity of non-DA neurons was recorded. SB 242084 was dissolved in 0.9% NaCl containing 8% hydroxypropyl-b-cyclodextrin by weight and 25 mM citric acid, and injected i.v. (200 mg/kg) 8±10 min prior to the administration of the ®rst dose of mCPP. Only one cell per animal was studied. All drug dosages refer to the weight of the salt. Statistical analysis Data acquisition and analysis were accomplished with an 83286-based PC and an integrated software package for electrophysiology (RISI, Symbolic Logic, Dallas, TX, USA). Dose± response curves were constructed by comparing the mean ®ring rate during 2 min, starting immediately after the injection of each dose, with the basal ®ring rate. The modi®cations in ®ring rate induced by microiontophoretic application of mCPP were calculated as percentages of drug-induced changes relative to the baseline. The data obtained were subjected to an ANOVA for repeated measures. When signi®cant effects were found, post hoc comparisons were made with Tukey's test. The interaction between SB
242084 and mCPP was analyzed by ANOVA (two-factor mixed design), followed by Tukey's test. In all cases, P , 0.05 was considered statistically signi®cant. Materials mCPP and hydroxypropyl-b-cyclodextrin were from Research Biochemicals Incorporated (RBI; Natick, MA, USA). SB 242084 was kindly provided by SmithKline Beecham Pharmaceuticals (Harlow, UK). RESULTS
Administration of mCPP (5±320 mg/kg, i.v.) caused a marked excitation of non-DA neurons in the P(0) group (n 10) of SNr neurons (Fig. 1A, C). A representative rate histogram is reported in Fig. 1A, which shows the typical excitatory action of mCPP. The effect of mCPP on P(0) neurons in the SNr was already evident at the cumulative dose of 20 mg/kg, which signi®cantly enhanced the basal ®ring rate by 31.8 ^ 19.9% (Fig. 1C). The maximum excitatory effect (59.0 ^ 15.0%) on this group of neurons was observed after administration of 320 mg/kg mCPP (Fig. 1C). The P(1) group (n 8) of SNr neurons was unaffected by the same dose range of mCPP (Fig. 1B, C). In contrast, all non-DA neurons tested in the VTA increased their ®ring rate following mCPP administration. The typical excitatory effect of mCPP on non-DA neurons in the VTA is shown in Fig. 2A. The effect of mCPP (5±320 mg/kg, i.v.) on non-DA cells in the VTA (n 11) was somewhat more marked than on P(0) neurons in the SNr (Fig. 2B). Thus, the dose of 320 mg/kg elicited a maximal excitation of 74.0 ^ 16.5% (Fig. 2B). Moreover, local application of mCPP by microiontophoresis caused a clear-cut excitation of
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Fig. 3. (A) Typical rate histogram showing the excitatory effect of microiontophoretically applied mCPP on non-DA neurons in the VTA. Numbers above each bar indicate the ejecting currents in nA. (B) Dose±response relationship for the effect of mCPP on non-DA neurons in the VTA (n 5). The dose (ejecting current in nA) is reported on the abscissa. Ordinate: mean percentage (^S.E.M.) of excitation of VTA non-DA neurons. *P , 0.05, **P , 0.01 compared to basal ®ring rate (one-way ANOVA, followed by Tukey's test).
non-DA VTA neurons (n 5; Fig. 3). As can be seen in Fig. 3B, the excitatory effect of mCPP was related to the amount of current applied, which ranged from 10 to 40 nA. The current±response curve shows that 40 nA mCPP caused a marked excitation (83.5 ^ 17.3%) of non-DA cells in the VTA (Fig. 3B). A similar excitatory effect was observed after microiontophoretic application of mCPP on P(0) non-DA neurons of the SNr (n 4; not shown). In one series of experiments, the effect of pretreatment with the potent and selective 5-HT2C receptor antagonist SB 242084 on mCCP-induced excitation of non-DA neurons was evaluated. Administration of SB 242084 (200 mg/kg, i.v.) 8±10 min before the injection of the ®rst dose of mCPP abolished the excitatory effect of this drug. Thus, Fig. 4A represents a typical rate histogram showing that pretreatment with SB 242084 prevents the stimulating effect of mCPP on this nonDA P(0) neuron in the SNr. Cumulative dose±response curves clearly show that SB 242084 abolishes the mCCPinduced excitation of P(0) non-DA neurons in the SNr (n 4; ANOVA, two-factor mixed design, F1,13 1.6; Fig. 4B). The maximal reduction was observed for the highest dose of mCPP injected (Tukey's test, P , 0.05 compared to the corresponding dose of mCPP alone; Fig. 4B). Administration of SB 242084 (200 mg/kg, i.v.) also antagonized the stimulatory effect of mCPP on non-DA neurons in the VTA (Fig. 5). As can be seen in Fig. 5, SB 242084 pretreatment completely blocked the effect of mCPP on non-DA neurons in the VTA (ANOVA,
Fig. 4. Pretreatment with SB 242084 blocks the excitatory effect of mCPP on non-DA neurons in the SNr. (A) Typical rate histogram showing that i.v. administration of SB 242084 (200 mg/kg, at arrow) prevents the effect of mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on this P(0) non-DA neuron. (B) Cumulative dose±response curves showing the mean percentage changes (^ S.E.M.) in ®ring rate of non-DA neurons after i.v. administration of mCPP alone (A; n 10) or SB 242084 1 mCPP (B; n 4). Pretreatment with SB 242084 abolished the stimulatory effect of mCPP on P(0) non-DA neurons. *P , 0.05, **P , 0.01 compared to basal ®ring rate of mCPP group (one-way ANOVA, followed by Tukey's test). 8P , 0.05 compared to the corresponding dose of mCPP alone (ANOVA, two-factor mixed design, followed by Tukey's test).
two-factor mixed design, F1,15 3.5; n 5). Also in this case, the maximal reduction was observed for the highest doses of mCPP injected (Tukey's test, P , 0.01 compared to the corresponding doses of mCPP alone). DISCUSSION
The present study shows that the non-selective 5-HT2C receptor agonist mCPP increases the basal ®ring rate of non-DA neurons in the SNr and VTA, recorded extracellularly in vivo. This effect is evident both after systemic administration and local microiontophoretic application of mCPP. Although mCPP is not a selective 5-HT2C receptor agonist, because it also acts on 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2B receptors, 1,11 its effects on non-DA neurons in the SNr and VTA are mediated by 5HT2C receptor, in as much as SB 242084, a potent and selective 5-HT2C antagonist, completely blocked the excitation elicited by mCPP. These ®ndings are consistent with in vitro electrophysiological studies showing that 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2B receptors are not involved in the excitatory effect exerted by 5-HT on non-DA neurons in the SNr. 20 Moreover, the inhibitory effect of mCPP on DA neurons in the VTA was completely prevented by SB 242084, a selective 5-HT2C
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Effect of mCPP on non-DA neurons in the SNr and VTA
Fig. 5. Pretreatment with SB 242084 blocks the excitatory effect of mCPP on non-DA neurons in the VTA. (A) Typical rate histogram showing that i.v. administration of SB 242084 (200 mg/kg, at arrow) prevents the effect of mCPP (5, 5, 10, 20, 40, 80 and 160 mg/kg, at arrows) on this non-DA neuron. (B) Cumulative dose±response curves showing the mean percentage changes (^S.E.M.) in ®ring rate of non-DA neurons after i.v. administration of mCPP alone (A; n 11) or SB 242084 1 mCPP (B; n 5). Pretreatment with SB 242084 abolished the stimulatory effect of mCPP on non-DA neurons. *P , 0.05, **P , 0.01 compared to basal ®ring rate of mCPP group (one-way ANOVA, followed by Tukey's test). 88P , 0.01 compared to the corresponding dose of mCPP alone (ANOVA, two-factor mixed design, followed by Tukey's test).
receptor antagonist. 3 Combined electrophysiological and immunocytochemical studies have shown that most nonDA neurons in the SNr and VTA are indeed GABAergic. 9,19,21 Therefore, it is conceivable that the neurons recorded in this study are GABAergic. Thus, our data are consistent with the evidence that 5-HT2C receptor mRNA is expressed by GABA-containing neurons within the SNr and VTA. 5 Moreover, our ®ndings con®rm and extend previous data, obtained in vitro, showing that 5HT exerts a direct excitatory effect on GABAergic neurons in the SNr by acting on 5-HT2C receptors. 20 One interesting ®nding of our study was the differential effect exerted by mCPP on subpopulations of SNr
neurons. Thus, mCPP caused a marked excitation of the so-called P(0) non-DA neurons in the SNr, whereas it did not affect the P(1) neurons. These neurons are identi®ed on the basis of the presence [P(1)] or the absence [P(0)] of an excitatory response to a noxious stimulus (footpinch). 6±8,23 There is evidence that P(1) neurons in the SNr are GABAergic interneurons that exert a direct inhibitory in¯uence on DA neurons in the substantia nigra, whereas P(0) cells represent SNr projection neurons. 6,8,23 All non-DA neurons in the VTA were equally excited by mCPP. However, it is important to consider that the maximal excitation exerted by mCPP in the VTA was 74 ^ 16% compared to 59 ^ 15% on the P(0) subpopulation of non-DA neurons in the SNr. It is tempting to speculate that this differential response to mCPP might be the basis of the preferential inhibitory effect of 5-HT2C agonists on the mesolimbic versus the nigrostriatal dopaminergic function. Thus, there is evidence that 5-HT2C receptor agonists such as mCPP, MK 212 and RO 60-0175 markedly inhibit the spontaneous ®ring rate of DA neurons in the VTA, whereas they do not signi®cantly affect the activity of DA neurons in the SNc. 3,4 Therefore, it is conceivable that 5-HT2C receptor agonists might be useful in clinical conditions where it is necessary to reduce the mesolimbic dopaminergic activity without affecting the nigrostriatal function. Moreover, since projection neurons originating in the SNr are involved in the control of basal ganglia function, 2 activation of 5-HT2C receptors in the SNr might induce motor effects which are independent of the nigrostriatal DA system. CONCLUSIONS
This study shows that mCPP stimulates non-DA neurons in the SNr and VTA by activating 5-HT2C receptors. However, mCPP excites only one subpopulation of non-DA cells in the SNr, which are referred to as P(0) neurons, whereas in the VTA all neurons sampled are affected. This is the ®rst in vivo electrophysiological study showing that stimulation of 5-HT2C receptors enhances the activity of non-DA (presumably GABAergic) neurons in the SNr and VTA.
AcknowledgementÐThis work was partially supported by the Italian National Research Council (CNR, Rome, Italy), ShortTerm Mobility Programme 2000.
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