Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8155-8159, September 1992 Neurobiology Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7-[3H]hydroxy-NN-di-n-propyl- 2-aminotetralin (guanine nucleotide-binding regulatory protein-coupled receptor/antipsychotics/islands of Calleja/cerebellum/nucleus accumbens) DANIEL LtVESQUE*, JORGE DIAZt, CATHERINE PILON*, MARIE-PASCALE MARTRES*, BRUNO GIROS*, EVELYNE SOUILt, DOMINIQUE SCHOTTt, JEAN-LOUIS MORGATt, JEAN-CHARLES SCHWARTZ*§, AND PIERRE SOKOLOFF* *Unite de Neurobiologie et Pharmacologie (U. 109) de l'Institut National de la Sante et de la Recherche Medicale, Centre Paul Broca, 75014 Paris, France; tLaboratoire de Physiologie, Facultd de Pharmacie, Universite Rene Descartes, 75006 Paris, France; and tD6partement de Biologie, Centre d'Etudes Nucleaires, 91191 Saclay, France Communicated by Jean-Pierre Changeux, May 20, 1992

ABSTRACT We have identified 7-[3Hlhydroxy-N,N-di-n- Therefore, there is an obvious interest in studying the propyl-2-aminotetralin ([3H]7-OH-DPAT) as a selective probe native receptor protein in brain, particularly in the case ofthe for the recently cloned dopamine D3 receptor and used it to D3 receptor, whose pharmacology in Chinese hamster ovary assess the presence ofthis receptor and establish its distribution (CHO) cells and cerebral localization of its mRNA suggest and properties in brain. In transfected Chinese hamster ovary that it may represent an important target for antipsychotics (CHO) cells, it binds to D3 receptors with subnanomolar (8, 12, 13). affinity, whereas its affinity is approximately 100-, 1000-, and Starting with the idea that designing a selective radioactive 10,000-fold lower at D2, D4, and D, receptors, respectively. probe for the D3 receptor would help to settle these issues, we Specific [3H]7-OH-DPAT binding sites, with aKd of0.8 nM and have screened a series of dopamine agonists and thereby a pharmacology similar to those at reference D3 receptors of identified 7-hydroxy-N, N-di-n-propyl-2-aminotetralin (7- CHO cells, were identified in rat brain. D3 receptors differ OH-DPAT) (14-16) as fulfilling this purpose. from D2 receptors in brain by their lower abundance (2 orders of magnitude) and distribution, restricted to a few mainly phylogenetically ancient areas-e.g., paleostriatum and ar- MATERIALS AND METHODS chicerebellum-as evidenced by membrane binding and auto- Brain Membranes. Male Wistar rats (IFFA Credo, Orldans, radiography studies. Native D3 receptors in brain are charac- France) weighing 180-200 g were used. They were exposed terized by an unusually high nanomolar affinity for dopamine to an artificial light cycle of 12 h/day and had free access to and a low modulatory influence of guanyl nucleotides on food and water. After decapitation, brain tissues were rapidly agonist binding. These various features suggest that D3 recep- dissected out, frozen on dry ice, and kept at -70°C until use. tors are involved in a peculiar mode of neurotransmission in a Tissues were homogenized with a Polytron (setting 7 for 10 restricted subpopulation of dopamine neurons. sec) in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA and centrifuged at 2500 x g for 30 sec; the supernatant was Dopamine is an important neurotransmitter in brain, being centrifuged at 35,000 x g for 15 min. The pellet was resus- involved physiologically in the control of cognitive, motor, pended and centrifuged again, and this washing procedure and endocrine processes and pathologically in Parkinson and was repeated twice to eliminate endogenous dopamine. The possibly mental diseases. final was sonication in a buffer con- Until recently, it was largely believed that its various pellet resuspended by actions were mediated by two receptor subtypes, termed D1 taining 50 mM NaHepes, 1 mM EDTA, 50 ,uM 8-hydroxy- and D2 (1, 2). Molecular biology approaches have led, how- quinoleine, 0.005% ascorbic acid, and 0.1% bovine serum ever, to the identification and cloning of the genes corre- albumin (pH 7.5) (incubation buffer). sponding not only to these two receptor subtypes (3-7) but Membranes from Cell Lines. CHO cell lines expressing rat also to additional and less expected ones, termed D3 (8), D4 D2 or D3 dopamine receptors (CHO-D2 or CHO-D3) have (9), and D5 (10, 11). For the latter receptors, the information been described (12). CHO-D1 were created by transfecting so far available derives from molecular biology approaches; pCD-BS plasmid containing the human D1 receptor gene (6), their pharmacology and signaling system have only been a generous gift of P. Seeman. COS-7 cells transiently ex- studied in transfected cells, generally fibroblasts, and their pressing the human D4 receptor were obtained by transfect- distribution has been indirectly approached by studies of ing pCD-PS plasmid, a generous gift of H. H. M. Van Tol, mRNA localization. Nevertheless, these otherwise valuable containing the human D4 receptor gene (9). Cells were grown approaches suffer from some limitations. When expressed in in Dulbecco's modified Eagle's medium containing 10% fetal fibroblasts, receptors might find a membrane environment, bovine serum. Cells were harvested by trypsin treatment which could modify their pharmacological specificity, and a (0.25%) for 4-5 min and centrifugation at 2000 x g for 5 min. repertoire of cellular components, particularly GTP-binding They were homogenized with a Polytron in 10 mM Tris HCl proteins (G proteins), which may differ from that found in (pH 7.5) containing 1 mM EDTA and were centrifuged at neurons. Regarding distribution, mRNA detection reveals 35,000 x g for 15 min. The pellet was then resuspended by the sites of receptor synthesis rather than receptor localiza- sonication in the NaHepes buffer described above. tion, which may be different. Abbreviations: 7-OH-DPAT, 7-hydroxy-N,N-di-n-propyl-2-amino- The publication costs of this article were defrayed in part by page charge tetralin; p[NH]ppG, 5'-guanylyl imidodiphosphate; G protein, GTP- payment. This article must therefore be hereby marked "advertisement" binding protein. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed.

8155 Downloaded by guest on October 2, 2021 8156 Neurobiology: Ldvesque et al. Proc. Natl. Acad. Sci. -USA 89 (1992) A 20 B [3H]70H-OPAT [ 125I]IODOSULPRIDE

I-I. CD3 60 - D3 D2 D04 D 010 cm

o -11 -10 -9 -8 -7 -6 -5 -4 -3 0 100 200 300 400 500 0 200 400 600 8 1000 LOG [70H-OPAT] (M) BOUND (fuol.ag protein-') BOUND (fmol.mg protein-1)

FIG. 1. (A) Inhibition by 7-OH-DPAT of ligand binding to D1, D2, D3, and D4 receptors in transfected CHO cells. The ligands were 0.3 nM [3H]SCH23390 at the D1 receptor, 0.1 and 0.2 nM (125I]iodosulpride at the D2 and D3 receptors, respectively, and 0.1 nM [3H]spiperone at the D4 receptor. Results are means from two experiments. (B) Scatchard analysis of [3H]7-OH-DPAT (Left) and [125Iliodosulpride (Right) specific binding to D2 and D3 receptors in transfected CHO cells. This is a representative example of two such experiments. Note that [3H]7-OH-DPAT binding to D2 receptors was not measurable. (Inset) Chemical structure of 7-OH-DPAT; *, tritium atom. Binding Assays. Membranes from brain (150-300 pg of 32P-labeled complementary RNA probe specific for the D3 protein) or cell lines (15-25 ,ug of protein) were added to receptor (13). polypropylene test tubes containing [3H]7-OH-DPAT for the Drugs. 7-OH-DPAT and its precursor 7-hydroxy-N,N- D3 receptor assay, [1251]iodosulpride for D2 and D3 receptors, diallylaminotetralin were custom synthesized at the Institut de [3H]spiperone for the D4 receptor, or [3H]SCH23390 for the Chimie des Substances Naturelles (Gif-sur-Yvette, France). D1 receptor. Competing drugs were dissolved in incubation [3H]7-OH-DPAT was prepared by catalytic reduction, with buffer, the final volume being 1 ml, except for [125I]iodosul- pure 3H gas, of the double bond located on the N,N-diallyl pride binding, for which it was 0.4 ml. Tubes were incubated precursor. The tritiated molecule was then purified by high- in triplicate for 1 h at room temperature. The incubations performance liquid chromatography through a preparative were stopped by rapid filtration under reduced pressure ,uBondapak column in a 0-50%o methanol gradient in water through Whatman GF/C glass filters coated with 0.1% bovine containing 0.05% triethanolamine. The specific activity was serum albumin (except for the [125I]iodosulpride binding estimated to be 158 Ci/mmol (1 Ci = 37 GBq). [M2IJIodosul- assay, for which the filters were Whatman GF/B-coated with pride (2000 Ci/mmol), [N-methyl-3H]SCH23390 (80 Ci/ 0.3% polyethyleneimine), followed by three rinses with 3-4 mmol), and [phenyl-4-3H]spiperone (60 Ci/mmol) were pur- ml of ice-cold buffer (50 mM TrisHCl, pH 7.5/120 mM chased from Amersham. Dopamine, apomorphine, and 5'- NaCl). Nonspecific binding was measured in the presence of guanylyl imidodiphosphate (p[NHflppG) were purchased from 1 ,uM dopamine (for [3H]7-OH-DPAT), 5 ,M (-)-sulpiride Sigma. (-)-Sulpiride was generously donated by Delagrange (for [1251]iodosulpride), 0.5 ,uM YM-09151-2 (for [3H]spiper- Laboratoires (Paris), haloperidol and domperidone were sup- one), and 1 ,uM SCH23390 (for [3H]SCH23390). Radioactiv- plied by Janssen Laboratoires (Paris), and SCH23390 was ity was counted by y scintigraphy for the 1251 ligand or by from Schering. scintillation spectrometry, at an efficiency of 41%, for 3H ligands. Saturation curves were analyzed by computer non- RESULTS linear regression using a one-site cooperative model to obtain equilibrium dissociation constants (Kd) and maximal density 7-OH-DPAT Binding to Ceil Line Membranes. The selec- of receptors (Bn.). Competition curves were also analyzed tivity of nonradioactive 7-OH-DPAT was first assessed in by computerized nonlinear regression using either a one-site competition experiments with transfected cell lines express- or a two-site cooperative model to estimate IC50 and the ing a single dopamine receptor subtype, labeled with an proportion of sites (17). The best fit was determined by appropriate radioactive ligand. 7-OH-DPAT was much more statistical analysis between computer calculations. Inhibition potent at the D3 than at the other receptor sites, its Ki values constants (Kj) were estimated according to the equation Ki = being 0.78 ± 0.02 nM, 61 ± 2 nM, 650 ± 80 nM, and 5300 ± IC50/(1 + S/Kd), where S represents the ligand concentra- 500 nM at D3, D2, D4, and D1 receptors, respectively (Fig. tion. Curves were drawn with the INPLOT program (Graph- Pad, San Diego). Autoradiography. Rats were killed by decapitation; brains were removed and immediately dipped into liquid monochlo- rodifluoromethane for 5 min. Frozen brains were sectioned with a Bright-Shandon cryostat and 10-,um sections were thaw-mounted onto precleaned and polylysine-coated glass slides. For [3H]7-OH-DPAT binding, slides were preincu- bated in 50 mM NaHepes (pH 7.5) containing 1 mM EDTA and 0.1% bovine serum albumin for 5 min three times at 22°C and then incubated in the same buffer containing 0.5 nM [3H]7-OH-DPAT for 1 h. Some slides were incubated in the presence of 1 ,uM dopamine to estimate nonspecific binding. 5 10 15 20 The slides were then rinsed four times in ice-cold NaHepes BOUND (fmol.mg protein-') buffer containing 100 mM NaCl. After brief dipping into ice-cold distilled water, the slides were dried and apposed to FIG. 2. Scatchard analysis of[3H]7-OH-DPAT specific binding to Hyperfilm-3H (Amersham) for 2 months. membranes of olfactory tubercle (Olf. Tub.), nucleus accumbens (N. In Situ Hybridization. Brain sections were fixed in 4% Acc.), and striatum (Str.). [3H]7-OH-DPAT binding in the olfactory paraformaldehyde, prehybridized, and hybridized with a tubercle was not affected by 100 jM p[NH]ppG (see Table 2). Downloaded by guest on October 2, 2021 Neurobiology: Uvesque et al. Proc. Natl. Acad. Sci. USA 89 (1992) 8157 Table 1. Regional distribution of [3H]7-OH-DPAT binding sites Table 2. Pharmacology of [3H]7-OH-DPAT binding to rat in rat brain olfactory tubercle compared to D2 and D3 receptor Binding at 0.7 nM, Bm, fmol pharmacology in transfected CHO cells fmol per mg of per mg Ki values, nM Region protein of protein Kd, nM Olfactory Olfactory tubercle 6.6 ± 0.4 (100) 17 ± 1 0.7 ± 0.2 Agent tubercle CHO-D3 CHO-D2 Archicerebellum 6.2 ± 0.4 (94) 11 ± 2 0.5 ± 0.1 Agonists ± Nucleus accumbens 3.6 ± 0.2 (55) 10 ± 1 1.0 0.2 Dopamine 8 + 1 25 474 Olfactory bulb 2.9 ± 0.5 (44) 13 ± 2 1.4 ± 0.4 Dopamine + p[NH]ppG 9 ± 2 27 1705 Hypothalamus 1.5 ± 0.4 (23) Quinpirole 5 ± 1 5.1 576 Striatum 1.4 ± 0.3 (21) 5.5 ± 0.4 1.0 ± 0.1 Apomorphine 14 ± 3 20 24 Prefrontal cortex 1.0 ± 0.5 (15) 7-OH-DPAT 0.7 ± 0.2* 0.78 61 Substantia nigra 0.8 ± 0.2 (12) 7-OH-DPAT + p[NH]ppG 0.9 ± 0.4* Anterior pituitary 0.8 ± 0.2 (12) 8-OH-DPAT 328 ± 44 Hippocampus 0.3 ± 0.1 (5) Antagonists Specific binding was evaluated either at a single [3H]7-OH-DPAT Haloperidol 5 ± 1 10 0.4 concentration or by analysis of saturation isotherms like those shown UH232 9 ± 1 9 40 in Fig. 2. Values are means ± SEM of three to six independent (-)-Sulpiride 12 ± 3 25 9 determinations. Values in parentheses represent percentage of ol- Domperidone 20 ± 3 10 0.3 factory tubercle binding. Clozapine 224 ± 99 180 56 1A). Then, the best conditions to obtain a selective binding SCH23390 1581 ± 415 780 720 of [3H]7-OH-DPAT to a D3 receptor with negligible binding Values for the olfactory tubercle were obtained by using 0.8 nM to D2 receptors were selected by using the corresponding cell [3H]7-OH-DPAT in three or four independent experiments. Values lines. In the absence of Mg2+ (and the presence of EDTA), for transfected CHO cells were obtained by using [125I]iodosulpride binding to D3 receptor was not affected, whereas specific and are from ref. 8 (or Fig. 1 for 7-OH-DPAT). binding to D2 receptors was nearly abolished; total binding at *Kd value. 0.1-5 nM did not significantly differ from nonspecific binding areas (Table 1); in olfactory tubercle, it represented 50-60% in CHO-D2 membranes to which the Bmax of [1251]iodosul- of total binding, but in striatum and hippocampus it repre- pride was 758 fmol per mg of protein (Fig. 1B). [3H]7-OH- and oftotal binding, respectively. DPAT binding to CHO-D3 membranes occurred monophasi- sented only 10-20% 5-10%o = (28% at 0.8 The pharmacological properties of [3H]7-OH-DPAT binding cally (nH 0.996) over low nonspecific binding the values of a series of nM) with a Kd of 0.67 + 0.03 nM and a Bma. of 403 ± 7 fmol sites were defined by evaluating Ki per mg of protein in the same range as the Bn. of [1251]io- dopamine agonists and antagonists with membranes of ol- dosulpride with CHO-D3 membranes-i.e., 485 + 5 fmol per factory tubercle (Table 2); these values were highly corre- mg ofprotein (Fig. 1B). Upon addition of 100 AM p[NH]ppG, lated (r = 0.996; n = 11; P < 0.001) with those derived from in the absence of Mg2+, the Kd value of [3H]7-OH-DPAT a previous study (8) with CHO-D3, but not with CHO-D2 (r became 0.80 + 0.03 nM; in the presence of 5 mM MgCl2, the = 0.218; n = 11). As in CHO-D3, the Ki of dopamine and the Kd values were 0.50 + 0.02 and 0.77 + 0.08 nM in the absence Kd of [3H]7-OH-DPAT were not significantly modified by and presence of p[NH]ppG, respectively (data not shown). p[NH]ppG (Table 2). Addition ofthe guanyl nucleotide in the [3H]7-OH-DPAT and [2'sI]Iodosuipride Binding to Rat presence of 2 mM MgCl2 affected only slightly the dopamine Brain Membranes. A specific saturable [3H]7-OH-DPAT inhibition curve of [125I]iodosulpride binding to membranes binding was detected in membranes from some brain areas. of the archicerebellum; the biphasic curve with components Saturation experiments with membranes from olfactory tu- of K, values of 0.33 ± 0.15 (30% of sites) and 20.2 + 0.1 nM bercle, nucleus accumbens, and striatum (Fig. 2) and a few was transformed into a monophasic curve (Ki = 10.5 ± 1.5 other areas (data not shown) indicated that the binding nM) (Fig. 3). [125I]Iodosulpride binding in the archicerebel- occurred with a rather similar Kd (Table 1). The specific lum was inhibited by 7-OH-DPAT with a K, of 3.7 ± 0.9 nM; binding of [3H]7-OH-DPAT was also evaluated at a single addition of p[NH]ppG did not modify this Ki value (3.5 + 0.8 concentration close to the Kd (0.7 nM) in additional brain nM), while clozapine had a Ki of2% ± 29 nM. In comparison, CEREBELLUM PITUITARY 2 2 co WI 8!

r-IN 9

be 0 -12 -11 -10 -9 -8 -7 -6 -5 0 -11 -10 -9 -8 -7 -6 -5 LOG [DOPAMINE] (M) LOG [DOPAMINEJ (M) FIG. 3. Effect ofp[NH]ppG (100 ,M) on inhibition of [1251]iodosulpride binding by dopamine in archicerebellum (Left) and anterior pituitary (Right). Means of two or three independent determinations are shown. [125I]Iodosulpride was used at 0.35 nM in a buffer containing 50 mM Tris*HCl, 5 mM KC1, 2 mM MgCl2, 2 mM CaCl2, 50 ,M 8-hydroxyquinoleine, 0.005% ascorbic acid, 0.1% bovine serum albumin, and 120 mM NaCl (pH 7.5). Nonspecific binding was estimated by using 5 ,uM (-)-sulpiride and 3 ,uM dopamine for anterior pituitary and cerebellum. o, With p[NH]ppG; *, without p[NH]ppG. Downloaded by guest on October 2, 2021 8158 Neurobiology: Ldvesque et al. Po.Nt.Aa.SiProc. Nad. Acad. Sci. USAS 899(92(1992)

the two components of dopamine inhibition of ["25Iliodosul- DISCUSSION pride binding to pituitary membranes had K, values of 3.3 +- 0.6 nM (52% of sites) and 376 ±83 nM, which became 12.3 The present identification of 7-OH-DPAT as a selective D3 ± 4.2 (30% of sites) and 1328 ±244 nM in the presence of receptor ligand and its consecutive use in tritiated form has p[NH]ppG (Fig. 3). allowed us to establish the occurrence, pharmacological Autoradiographic Loaiain of [3H17-OH-DPAT Binding properties, and regional distribution of the D3 receptor pro- Sites and D3 Receptor mRNA in Rat Brain. Autoradiograms tein in rat brain. indicated a restricted distribution of [3H]7-OH-D)PAT binding The compound was selected through screening of various sites, with very high levels on the islands of Calleja and in the putative dopamine "autoreceptor-selective" agents, since a molecular layer of lobules 9 and 10 of the cerebellum. fraction of )3 receptor mRNAs is present within dopaminer- Distinct, although lower, labeling was observed in nucleus gic perikarya, suggesting that this receptor, as well as the 1)2 accumbens and with low in the receptor, may play an autoreceptor function (8, 13). Indeed, olfactory bulb, very labeling 7-OH-DPAT had been shown to potently inhibit dopamine striatum. was inhibited in the of Binding completely presence synthesis in vivo (14) and in vitro (15) in slices of rat striatum. 1 u.M a and uniform dopamine, leaving negligible nonspecific In transfected cell lines expressing a single dopanune labeling (data not shown). This distribution of [3H]7-OH- receptor subtype, 7-OH-DPAT displayed subnanomolar af- DPAT sites was similar to that of mRNA D3 receptor (Fig. 4). finity at the 1)3receptor (K, = 0.7 nM) and 100-, 1000-, and

10,000-fold lower affinity at 1)2, D4, and D1 receptors, re- spectively. In addition, the experimental conditions ensuring A B a selective binding of [3H]7-OH-1)PAT at the 1)3 receptor, without significant 1)2 receptor binding, were optimized in studies with CHO-1)2 and CHO-1)3 cell lines. Under the conditions selected to minimize its binding to the high-affinity

form of the 1)2 receptor, [3H]7-OH-1)PAT labeled the full

population of D3 receptors but only a negligible fraction of 1)2 receptors. Under the same conditions, binding of [3H]7-OH-DPAT to

membranes of some rat brain areas was evidenced. That the I #-Acb binding sites correspond selectively to 1)3receptors is shown i by the Kd of the ligand, similar in several areas to its Kd in the reference CHO-D)3 cells. In addition, the pharmacology of

,.a binding sites was similar in rat olfactory tubercle and in IV * *,%,.r I., CHO-)3 cells. Finally, the restricted localization of binding 7 2mm sites in few dopaminoceptive areas expressing 1)3 receptor lcj mRNA also supports the selectivity of labeling. c Both membrane binding and autoradiographic studies show us that the 1)3receptor protein is less abundant and is

expressed in a much more restricted manner than the 1)2 receptor protein. Overall, the number of 1)3receptors in the

whole brain can be estimated to be lower than that of12

receptors by =%2 orders of magnitude, a ratio in the same range as that of corresponding mRNAs (8, 13). Indeed, whereas the receptor occurs in all major dopamine pro- jection fields, particularly the neostriatum, and its abundance

more or less parallels the density of dopamine axons, 1)3 receptors are expressed in an appreciable manner in only a few brain areas, among which the ventral (lImbic) parts of the D striatal complex (i.e., the shell and anterior part of the nucleus accumbens, olfactory tubercle, and islands of Calleja) and lobules 9 and 10 of the cerebellum (i.e., archicer- ebellum) are the most prominent. Since the same areas selectively express 1)3receptor mRNA (refs. 8 and 13 and this ,CPU study), these localizations should correspond to dendrites, perikarya, or short axons rather than to axons of distant

neurons. These various areas enriched in 1)3 receptors gen- Lob.1O to ~4AcbB erally correspond well established, although relatively minor, dopamine projections, except in archicerebellum I~j 2mm where histochemical (18, 19) but not biochemical approaches (20) suggest their absence. Nevertheless, these sites in the FIG. 4. Comparison of autoradiographic localization of [3H]7- cerebellum were previously evidenced by using [1251I]iodosul- OH-DPAT specific binding to rat brain sections (A and C) with the pride (21), which binds with subnanomolar affinity to )2 and distribution of D3 receptor mRNA measured by in situ hybridization 1)3receptors and with 10 times lower affinity to 1)4receptors (B and D). Frontal (A and B) or sagittal (C and D) sections at (data not shown). These features, together with the pharma- approximately similar levels were either incubated with [3H]7-OH- cology of these sites (see Results) as well as with the absence DPAT (0.5 nM) or hybridized with a 32p-labeled complementary and low level of 1)2 and 1)4 receptor mRNA, respectively RNA probe. In sections incubated with [3H]7-OH-DPAT and dopa- (unpublished observattion), indic-ate- that cerebellar (1f'I5io- mine (1 AM), labeling in nucleus accumbens (Acb), islands of Calleja sites to In (ICj), and cerebellum was completely abolished. Lob. 10, lobule 10; dosulpride binding correspond 1)3 receptors. OR, olfactory bulb; Cpu, caudate putamen; IQJM, islands of Calleja, substantia nigra. and neostriatum, 1)3 receptor abundance is major island. extremely low but may largely reflect the expression of 1)3 Downloaded by guest on October 2, 2021 Neurobiology: Uvesque et al. Proc. Natl. Acad. Sci. USA 89 (1992) 8159 autoreceptors on perikarya and terminal areas of a subpop- 1. Spano, P. F., Govoni, S. & Trabucchi, M. (1978) Adv. Bio- ulation of dopamine neurons. chem. Psychopharmacol. 19, 155-165. Two major properties of the native D3 receptor protein in 2. Kebabian, J. W. & Calne, D. B. (1979) Nature (London) 277, brain could be established through the use of [3H]7-OH- 93-96. DPAT: its unusually high affinity for the neurotransmitter 3. Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., dopamine and the low modulatory effect Salon, J., Christie, M., Machida, C. A., Neve, K. A. & Civelli, of guanyl nucleo- 0. (1988) Nature (London) 336, 783-787. tides on its binding. Both properties, which suggest a peculiar 4. Dearry, A., Gingrich, J. A., Falardeau, P., Fremeau, R. T., mode of dopaminergic transmission, were previously de- Bates, M. D. & Caron, M. G. (1990) Nature (London) 347, tected in transfected CHO cells but they could have been 72-76. related to differences in membrane composition or absence of 5. Zhou, Q. Z., Grandy, D. K., Thambi, L., Kushner, J. A., Van a cellular component, particularly a G protein, in the trans- Tol, H. H. M., Cone, R., Pribnow, D., Salon, J., Bunzow, formed fibroblast as compared to cerebral neurons (8, 12). J. R. & Civelli, 0. (1990) Nature (London) 347, 76-80. The nanomolar affinity ofthe neurotransmitter, quite unusual 6. Sunahara, R. K., Niznik, H. B., Weiner, D. M., Stormann, for an amine, suggests that dopamine might act, through D3 T. M.,Brann,M. R.,Kennedy,J. L.,Gelernter,J. E.,Rozma- receptors, at some distance from the terminals that release it hel, R., Yang, Y., Israel, Y., Seeman, P. & O'Dowd, B. F. (1990) Nature (London) 347, 80-83. and/or occupy the D3 receptor for longer periods oftime than 7. Monsma, F. J., Mahan, L. C., McVittie, L. D., Gerfen, C. R. in the case ofother receptor subtypes. Such a quasi-hormonal & Sibley, D. R. (1990) Proc. Natl. Acad. Sci. USA 87, 6723- mode of action might be consistent with the high expression 6727. of the D3 receptor within the core of the islands of Calleja, 8. Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L. & whereas dopamine axons surround these islands, making Schwartz, J.-C. (1990) Nature (London) 347, 146-151. only few contacts with granule cells (22). In addition, since 9. Van Tol, H. H. M., Bunzow, J. R., Guan, H. C., Sunahara, the background dopamine concentration in the extracellular R. K., Seeman, P., Niznik, H. B. & Civelli, 0. (1991) Nature space of the striatum is 5-20 nM (23), D3 receptors should be (London) 350, 610-614. subjected to a tonic stimulation. 10. Sunahara, R. K., Guan, H. C., O'Dowd, B. F., Seeman, P., Laurier, L. G., Ng, G., George, S. R., Torchia, J., Van Tol, The weak modulatory effect of a guanyl nucleotide on H. H. M. & Niznik, H. B. (1991) Nature (London) 350, 614- dopamine binding at native D3 receptors was first evidenced 619. by the absence ofsignificant change in dopamine competition 11. Tiberi, M., Jarvie, K. R., Silvia, C., Falardeau, P., Gingrich, of [3H]7-OH-DPAT binding to membranes from the olfactory J. A., Godinot, N., Bertrand, L., Yang-Feng, T. L., Fremeau, tubercle (Table 2). This observation was made, however, in R. T., Jr., & Caron, M. G. (1991) Proc. Natl. Acad. Sci. USA conditions in which coupling of the G protein was possibly 88, 7491-7495. not favored-i.e., in the absence ofMg2+ (24). Nevertheless, 12. Sokoloff, P., Andrieux, M., Besancon, R., Pilon, C., Martres, it was confirmed by using membranes ofthe archicerebellum M.-P., Giros, B. & Schwartz, J.-C. (1992) Eur. J. Pharmacol. and [125I]iodosulpride as the ligand. The marginal effect of Mol. Biol. 225, 331-337. 13. Bouthenet, M.-L., Souil, E., Martres, M.-P., Sokoloff, P., p[NH]ppG in this prototypic D3 receptor area contrasts with Giros, B. & Schwartz, J.-C. (1991) Brain Res. 564, 203-219. its well-established effect in pituitary, where the vast major- 14. Feenstra, M. G., Sumners, C., Goedemoed, J. H., De Vries, ity of [125I]iodosulpride binding sites correspond to D2 re- J. B., Rollema, H. & Horn, A. S. (1983) Naunyn Schmiede- ceptors (Fig. 3). Hence, these data support the view that the bergs Arch. Pharmacol. 324, 108-115. two native receptor subtypes are coupled to distinct G 15. El Mestikawi, S., Glowinski, J. & Hamon, M. (1986) J. Neu- proteins or do not interact similarly with a single G protein. rochem. 46, 12-22. This is consistent with observations in transfected CHO cells 16. Mulder, T. B., De Vries, J. B., Dijkstra, D., Wiechers, J. W., showing that, in contrast with D2, D3 receptors mediate only Grol, C. J. & Horn, A. S. 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