F. LEJEUNE, A. NEWMAN-TANCREDI, V. AUDINOT and M. J. MILLAN

Transcription

1 /97/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 280, No. 3 Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 280: , 1997 Interactions of ( )- and ( )-8- and 7-Hydroxy-2-(Di-n- Propylamino)tetralin at Human (h)d 3,hD 2 and h Serotonin 1A Receptors and Their Modulation of the Activity of Serotoninergic and Dopaminergic Neurones in Rats F. LEJEUNE, A. NEWMAN-TANCREDI, V. AUDINOT and M. J. MILLAN Institut de Recherches Servier, Centre de Recherches de Croissy, Psychopharmacology Department, 78290, Croissy-sur-Seine, France Accepted for publication November 7, 1996 ABSTRACT The aminotetralins, 8-hydroxy-2-(di-n-propylamino)tetralin (8- OH-DPAT) and 7-OH-DPAT behave as preferential agonists at serotonin (5-HT) 1A and dopamine D 3 and D 2 receptors, respectively. In our study, we evaluated the influence of their ( )- and (-) isomers on the electrical activity of serotoninergic neurones of the dorsal raphe nucleus (DRN), which bear 5-HT 1A autoreceptors, and of dopaminergic neurones of the ventral tegmental area (VTA), which possess inhibitory D 3 and D 2 receptors. These actions were compared to their in vitro interactions with cloned, human (h)5-ht 1A,hD 3 and hd 2 receptors. In binding studies, racemic 8-OH-DPAT showed 100-fold selectivity for h5-ht 1A vs. hd 2 and hd 3 receptors and there was little difference between its ( )- and (-)-isomers either in terms of their potency at 5-HT 1A receptors or of their selectivity at 5-HT 1A vs hd 2 /hd 3 sites. Nevertheless, the ( )-isomer was markedly more efficacious than its (-)-counterpart in stimulating the binding of guanosine 5 -O-(3-[ 35 S]thiotriphosphate) ([ 35 S]-GTP S) at h5-ht 1A receptors, a measure of coupling to G-proteins; 90 vs. 57% maximal stimulation respectively, relative to 5-HT 100%. Also the ( )-isomer was ca. 3-fold more potent than the (-)-isomer in inhibiting the firing rate of DRN neurones. These actions were abolished by the 5-HT 1A antagonist, (-)-tertatolol, but unaffected by the hd 2 /hd 3 antagonist, haloperidol. Whereas ( )-8-OH-DPAT stimulated VTA neurone firing with a bell-shaped dose response curve, the (-)-isomer only inhibited VTA firing. The ( )-isomer-induced stimulation was blocked by (-)-tertatolol but not haloperidol, whereas the (-)-isomer-induced inhibition was abolished by haloperidol and unaffected by (-)-tertatolol. In contrast to 8-OH-DPAT, the ( )- and (-)- isomers of 7-OH-DPAT showed marked stereoselectivity inasmuch as the latter bound with 20-fold less potency than the former at hd 3 and, at higher concentrations, hd 2 receptors. Correspondingly, ( )-7-OH-DPAT was 20-fold more potent than (-)-7-OH-DPAT in reducing VTA firing. Concerning 5-HT 1A receptors, the ( )-isomer showed 20-fold lower affinity than at hd 3 receptors and, accordingly, it inhibited DRN firing at 20- fold higher doses than for inhibition of VTA firing. (-)-7-OH- DPAT showed even less (5-fold) selectivity for hd 3 vs. 5-HT 1A sites and for inhibition of VTA vs. DRN firing. The inhibition of VTA and DRN neurone firing by ( )-7-OH-DPAT was abolished by haloperidol and (-)-tertatolol, respectively. Finally, in line with this inhibition of DRN firing, both ( )- and (-)-7-OH-DPAT showed substantial efficacy ([ 35 S]-GTP S binding, 76 and 53%, respectively) at h5-ht 1A receptors. In conclusion, for these substituted aminotetralins, stereospecificity is a more marked feature of interactions at hd 3 (and hd 2 ) than at h5-ht 1A receptors and is more pronounced for 7- as compared to 8-OH-DPAT. Neither ( )- nor (-)-7-OH-DPAT show substantial selectivity for hd 3 vs. 5-HT 1A receptors and their inhibition of the firing of VTA as compared to DRN neurones is mediated by hd 3 /hd 2 and 5-HT 1A receptors, respectively. Finally, VTA neurones are stimulated by ( )-8-OH-DPAT via 5-HT 1A receptors and inhibited by (-)-8-OH-DPAT via hd 3 and/or hd 2 receptors. Ascending serotoninergic pathways from the DRN innervate about 50% of dopaminergic neurones in the VTA (Hervé et al., 1987) and provide a major dopaminergic input to the (SNPC) (Dray et al., 1978; Vertes, 1991). Further, serotoninergic terminals form excitatory synapses with VTA-localized dopaminergic neurons (Van Bockstaele et al., 1994) and Received for publication June 25, HT 1A receptors have been implicated in the modulatory influence of serotoninergic pathways on dopaminergic transmission. However, 5-HT 1A receptors are virtually absent from the SNPC (Miquel et al., 1991) and their concentration in the VTA itself is modest (Pazos and Palacios, 1985). Accordingly, the ability of systemic administration of the 5-HT 1A agonist, 8-OH-DPAT, to increase DA turnover in the VTA (Ahlenius et al., 1990; Chen and Reith, 1995) and to ABBREVIATIONS: DA, dopamine; 5-HT, serotonin; DRN, dorsal raphe nucleus; SNPC, substantia nigra pars compacta; VTA, ventral tegmental area; CHO, Chinese hamster ovary; [ 35 S]GTP S, guanosine 5 -O-(3-[ 35 -S]thiotriphosphate). 1241

2 1242 Lejeune et al. Vol. 280 facilitate DA release in the striatum (Benloucif et al., 1993) may reflect activation of inhibitory 5-HT 1A autoreceptors on serotoninergic perikarya localized in the DRN. Indeed, electrolytic destruction of raphe nuclei accelerates DA turnover in both the accumbens (Hervé et al., 1987) and the striatum (Giambalo and Snodgrass, 1978) whereas stimulation of raphe neurones inhibits the activity of SNPC-localized dopaminergic neurones (Dray et al., 1978). However, De Simoni et al. (1987) suggested that DRN stimulation enhanced the activity of dopaminergic pathways in the striatum. Indeed, there are also other contradictory data concerning the influence of 8-OH-DPAT on dopaminergic transmission. For example, it has both inhibitory and stimulatory influences upon VTA dopaminergic neurones in biochemical (Arborelius et al., 1993a; Ahlenius et al., 1990; Ichikawa et al., 1995) and electrophysiological studies (Arborelius et al., 1993b; Prisco et al., 1994). Further, microinjection of 8-OH-DPAT (or 5-HT) into the DRN decreases extracellular levels of DA in the nucleus accumbens (Yoshimoto and McBride, 1992). Finally, as concerns the influence of local administration of 8-OH-DPAT into the VTA on the activity of dopaminergic neurones, there are reports of both an increase in electrical activity (Arborelius et al., 1993a) and of a lack of effect (Zhang and Freeman, 1993). Interestingly [with two exceptions (Arborelius et al., 1993b; Ichikawa et al., 1995)] the above studies were performed with racemic 8-OH-DPAT, which may be resolved into ( )- and (-)-isomers. Although these do not present a marked difference in terms of their affinity at rat postsynaptic 5-HT 1A sites, the ( )-isomer possesses greater intrinsic activity (Cornfield et al., 1991). Currently, no information is available concerning their relative activities at presynaptic 5-HT 1A receptors. In addition, racemic 8-OH-DPAT exerts significant (partial agonist) activity at dopamine (D) 2 receptors (Gobert et al., 1995a; Smith and Cutts, 1989), which control the activity of ascending dopaminergic projections (see Gobert et al., 1995b). To date, the activity of both ( )- and (-)-8-OH- DPAT at hd 2 sites remains undefined. Further, the closely related dopamine hd 3 (auto)receptor may also be involved in the modulation of the activity of mesolimbic dopaminergic neurones (Bergstrom et al., 1994; Gobert et al., 1995b; Tang et al., 1994) and the putative activity of 8-OH-DPAT at these sites is unknown. It is, thus, intriguing that the aminotetralin, 7-OH-DPAT, which is closely related to 8-OH-DPAT, has been extensively used as a selective agonist at hd 3 receptors (Sokoloff et al., 1992; Sokoloff and Schwartz, 1995), although its selectivity for hd 3 vs. hd 2 sites may be less than originally claimed (Burris et al., 1995; Chio et al., 1993; Gonzalez and Sibley, 1995). D 2 as well as D 3 (auto)receptors may contribute to the ability of 7-OH-DPAT to inhibit the electrical activity of VTA dopaminergic neurones and to suppress DA release and turnover in the nucleus accumbens, striatum and cortex (Gobert et al., 1995b and 1996; Lejeune and Millan, 1995). Concerning the stereospecificity of its actions, the ( )-isomer possesses markedly higher affinity for hd 3 sites than the (-)-isomer (Damsma et al., 1993; Malmberg et al., 1994; Newman-Tancredi et al., 1995) and more potently reduces mesolimbic dopamine release (Rivet et al., 1994). In view of the striking structural homology between 7- and 8-OH-DPAT, an important question that arises is the selectivity of ( )- and (-)-7-OH-DPAT for hd 3 vs. 5-HT 1A sites. Indeed, the influence of 7-OH-DPAT on the activity of DRN neurones has not been documented. This question is also of interest in the light of scarce information concerning a possible reciprocal influence of dopaminergic pathways upon serotoninergic perykarya themselves. Despite the apparent dopaminergic innervation of the DRN by projections from the VTA (Kalén et al., 1988) and the substantia nigra (Beckstead et al., 1979; Stern et al., 1981), and the presence therein of D 2 -like receptors (Bouthenet et al., 1987; Palacios and Pazos, 1987), only few and contradictory data are available as concerns a putative dopaminergic modulation of the DRN (Ferré and Artigas, 1993; Ferré et al., 1994; Lee and Geyer, 1984). Our study was carried out to clarify the comparative influence of ( )- and (-)-isomers of 8- and 7-OH-DPAT on the activity of DRN-localized serotoninergic as compared to VTAlocalized dopaminergic neurones. First, we determined the in vitro affinities of ( )- and (-)-7- and 8-OH-DPAT at cloned, human (h)5-ht 1A vs.hd 3 and hd 2 receptors. Second, their in vitro efficacies for stimulation of h5-ht 1A receptor-mediated G-protein activation was determined by [ 35 S]GTP S binding (Newman-Tancredi et al., 1996). Third, their in vivo ability to modulate the electrical activity of DRN-localized serotoninergic neurones as compared with VTA-localized dopaminergic cells was determined. Fourth, we examined the influence of the selective 5-HT 1A antagonist, (-)-tertatolol (Jolas et al., 1993; Lejeune et al., 1994) which possesses negligible (K i 10 M) affinity at hd 2 and hd 3 receptors (see Results ), and of the selective hd 2 /hd 3 antagonist, haloperidol, respectively, on their actions (Lejeune et al., 1994; Lejeune and Millan, 1995; Millan et al., 1994, 1995). Materials and Methods Binding at cloned, human 5-HT 1A receptors. Binding was carried out as previously described (Newman-Tancredi et al., 1992) using [ 3 H]8-OH-DPAT (212 Ci/mmol, Amersham corp., Arlington Heights, IL) as a radioligand. Membranes (20 g protein) prepared from CHO cells stably transfected with the cloned human 5-HT 1A receptor (CHO-h5-HT 1A ) were incubated (2.5 hr, 22 C) in triplicate with competing ligands in a buffer containing HEPES 20 mm (ph 7.5) and MgSO 4 5 mm. Nonspecific binding was defined with 10 M 5-HT. Binding at cloned, human dopamine hd 2 and hd 3 receptors. Binding was carried out as previously described (Millan et al., 1994; Sokoloff et al., 1992). Membranes prepared from CHO cells stably transfected with cdna coding for human hd 2 and hd 3 receptors were incubated with [ 125 I]-iodosulpride at 0.1 and 0.2 nm for hd 2 and hd 3 sites, respectively. Nonspecific binding was defined with raclopride (10 M) and specific binding was 90% for both hd 2 and hd 3 sites. Inhibitory concentration 50 (IC 50 ) were calculated by nonlinear regression analysis and K i were computed according to K i IC 50 /(1 L/K D ) where L is the concentration of radioligand and K D is its apparent dissociation constant. Agonist efficacy at h5-ht 1A receptors was determined by measuring receptor-linked G-protein activation by [ 35 S]GTP S binding. CHO-h5-HT 1A membranes (50 g protein) and agonists were incubated (20 min, 22 C) in triplicate in a buffer containing HEPES 20 mm (ph 7.4), GDP 3 M, MgSO 4 3 mm and [ 35 S]GTP S (1300 Ci/mmol, NEN) 0.1 nm. Nonspecific binding was defined with 10 M GTP S. Incubations were terminated by rapid filtration and radioactivity determined by liquid scintillation counting. Binding isotherms were analyzed by nonlinear regression to yield estimates of effective concentration 50 (EC 50 ) and efficacy (E max ). The latter was expressed as the percentage of the maximal effect produced by the endogenous agonist, 5-HT ( 100%).

3 HT 1A Receptors and Dopamine 1243 Electrophysiological analysis. Male Wistar rats (Iffa Credo, Illskirchen, France), weighing 275 to 325 g, were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic apparatus after femoral vein cannulation. Additional doses of chloral hydrate were administered i.p. to maintain surgical anesthesia throughout the experiment. Rectal temperature was maintained at 37 1 C using a homeothermic heating pad. A burr hole was made over the VTA or the DRN. A tungsten microelectrode was lowered, according to the atlas of Paxinos and Watson (1986), into the VTA (AP: -5.5 from bregma, L: 0.7, H: -7/-8.5 from dura) or the DRN (AP: -7.8 from bregma, L: 0, H: -5/-6.5 from dura) for recording of extracellular unit activity. Neurones were identified as described elsewhere [Wang (1981) for dopaminergic and Aghajanian et al. (1978) for serotoninergic neurones[. Only one cell was studied in each animal. After base-line recording ( 5 min) and a first injection of vehicle, drugs were administered by i.v. route in increasing cumulative doses for evaluation of dose-response relationships. For agonistantagonist interactions, one dose of antagonist was given after a single dose of agonist. The interval between injections was 2 to 5 min. The peak drug action was attained within 1 to 2 min after injection for all drugs excepted haloperidol that had a delay time to maximal efficacy between 3 to 5 min. Drugs were dissolved in sterile water and injected i.v. in a volume of 0.5 ml/kg, followed by 0.1 ml of saline to flush the catheter. Data acquisition was accomplished by using Spike2 software (C.E.D., Cambridge, England). Interspike time interval histograms were calculated over 500 consecutive spikes as previously described (Arborelius et al., 1993b). Burst firing was the percentage ration of spikes in bursts to the total number of spikes. Results were expressed as firing rate (60-sec bins at time of peak drug action) in percent change from baseline ( mean of predrug values) and presented as means S.E.M. (3 n 8). Data were analyzed by two-way analysis of variance followed by Newman- Keuls test (for paired data) or Student s t test (for unpaired data). Inhibitory dose (ID) 50 and 95% confidence limits were calculated. Results In vitro binding affinities of agonists and antagonists at cloned h5-ht 1A,hD 2 and hd 3 receptors. The affinities of ( )-8-OH-DPAT, ( )-8-OH-DPAT and (-)-8-OH- DPAT at cloned h5-ht 1A receptors were almost identical (table 1). Racemic 8-OH-DPAT, as well as its ( )- and (-)- isomers, all showed markedly lower affinity at hd 2 and hd 3 receptors. However, at both these sites, the ( )-isomer showed about 2-fold higher affinity than the racemate and about 10-fold higher affinity than the (-)-isomer. ( )-7-OH- DPAT displayed higher affinity than (-)-7-OH-DPAT at both hd 3 and hd 2 receptors. Notably, the affinity of each of these TABLE 1 Ligand affinities at cloned, human dopamine hd 2 and hd 3 and h5-ht 1A receptors K i (nm) Drug hd 2 hd 3 h5-ht 1A ( )-8-OH-DPAT 1, ( )-8-OH-DPAT 1, ( )-8-OH-DPAT 12, , ( )-7-OH-DPAT ( )-7-OH-DPAT 1, Haloperidol , ( )-Tertatolol 10,000 10, Ligand binding affinity at cloned hd 2, hd 3 and h5-ht 1A receptors stably expressed in CHO cells was determined in competition binding assays with [ 125 I]iodosulpride and [ 3 H]( )8-OH-DPAT (see Materials and Methods ). Values are based on at least three independent determinations performed in triplicate (means S.E.M.). isomers was even higher at h5-ht 1A than at hd 2 receptors. The separation was only 5-fold for (-)-7-OH-DPAT at hd 3 vs. h5-ht 1A sites. Haloperidol displayed high affinity for hd 2 receptors and about 5-fold lower affinity for hd 3 sites with its affinity at h5-ht 1A receptors being very low. In contrast, (-)-tertatolol manifested high affinity at h5-ht 1A receptors and negligible affinity at hd 2 and hd 3 receptors. Influence of ( )- and (-)-7- and 8-OH-DPAT on G- protein activation. ( )-8-OH-DPAT potently and markedly activated h5-ht 1A receptor-mediated stimulation of [ 35 S]GTP S binding with a maximal effect of % relative to that of 5-HT (fig. 1) and an EC 50 of nm. (-)-8-OH-DPAT similarly stimulated [ 35 S]GTP S binding with a potency similar (EC nm) to that of its ( )-counterpart, though with significantly (P.05) less efficacy ( %). ( )-8-OH-DPAT had intermediate efficacy ( %). As concerns 7-OH-DPAT, in line with its higher affinity at h5-ht 1A receptors, the ( )-isomer more potently stimulated [ 35 S]GTP S binding than the (-)-isomer (EC 50 were 1, nm and 10,300 4,400 nm, respectively). It also showed somewhat higher efficacy ( % vs %), although this difference was not significant (fig. 1). Influence of ( ), ( ) and (-) 8-OH-DPAT on the electrical activity of DRN serotoninergic neurones. 8-OH- DPAT potently and markedly inhibited the firing rate of DRN cells with the (-)-isomer displaying about 3-fold lower potency and the racemate showing intermediate activity as compared with the ( )-isomer (table 2; fig. 2). Indeed, the inhibitory actions of both ( )- and (-)-8-OH-DPAT were completely antagonized (figs. 4 and 5) by administration of the 5-HT 1A antagonist, (-)-tertatolol (2 mg/kg) that did not itself modify the firing rate of these DRN neurones (see also Lejeune et al., 1994). Haloperidol did not affect their actions and did not modify DRN firing rate alone (fig. 5). Influence of ( )-, ( )- and (-)-8-OH-DPAT on the electrical activity of VTA dopaminergic neurones. At a higher dose range ( 10 g/kg), racemic 8-OH-DPAT elicited a dose-dependent and biphasic influence on dopaminergic VTA cell activity with an increase in firing at modest doses and a decrease at high doses (fig. 2). At these modest doses, ( )-8-OH-DPAT increased VTA firing rate and transformed the regular firing pattern into an irregular burst mode (fig. 3); the percentage of bursts increased after ( )-8-OH-DPAT treatment from to (P.026, paired Student s t test). However, (-)-8-OH-DPAT only inhibited the activity of VTA dopaminergic neurones (fig. 2) at Fig. 1. Stimulation of h5-ht 1A receptor-activated [ 35 S]-GTP S binding by ( )- and (-)-7- and 8-OH-DPAT. Agonist efficacy is expressed relative to 5-HT-induced stimulation ( 100%).

4 1244 Lejeune et al. Vol. 280 TABLE 2 Modulation of the firing rate of dopaminergic (VTA) and serotoninergic neurones (DRN) Drug VTA ID 50 (C.L.) ( )-8-OH-DPAT NC 0.59 ( ) ( )-8-OH-DPAT NC 0.29 ( ) ( )-8-OH-DPAT 1,379 ( ) 0.84 ( ) ( )-7-OH-DPAT 0.71 ( ) 26.6 ( ) ( )-7-OH-DPAT 15.0 ( ) 198 ( ) Haloperidol ( )-Tertatolol ID 50 (95% C.L.): inhibitory dose 50 (95% confidence limits); VTA, ventral tegmental area; DRN, dorsal raphe nucleus; NC, Not computable. Values are in g/kg, i.v. DRN high doses ( 0.25 mg/kg) and did not induce a bursting pattern in regular firing cells (not shown). The stimulation of VTA activity by ( )-8-OH-DPAT (40 g/kg), as well as the induction of a bursting pattern, were blocked by (-)-tertatolol (2 mg/kg; % bursts , P.014 vs. ( )-8-OH- DPAT and P.113 vs. baseline, paired Student s t test) but not influenced by haloperidol (16 g/kg) (figs. 4 and 5). In contrast, (-)-tertatolol (2 mg/kg) did not affect the inhibition of VTA firing induced by (-)-8-OH-DPAT (2.5 mg/kg) whereas this action was antagonized by haloperidol (16 g/kg) (figs. 4 and 5). Although haloperidol slightly stimulated VTA firing rate alone, (-)-tertatolol was without effect (fig. 5). Influence of ( )- and (-)-7-OH-DPAT on the electrical activity of VTA dopaminergic neurones. Both ( )-7-OH- DPAT and (-)-7-OH-DPAT induced a dose-dependent and complete inhibition of the firing rate of DA cells in the VTA (fig. 2), the ( )-isomer displaying about 20-fold higher potency than its (-) counterpart (table 2). Haloperidol abolished this action of ( )- and (-)-7-OH-DPAT whereas (-)-tertatolol was inactive (figs. 4 and 5). Influence of ( )- and (-)-7-OH-DPAT on the electrical activity of DRN serotoninergic neurones. Over a higher dose range, both ( )-7-OH-DPAT and (-)-7-OH-DPAT also dose-dependently and completely inhibited the firing rate of serotoninergic cells in the DRN (table 2; fig. 2) with the ( )- isomer being only 7-fold more potent than the (-). This inhibition was antagonized by (-)-tertatolol and unaffected by haloperidol (figs. 4 and 5). Correlation analysis between in vitro affinities and in vivo potencies. Across all ligands tested, there was a highly significant correlation between 5-HT 1A affinity and ID 50s for inhibition of DRN serotoninergic cell firing and between hd 3 affinity and ID 50s for inhibition of VTA neuronal activity (table 3). No other significant correlations were observed. Discussion Actions of ( )-, ( )- and (-)-8-OH-DPAT at h5-ht 1A receptors. The affinities of the ( )- and (-)-isomers of 8-OH- DPAT for cloned h5-ht 1A receptors did not differ markedly, in agreement with studies in rat hippocampus (Cornfield et al., 1991; Foreman et al., 1995). Nevertheless, ( )-8-OH- DPAT displayed higher efficacy than (-)-8-OH-DPAT in activating h5-ht 1A receptor-mediated [ 35 S]GTP S binding, an in vitro measure of G-protein activation. This observation is analogous to the greater efficacy of ( )- as compared to Fig. 2. Dose-dependent effects of 8-OH-DPAT (upper graph) and 7-OH-DPAT (lower graph) isomers on firing rates of serotoninergic neurones of DRN (open symbols) and dopaminergic neurones of VTA (filled symbols). Data are means S.E.M. (n 4 recorded cells in each condition). Analysis of variance with repeated measures as follows: DRN, ( )-8-OH-DPAT, F (5,30) , P.001; DRN, ( )-8-OH- DPAT, F (5,20) 49.8, P.001; DRN, (-)-8-OH-DPAT, F (4,20) 45.9, P.001; VTA, ( )-8-OH-DPAT, F (9,27) 7.7, P.001; VTA, ( )-8- OH-DPAT, F (8,24) 3.5, P.02; VTA, (-)-8-OH-DPAT, F (9,27) 2.0, P.05; DRN, ( )-7-OH-DPAT, F (5,20) 76.4, P.001; DRN, (-)-7-OH-DPAT, F (5,15) 93.6, P.001; VTA, ( )-7-OH-DPAT, F (5,20) 141.4, P.001; VTA, (-)-7-OH-DPAT, F (6,18) 51.4, P.001. Asterisks indicate significance (P.05) of differences to vehicle in Newman-Keuls test (paired data). For clarity, asterisks are omitted for the 8-OH-DPAT-DRN curves for which all values, except the first, were significantly (P.05) different from vehicle. (-)-8-OH-DPAT in inhibiting forskolin-stimulated adenylyl cyclase activity in rat hippocampal membranes (Cornfield et al., 1991; Foreman et al., 1995). Such isomeric differences may be an intrinsic property of 5-HT 1A receptors (Björk et al., 1989), although the G-protein coupling of recombinant human 5-HT 1A receptors in CHO cells may differ from that of native rat 5-HT 1A autoreceptors, with respect to G-protein subtypes and subcellular localisation. Further, due to their high receptor reserve in the DRN, even weak partial agonists suppress serotoninergic transmission (Gobert et al., 1995a; Meller et al., 1990). Thus, the higher efficacy, but not potency, of ( )- vs. (-)-8-OH-DPAT at h5-ht 1A receptor, may underlie its more potent inhibition of the firing of DRN serotoninergic neurones (see below). Similar findings at 5-HT 1A

5 HT 1A Receptors and Dopamine 1245 Fig. 3. ( )-8-OH-DPAT augments burst firing activity in dopaminergic neurones of the VTA. Upper panel, Regular firing pattern before treatment. Middle panel, 2 min after ( )-8-OH-DPAT (40 g/kg, i.v.), the interspike interval histogram reflects the increased proportion of burst firing. Lower panel, 3 min after (-)-tertatolol (2000 g/kg, i.v.), the firing mode has returned to a regular pattern. The three traces were from the same neurone; (-)-tertatolol was injected 2 min after ( )-8-OH-DPAT autoreceptors controlling 5-HT synthesis were noted, using other stereoresolved ligands, by Foreman et al. (1995) and Malmberg et al. (1994). Actions of ( )-, ( )- and (-)-8-OH-DPAT at hd 2 and hd 3 receptors. Racemic 8-OH-DPAT possesses modest affinity at native rat D 2 receptors (Gobert et al., 1995a; Van Wijngaarden et al., 1990), and we demonstrate that it displays low affinity at cloned hd 2 receptors. Further, its hd 3 receptor affinity was 4-fold higher than at hd 2 sites suggesting that certain effects of 8-OH-DPAT previously attributed to D 2 sites (Gobert et al., 1995b; Smith and Cutts, 1989) might in fact be mediated by D 3 receptors. In terms of affinity, (-)-8-OH-DPAT was more selective than ( )-8-OH-DPAT for h5-ht 1A vs. hd 2 /hd 3 receptors. The greater separation between ( )- and (-)-8-OH-DPAT as concerns their affinities at hd 3 as compared to h5-ht 1A receptors indicates that stereospecificity may be a more pronounced feature of hd 3 than h5-ht 1A receptors. Actions of ( )- and (-)-7-OH-DPAT at hd 2 and hd 3 receptors. The ( )-isomer of 7-OH-DPAT showed 20-fold higher affinity than its (-) counterpart at cloned hd 3 (and hd 2 ) receptors suggesting that pharmacological activity at hd 3 receptors resides primarily in the former. These data corroborate the findings of Damsma et al., (1993) who used a different radioligand, [ 3 H]-spiperone. Further, Baldessarini et al. (1993) found that ( )-7-OH-DPAT was 2-fold more potent than racemic 7-OH-DPAT in binding to hd 3 receptors transfected into fibroblasts. In addition, in an extensive study of substituted 2-aminotetralins, Malmberg et al. (1994) revealed that, although hydroxy-substitued ligands (at position 7 and elsewhere) behave as agonists at hd 3 and hd 2 receptors, the ( )- and (-)-isomers may display different binding modes with the latter consistently presenting lower affinities. It must be mentioned that the hd 2 affinities given here may be underestimated owing to the existence of multiple affinity states (Burris et al., 1995; Chio et al., 1993; Gonzalez and Sibley, 1995; Millan et al., 1995; Newman- Tancredi et al., 1995) and a role of hd 2 receptors in the actions of ( )- and (-)-7-OH-DPAT cannot be excluded (Gobert et al., 1995b; Lejeune et al., 1995). Actions of ( )- and (-)-7-OH-DPAT at h5-ht 1A receptors. This study extends to cloned h5-ht 1A receptors our previous observation (Millan et al., 1995) that ( )-7-OH- DPAT possesses significant affinity for rat 5-HT 1A receptors and demonstrates that the (-)-isomer displays even less (5- fold) selectivity for hd 3 vs. h5-ht 1A receptors. In line with its higher affinity, ( )-7-OH-DPAT was 8-fold more potent than the (-)-isomer in enhancing 5-HT 1A receptor-stimulated [ 35 S]GTP S binding. In analogy to the differential efficacy of ( )- vs. (-)-8-OH-DPAT at h5-ht 1A receptors, the efficacy of ( )-7-OH-DPAT was higher than that of (-)-7-OH-DPAT in [ 35 S]GTP S binding. Nevertheless, both isomers of 7-OH- DPAT completely inhibited the firing of DRN serotoninergic neurones (see below). Actions of ( )-, ( )- and (-)-8-OH-DPAT upon dopaminergic vs. serotoninergic transmission in vivo. In our study, the robust influence of ( )-8-OH-DPAT on DRN firing rate (fig. 2) (Blier et al., 1988; Lejeune et al., 1994; Lum and Piercey, 1988; Sinton and Fallon, 1988; Sprouse and Aghajanian, 1986) was confirmed and extended to its isomers. The involvement of 5-HT 1A autoreceptors was revealed by the antagonist activity of (-)-tertatolol and by the lack of effect of haloperidol. In contrast, the modulation by 8-OH-DPAT of the electrical activity of dopaminergic cells has proven vari-

6 1246 Lejeune et al. Vol. 280 Fig. 4. Modulation of spontaneous unit activity of DRN serotoninergic and VTA dopamergic neurones by ( )- and (-)-isomers of 8- and 7-OH-DPAT. 1, Antagonism of ( )-8-OH-DPAT, 5 g/kg (( )-8)-induced inhibition of DRN firing by (-)-tertatolol, 2 mg/kg (TER) and not haloperidol (HAL), 31 g/kg. 2, Antagonism of ( )-8-OH-DPAT, 40 g/kg-induced stimulation of VTA firing by (-)-tertatolol, 2 mg/kg and not haloperidol 16 g/kg. 3, Antagonism of (-)-8-OH-DPAT, 2.5 mg/kg-induced inhibition of VTA firing by haloperidol, 16 g/kg and not (-)-tertatolol, 2 mg/kg. 4, antagonism of ( )-7-OH-DPAT, 5 g/kg-induced inhibition of VTA firing by haloperidol, 16 g/kg but not (-)-tertatolol, 2 mg/kg. 5, Antagonism of ( )-7-OH-DPAT, 125 g/kg-induced inhibition of DRN firing by (-)-tertatolol, 2 mg/kg, not haloperidol, 31 g/kg. able (Lum and Piercey, 1988; Sinton and Fallon, 1988): an inhibitory (Gervais and Rouillard, 1993; Yoshimoto and McBride, 1992) or excitatory (Kelland et al., 1990; Prisco et al., 1994; Zhang and Freeman, 1993) action has been observed. In all these cases racemic 8-OH-DPAT was used but the study of Arborelius et al. (1993b) reported a biphasic modulation of DA cell firing rate after systemic administration of ( )-8-OH-DPAT. Our results corroborate this biphasic influence of systemically administered ( )-8-OH-DPAT on VTA firing (fig. 2). Further, (-)-8-OH-DPAT had no excitatory effect but only inhibited VTA cell firing. This dissociation between the stimulation (( )-isomer) and the inhibition ((-)- isomer) of VTA electrical activity provides an explanation for conflicting results previously reported for racemic 8-OH- DPAT and emphasizes the importance of examining both stereoisomers. In addition, we show that the stimulation of VTA firing by ( )-8-OH-DPAT is blocked by (-)-tertatolol, but unaffected by haloperidol, implicating the involvement of 5-HT 1A receptors in the activation of dopaminergic neurones by ( )-8-OH-DPAT. Conversely, the inhibition of VTA firing by (-)-8-OH-DPAT was abolished by haloperidol, consistent with a direct interaction at inhibitory D 3 and/or D 2 (auto)receptors. These findings support the hypothesis that stimulation of 5-HT 1A receptors increases the activity of VTA-localized dopaminergic neurones. Our data do not address the issue of whether pre- or postsynaptic 5-HT 1A receptors are involved; however, ( )-8-OH-DPAT was more potent than (-)-8-OH-DPAT in stimulating VTA cells and inhibiting DRN firing. Thus, the excitatory effect of ( ) vs. (-)-8-OH-DPAT on dopaminergic neurones more likely reflects its greater potency in inhibiting the firing of DRN neurones via stimulation of 5-HT 1A presynaptic autoreceptors. Further, we have recently observed that the selective 5-HT 1A ligand, S 15535, which behaves as an agonist and antagonist at pre and postsynaptic 5-HT 1A receptors, respectively (Millan et al., 1994), also activates dopaminergic neurones (Gobert et al., 1995c; Lejeune et al., 1996; Millan et al., 1996, submitted for publication). The inhibitory effect of (-)-8-OH-DPAT on VTA firing was reversed by haloperidol, indicating the involvement of dopaminergic autoreceptors, in agreement with the dopaminergic agonist effects of ( )-8-OH-DPAT previously observed by Smith and Cutts (1989) in vitro. Actions of ( )- and (-)-7-OH-DPAT on dopaminergic vs. serotoninergic transmission in vivo. ( )- and (-)-7- OH-DPAT haloperidol-reversibly inhibited VTA-localized dopaminergic neurones; the ( )-isomer was 20-fold more potent than its (-)-counterpart. These observations are paralleled by previous reports of the more potent influence of ( )- vs.

7 HT 1A Receptors and Dopamine 1247 Fig. 5. Antagonism of a single, maximally effective dose of ( )- and (-)-isomers of 8- and 7-OH-DPAT. VEH, vehicle; TER, (-)- tertatolol and HAL, haloperidol. Doses are the same as in figure 3 plus (-)-7-OH-DPAT (160 g/kg for DRN and 1000 g/kg for VTA). Data are means S.E.M. (n 3 recorded cells in each condition). Analysis of variance was as follows: DRN, ( )-8-OH-DPAT, F (2,12) 22.6, P.001; DRN, (-)-8- OH-DPAT, F (2,12) 21.2, P.001; VTA, ( )-8-OH-DPAT, F (2,13) 10.8, P.002; VTA, (-)-8-OH-DPAT, F (2,14) 17.8, P.001; DRN, ( )-7-OH-DPAT, F (2,13) 22.3, P.001; DRN, (-)-7-OH-DPAT, F (2,6) 618.4, P.001; VTA, ( )-7-OH-DPAT, F (2,16) 74.3, P.001; VTA, (-)-7-OH-DPAT, F (2,13) 42.4, P.001. Asterisks indicate significance (P.01) of differences in Dunnett s test as compared with respective vehicle values. TABLE 3 Correlations analysis for in vitro binding affinities versus in vivo potencies for inhibition of the firing rate of dopaminergic (VTA) and serotoninergic neurones (DRN) Receptor h5-ht 1A hd 2 hd 3 ID 50 (DRN) 0.98 a ID 50 (VTA) a VTA, Ventral tegmental area; DRN, dorsal raphe nucleus; ID 50, inhibitory dose 50. Values are product moment correlation coefficients. a P (-)-7-OH-DPAT on release of DA from mesolimbic dopaminergic terminals in vivo, an action reflecting activation of D 3 and, possibly, D 2 autoreceptors (Rivet et al., 1994; Gobert et al., 1995b and 1996; Patel et al., 1995; Tang et al., 1994). ( )- and (-)-7-OH-DPAT (-)-tertatolol-reversibly inhibited the firing rate of serotoninergic neurones in the DRN, suggesting that they activate 5-HT 1A autoreceptors. This is in line with the activation of h5-ht 1A receptor-coupled [ 35 S]GTP S binding. Further, ( )-7-OH-DPAT showed 100-fold lower potency than ( )-8-OH-DPAT in this binding model, a ratio identical to that for their respective potencies in inhibiting DRN firing. To date, there is no evidence for the occurrence of dopamine D 3 receptors in the DRN (Ariano and Sibley, 1994; Herroelen et al., 1994; Sokoloff and Schwartz, 1995) and the contribution of dopaminergic neurones to projections from the VTA (and SNPC) to the DRN is minor (Geffard et al., 1987; Kalén et al., 1988; Swanson, 1982). These observations are consistent with our study in suggesting that dopamine D 3 receptors are unlikely to play a major role in the effect of 7-OH-DPAT on DRN firing rate. This conclusion is supported by recent studies with CGS-15855, a high affinity agonist at hd 3 (K i 5 nm) vs. h5-ht 1A (K i 620 nm) receptors (Millan et al., 1995). CGS fails to modify the activity of DRN-localized serotoninergic neurones (unpublished observation) even at high doses relative to those suppressing dopaminergic transmission (Gobert et al., 1995b). General Discussion. We did not observe systematic differences between slow and fast VTA-localized dopaminergic neurones in terms of the excitatory influence of ( )-8-OH- DPAT, an observation consistent with the remark of Arborelius et al. (1993a) that the stimulatory influence of 5-HT 1A agonists on dopaminergic neurones is independent of basal firing rate. In contrast, Kelland et al. (1990) suggested that slow SNPC-localized dopaminergic neurones were more susceptible to the excitatory actions of systemic 8-OH-DPAT. A further observation, in common with the study of Arborelius et al. (1993b), is that ( )-8-OH-DPAT transformed regularly firing cells into a bursting pattern of firing, a change associated with an increase in DA release and synaptic transmission (Svensson et al., 1995). Further, the reinforcement by 5-HT 1A agonists of cortical dopaminergic transmission via preferential activation of this subset of VTA-localized dopaminergic neurones is likely relevant to their ability to relieve the cortical hypofrontality common to both depressive states and the negative symptomatology of schizophrenia (Svensson et al., 1995; Tanda et al., 1994). Conclusions. Our data demonstrate that 7-OH-DPAT shows marked stereospecificity in its interactions at D 3,D 2 and 5-HT 1A receptors. Previous authors (e.g., Burris et al., 1995) have pointed out that the selectivity of 7-OH-DPAT for hd 3 vs. hd 2 sites is not as marked as originally claimed (Sokoloff et al., 1992), and our study emphasizes that potential actions at h5-ht 1A receptors should not be ignored. Indeed, the (-)-isomer shows only 5-fold selectivity for hd 3 vs. h5-ht 1A sites suggesting that studies of this ligand must be restricted to the ( )-isomer. As concerns 8-OH-DPAT, the data also reveal potential actions at high doses at D 3 receptors. Nevertheless, ( )-8-OH-DPAT shows marked in vivo selectivity for 5-HT 1A receptors and the activation of 5-HT 1A autoreceptors underlies its disinhibition of VTA-localized dopaminergic neurones. As concerns stereospecificity of actions, this appears to be a more marked feature of D 3 and D 2 vs. 5-HT 1A receptors and of 7- as compared to 8-OH-DPAT. Our data help resolve several discrepancies in the literature. Finally, they underscore the importance of 5-HT 1A (auto)re-

8 1248 Lejeune et al. Vol. 280 ceptor-mediated modulation of the activity of VTA-localized dopaminergic neurones, an action of potential importance to the therapeutic profiles of novel antidepressant and antipsychotic agents (Broekkamp et al., 1995; Deutch et al., 1991; Meltzer, 1992). Acknowledgments The authors thank C. Melon, V. Jacques, C. Chaput and L. Verrièle for technical assistance. References AGHAJANIAN, G. K., WANG, R. Y. AND BARABAN, J.: Serotonergic and nonserotonergic neurons of the dorsal raphe: Reciprocal changes in firing induced by peripheral nerve stimulation. Brain Res. 153: , AHLENIUS, S., HILLEGAART, V. AND WIJKSTRÖM, A.: Increased dopamine turnover in the ventral striatum by 8-OH-DPAT administration in the rat. J. Pharm. Pharmacol. 42: , ARBORELIUS, L., NOMIKOS, G. G., HACKSELL, U. AND SVENSSON, T. H.: (R)-8-OH- DPAT preferentially increases dopamine release in the rat medial prefrontal cortex. Acta Physiol. Scand. 148: , 1993a. ARBORELIUS, L., CHERGUI, K., MURASE, S., NOMIKOS, G. G., HÖÖK, B. B., CHOUVET, G., HACKSELL, U.AND SVENSSON, T. H.: The 5-HT 1A receptor selective ligands, (R)-8-OH-DPAT and (S)-UH-301, differentially affect the activity of midbrain dopamine neurons. Naunyn-Schmiedeberg Arch. Pharmacol. 347: , 1993b. ARIANO, M. A. AND SIBLEY, D. R.: Dopamine receptor distribution in the rat CNS: Elucidation using anti-peptide antisera directed against D 1A and D 3 subtypes. Brain Res. 649: , BALDESSARINI, R. J., KULA, N. S., MCGRATH, C. R., BAKTHAVACHALAM, V., KEBA- BIAN, J.W.AND NEUMAYER, J. L.: Isomeric selectivity at dopamine D 3 receptors. Eur. J. Pharmacol. 239: , BECKSTEAD, R. M., DOMESICK, V. B. AND NAUTA, W. J. H.: Efferent connections of the substantia nigra and ventral tegmental area in the rat. Brain Res., 175: , BENLOUCIF, S., KEEGAN, M. J. AND GALLOWAY, M. P.: Serotonin-facilitated dopamine release in vivo: Pharmacological characterization. J. Pharmac. Exp. Ther. 265: , BERGSTRÖM, D. A., KREISS, D. S., GONZALES, A. M., HUANG, K.-X., SIBLEY, D. R. AND WALTERS, J. R.: D 3 selective agonists potently inhibit firing rates of substantia nigra dopamine neurons; agonist potencies correlate with D 3 receptor binding affinities but not D 2. Neuropsychopharmacology 10: 237S, BJÖRK, L., BACKLUND HÖÖK, B., NELSON, D. L., ANDÉN, N. E. AND HACKSELL, U.: Resolved N, N-dialkylated 2-amino-8-hydroxytetralins: Stereoselective interactions with 5-HT 1A receptors in the brain. J. Med. Chem. 32: , BLIER, P., STEINBERG, S., CHAPUT, Y. AND DE MONTIGNY, C.: Electrophysiological assessment of putative antagonists of 5-hydroxytryptamine receptors: A single-cell study in the rat dorsal raphe nucleus. Can. J. Physiol. Pharmacol. 67: 98 10, BOUTHENET, M.-L., MARTRES, M.-P., SALES, N. AND SCHWARTZ, J.-C.: A detailed mapping of dopamine D 2 receptors in rat central nervous system by autoradiography with [ 125 I]iodosulpride. Neuroscience 20: , BROEKKAMP, C. L. E., LEYSEN, D., PEETERS, B. W. M. M. AND PINDER, R. M.: Prospect of improved antidepressants. J. Med. Chem. 38: , BURRIS, K. D., PACHECO, M. A., FILTZ, T. M., KUNG, M.-P., KUNG, H.F. AND MOLINOFF, P. B.: Lack of discrimination by agonists for D 2 and D 3 dopamine receptors. Neuropsychopharmacology 12: , CHEN, N.-H. AND REITH, E. A.: Monoamine interactions measured by microdialysis in the ventral tegmental area of rats treated systemically with ( )-8- hydroxy-2-(di-n-propylamino)tetralin. J. Neurochem. 64: , CHIO, C. L., LAJINESS, M. E. AND HUFF, R. M.: Activation of heterologously expressed D 3 dopamine receptors: Comparison with D 2 dopamine receptors. Mol. Pharmacol. 45: 51 60, CORNFIELD, L. J., LAMBERT, G., ARVIDSSON, L.-E., MELLIN, C., VALLGARDA, J., HACKSELL, U. AND NELSON, D. L.: Intrinsic activity of eniantiomers of 8-hydroxy-2-(di-n-propylamino)tetralin and its analogs at 5-hydroxytryptamine 1A receptors that are negatively coupled to adenylate cyclase. Molecular Pharmacol. 39: DAMSMA, G., BOTTEMA, T., WESTERINK, B. H. C., TEPPER, P. C., DIJKSTRA, D., PUGSLEY, T. A., MACKENZIE, R. G., HEFFNER, T. G. AND WIKSTRÖM, H.: Pharmacological aspects of R-( )-7-OH-DPAT, a putative dopamine D 3 receptor ligand. Eur. J. Pharmacol. 249: R9 R10, DE SIMONI, M. G., DAL TOSO, G., FODRITTO, F., SOKOLA, A. AND ALGERI, S.: Modulation of striatal dopamine metabolism by the activity of dorsal raphe serotonergic afferences. Brain Res. 411: 81 88, DEUTCH, A. Y., MOGHADDAM, B., INNIS, R. B., KRYSTAL, J. H., AGHAJANIAN, G. K., BUNNEY, B. S. AND CHARNEY, D. S.: Mechanisms of action of atypical antipsychotic drugs. Implications for novel therapeutic strategies for schizophrenia. Schizophrenia Res. 4: , DRAY, A., DAVIES, J., OAKLEY, N. R., TONGROACH, P. AND VELUCCI, S.: The dorsal and medial projections to the substantia nigra in the rat: Electrophysiological, biochemical and behavioral observations. Brain Res. 151: , FERRÉ, S.AND ARTIGAS, F.: Dopamine D 2 receptor-mediated regulation of serotonin extracellular concentration in the dorsal raphe nucleus of freely moving rats. J. Neurochem. 61: , FERRÉ, S., CORTÈS, R. AND ARTIGAS, F.: Dopaminergic regulation of the serotonergic raphe-striatal pathway: Microdialysis studies in freely moving rats. J. Neurosci. 14: , FOREMAN, M. M., FULLER, R. W., LEANDER, J. D., NELSON, D. L., CALLIGARO,D.O., LUCAITES, V. L.., WONG, D. T., ZHANG, L., BARRET, J. E. AND SCHAUS, J. M.: Pharmacological characterisation of enantiomers of 8-thiomethyl-2-(di-npropylamino)tetralin, potent and selective 5-HT 1A receptor agonists. Drug Dev. Res. 34: 66 85, GEFFARD, M., TUFFET, S., MONS, N. AND CHAGNAUD, J.-L.: Simultaneous detection of indolamines and dopamine in rat dorsal raphe nuclei using specific antibodies. Histochemistry 88: 61 64, GERVAIS, J.AND ROUILLARD, C.: Electrophysiological responses of mesencephalic dopamine neurons to dorsal raphe stimulation in 5-HT-depleted rats. Soc. Neurosci. Abstr. 19: 1372, GIAMBALO, G. T. AND SNODGRASS, S. R.: Biochemical and behavioral effects of serotonin neurotoxins on the nigrostriatal dopamine system: Comparison of injection sites. Brain Res. 152: , GOBERT, A., LEJEUNE, F., RIVET, J.-M., AUDINOT, V., NEWMAN-TANCREDI, A. AND MILLAN, M.. J.: Modulation of the activity of central serotoninergic neurons by novel serotonin 1A receptor agonists and antagonists: A comparison to adrenergic and dopaminergic neurons in rats. J. Pharmacol. Exp. Ther. 273: , 1995a. GOBERT, A., RIVET, J.-M., AUDINOT, V., CISTARELLI, L., SPEDDING, M., VIAN, J., PEGLION, J.-L. AND MILLAN, M. J.: Functional correlates of dopamine D 3 receptor activation in the rat in vivo and their modulation by the selective antagonist, ( )-S II: Both D 2 and silent D 3 autoreceptors control synthesis and release in mesolimbic, mesocortical and nigrostriatal pathways. J. Pharmacol. Exp. Ther. 275: , 1995b. GOBERT, A., RIVET, J.-M., CISTARELLI, L. AND MILLAN, M. J.: Simultaneous modulation of serotonin, dopamine and noradrenaline release in frontal cortex of freely-moving rats by the novel 5-HT 1A receptor ligand, S Eur. J. Neurosci. 8: 16, 1995c. GOBERT, A., LEJEUNE, F., RIVET, J.-M., CISTARELLI, L. AND MILLAN, M. J.: The selective dopamine D 3 agonist, ( )-PD 128,907, decreases the electrical activity of ventrotegmental dopaminergic neurones and inhibits dopamine release in the frontal cortex in vivo: Stereospecific blockade of its action by the selective dopamine D 3 antagonist, ( )-S J. Neurochem. 66: , GONZALEZ, A.M. AND SIBLEY, D. R.: [ 3 H]7-O. H.-Dpat is capable of labeling dopamine D 2 as well as D 3 receptors. Eur. J. Pharmacol. 272: R1 R3, 1995 HERROELEN, L., DE BACKER, J.-P., WILCZAK, N., FLAMEZ, A., VAUQUELIN, G. AND DE KEYSER, J.: Autoradiographic distribution of D 3 -type dopamine receptors in human brain using [ 3 H]7-hydroxy-N, N-di-n-propyl-2-aminotetralin. Brain Res. 648: , HERVÉ, D., PICKEL, V. M., JOH, T.H.AND BEAUDET, A.: Serotonin axon terminals in the ventral tegmental area of the rat: Fine structure and synaptic input to dopaminergic neurons. Brain Res. 435: 71 83, ICHIKAWA, J., KUROKI, T., KITCHEN, M.T.AND MELTZER, H. Y.: R( )-8-OH-DPAT, a 5-HT 1A receptor agonist, inhibits amphetamine-induced dopamine release in rat striatum and nucleus accumbens. Eur. J. Pharmacol. 287: , JOLAS, T., HAJ-DAHMANE, S., LANFUMEY, L., FATTACINI, C. M., KIDD, E. J., ADRIEN, J., GOZLAN, H., GUARDIOLA-LEMAITRE, B. AND HAMON, M.: (-)-Tertatolol is a potent antagonist at pre- and postsynaptic serotonin 5-HT 1A receptors in the rat brain, Naunyn-Schmiedeberg Arch. Pharmacol. 347: , KALÉN, P., SKAGERBERG, G. AND LINDVALL, O.: Projections from the ventral tegmental area and mesencephalic raphe to the dorsal raphe nucleus of the rat. Exp. Brain Res. 73: 69 77, KELLAND, M. D., FREEMAN, A. S. AND CHIODO, L. A.: Serotonergic afferent regulation of the basic physiology and pharmacological responsiveness of nigrostriatal dopamine neurons. J. Pharmac. Exp. Ther. 253: , LEE,E.H.Y.AND GEYER, M. A.: Dopamine autoreceptor mediation of the effects of apomorphine on serotonin neurons. Pharmac. Biochem. Behav. 21: , LEJEUNE, F.AND MILLAN, M. J.: Activation of dopamine D 3 autoreceptors inhibits firing of ventral tegmental dopaminergic neurons in vivo. Eur. J. Pharmacol. 275: R7 R9, LEJEUNE, F., AUDINOT, V., GOBERT, A., RIVET, J.-M., SPEDDING,M.AND MILLAN,M. J.: Clozapine inhibits serotoninergic transmission by an action at 1 - adrenoceptors not at 5-HT 1A receptors. Eur. J. Pharmacol. 260: 79 83, LEJEUNE, F., GOBERT, A., RIVET, J.-M., NEWMAN-TANCREDI, A., AUDINOT, V. AND MILLAN, M. J.: Stimulation of serotonin (5-HT) 1A autoreceptors enhances dopamine (DA) and noradrenaline (NAD) release in rat FCx: Influence of S 15535, WAY 100,635 and 8-OH-DPAT. Eur. J. Neurosci. 6: S4 121, LUM, J. T. AND PIERCEY, M. F.: Electrophysiological evidence that spiperone is an antagonist of 5-HT 1A receptors in the dorsal raphe nucleus. Eur. J. Pharmacol. 149: 9 15, MALMBERG, A. H., NORDVALL, G., JOHANSSON, A. M., MOHELL, N. A. AND HACKSELL,

9 HT 1A Receptors and Dopamine 1249 U.: Molecular basis for the binding of 2-aminotetralins to human dopamine D 2A and D 3 receptors. Mol. Pharmacol. 46: , MELLER, E., GOLDSTEIN, M. AND BOHMAKER, K.: Receptor reserve for 5-hydroxytryptamine 1A -mediated inhibition of serotonin synthesis: Possible relationship to anxiolytic properties of 5-hydroxytryptamine 1A agonists. Mol. Pharmacol. 37: , MELTZER, H. Y.: The importance of serotonin-dopamine interactions in the action of clozapine. Br. J. Psychiatry 160: 22 29, MILLAN, M. J., CANTON, H., GOBERT, A., LEJEUNE, F.,. RIVET, J.-M., BERVOETS, K., BROCCO, M., WIDDOWSON, P., MENNINI, T., AUDINOT, V., HONORÉ, P., RENOUARD, A., LE MAROUILLE-GIRARDON, S., VERRIÈLE, L., GRESSIER, H.AND PEGLION, J.-L.: Novel benzodioxopiperazines acting as antagonists at postsynaptic 5-HT 1A receptors and as agonists at 5-HT 1A autoreceptors: A comparative pharmacological characterization with proposed 5-HT 1A antagonists. J. Pharmacol. Exp. Ther. 268: , MILLAN, M. J., PEGLION, J.-L., VIAN, J., RIVET, J.-M., BROCCO, M., GOBERT, A., NEWMAN-TANCREDI, A., DACQUET, C., BERVOETS, K., GIRARDON, S., JACQUES, V., CHAPUT, C.AND AUDINOT, V.: Functional correlates of dopamine D 3 receptor activation in the rat in vivo and their modulation by the selective antagonist, ( )-S 14297: I. Activation of postsynaptic D3 receptors mediates hypothermia while blockade of D2 receptors elicits prolactin secretion and catalepsy. J. Pharmac. Exp. Ther. 275: , MILLAN, M. J., NEWMAN-TANCREDI, A., RIVET, J.-M., BROCCO, M., LACROIX, P., AUDINOT, V. AND GOBERT, A.: S 15535, a novel benzodioxopiperazine ligand of serotonin (5-HT) 1A receptors: I. Interaction with cloned human (h)5-ht1a, dopamine hd2/d3 and h 2A-adrenergic receptors in relation to modulation of cortical monoamine release and potential antidepressant properties. Submitted, MIQUEL, M.-C., DOUCET, E., BONI, C., EL MESTIKAWY, S., MATTHIESSEN, L., DAVAL, G., VERGÉ, D. AND HAMON, M.: Central serotonin 1A receptors: Respective distributions of encoding mrna, receptor protein and binding sites by in situ hybridization histochemistry, radioimmunohistochemistry and autoradiographic mapping in the rat brain. Neurochem. Int. 19: , NEWMAN-TANCREDI, A., AUDINOT, V., JACQUES, V., PEGLION, J.-L. AND MILLAN, M. J.: [ 3 H]( )S 14297: A novel, selective radioligand at cloned human dopamine D3 receptors. Neuropharmacology 34: , NEWMAN-TANCREDI, A., CHAPUT, C., VERRIÈLE, L. AND MILLAN, M. J.: Clozapine is a partial agonist at cloned, human serotonin 5-HT 1A receptors. Neuropharmacology 35: , NEWMAN-TANCREDI, A., WOOTTON, R. AND STRANGE, P. G.: High-level stable expression of recombinant 5-HT 1A receptors in Chinese hamster ovary cells. Biochem. J. 285: , PALACIOS, J. M. AND PAZOS, A.: Visualization of dopamine receptors: A progress review. In Dopamine Receptors, ed. by I. Creese and C. Fraser, pp Alan R. Liss Inc., New York, PATEL, J., TROUT, S. J., PALIJ, P., WHELPTON, R. AND KRUK, Z. L.: Biphasic inhibition of stimulated endogenous dopamine release by 7-OH-DPAT in slices of rat nucleus accumbens. Br. J. Pharmacol. 115: , PAXINOS, G. AND WATSON, C.: The Rat Brain in Stereotaxic Coordinates. Academic Press, New York, PAZOS, A. AND PALACIOS, J. M.: Quantitative autoradiographic mapping of serotonin receptors in the rat brain. Serotonin-1 receptors. Brain Res. 346: , PRISCO, S., PAGANNONE, S. AND ESPOSITO, E.: Serotonin-dopamine interaction in the rat ventral tegmental area: An electrophysiological study in vivo. J. Pharmac. Exp. Ther. 271: 83 90, RIVET, J.-M., AUDINOT, V., GOBERT, A., PEGLION, J.-L. AND MILLAN, M. J.: Modulation of mesolimbic dopamine release by the selective dopamine D 3 receptor antagonist, ( )-S Eur. J. Pharmacol. 265: , SINTON, C. M. AND FALLON, S. L.: Electrophysiological evidence for a functional differentiation between subtypes of the 5-HT 1 receptor. Eur. J. Pharmacol. 157: , SMITH, C. F. C. AND CUTTS S. D.: Dopamine agonist activity of 8-OH-DPAT. Br. J. Pharmacol. 98: 755P, SOKOLOFF, P. AND SCHWARTZ, J.-C.: Novel dopamine receptors half a decade later. Trends Pharmacol. Sci. 16: , SOKOLOFF, P., ANDRIEUX, M., BESANÇON, R., PILON, C., MARTRES, M.-P., GIROS, B. AND SCHWARTZ, J.-C.: Pharmacology of human dopamine D 3 receptor expressed in mammalian cell line: Comparison with D 2 receptor. Eur. J. Pharmacol. 225: ; SPROUSE, J. S. AND AGHAJANIAN, G. K.: (-)-Propranolol blocks the inhibition of serotonergic dorsal raphe cell firing by 5-HT 1A rdselective agonists. Eur. J. Pharmacol. 128: , STERN, W. C., JOHNSON, A., BRONZINO, J. D. AND MORGANE, P. J.: Neuropharmacology of the afferent projections from the lateral hebenula and substantia nigra to the anterior raphe in the rat. Neuropharmacology 20: , SVENSSON, T. H., MATHÉ, J. M., ANDERSSON, J. L., NOMIKOS, G. G., HILDEBRAND, B. E. AND MARCUS, M.: Mode of action of atypical neuroleptics in relation to the phencyclidine model of schizophrenia: Role of 5-HT 2 receptor and 1- adrenoceptor antagonism. J. Clin. Psychopharmacol. 15: 11S 18S, SWANSON, L. W.: The projections of the ventral tegmental area and adjacent regions: A combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res. Bull. 9: , TANDA, G., CARBONI, E., FRAU, R. AND DI CHIARA, G.: Increase of extracellular dopamine in the prefrontal cortex: A trait of drugs with antidepressant potential? Psychopharmacol. 115: , TANG, L., TODD, R.D. AND O MALLEY, K. L.: Dopamine D 2 and D 3 receptors inhibit dopamine release. J. Pharmac. Exp. Ther. 270: , VAN BOCKSTAELE, E. J., CESTARI, D. M. AND PICKEL, V. M.: Synaptic structure and connectivity of serotonin terminals in the ventral tegmental area: Potential sites for modulation of mesolimbic dopamine neurons. Brain Res. 647: , VAN WIJNGAARDEN, I., TULP, M. TH. M. AND SOUDIJN, W.: The concept of selectivity in 5-HT receptor research. Eur. J. Pharmacol. 188: , VERTES, R. P.: A PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat. J. Comp. Neurol. 313: , WANG, R. Y.: Dopaminergic neurons in the rat ventral tegmental area. I. Identification and characterization. Brain Res. Rev. 3: , YOSHIMOTO,K.AND MCBRIDE, W. J.: Regulation of nucleus accumbens dopamine release by the dorsal raphe nucleus in the rat. Neurochem. Res. 17: , ZHANG, J. AND FREEMAN, A. S.: Electrophysiological effects of the selective 5-HT 1A agonist, 8-OH-DPAT, on midbrain DA neurons: A9/A10 differences. Soc. Neurosci. Abstr. 19: 1373, Send reprint requests to: Dr. Mark J. Millan, Institut de Recherches Servier, Centre de Recherches de Croissy, Psychopharmacology Department, 125, Chemin de Ronde, Croissy-sur-Seine, France.