Proc. Nati. Acad. Sci. USAVol. 89, pp. 8155-8159, September 1992Neurobiology

Identification, characterization, and localization of the dopamine D3receptor 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'EtudesNucleaires, 91191 Saclay, France

Communicated by Jean-Pierre Changeux, May 20, 1992

ABSTRACT We have identified 7-[3Hlhydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) as a selective probefor the recently cloned dopamine D3 receptor and used it toassess the presence of this receptor and establish its distributionand properties in brain. In transfected Chinese hamster ovary(CHO) cells, it binds to D3 receptors with subnanomolaraffinity, whereas its affinity is approximately 100-, 1000-, and10,000-fold lower at D2, D4, and D, receptors, respectively.Specific [3H]7-OH-DPAT binding sites, with a Kd of0.8 nM anda pharmacology similar to those at reference D3 receptors ofCHO cells, were identified in rat brain. D3 receptors differfrom D2 receptors in brain by their lower abundance (2 ordersof magnitude) and distribution, restricted to a few mainlyphylogenetically ancient areas-e.g., paleostriatum and ar-chicerebellum-as evidenced by membrane binding and auto-radiography studies. Native D3 receptors in brain are charac-terized by an unusually high nanomolar affinity for dopamineand a low modulatory influence of guanyl nucleotides onagonist binding. These various features suggest that D3 recep-tors are involved in a peculiar mode of neurotransmission in arestricted subpopulation of dopamine neurons.

Dopamine is an important neurotransmitter in brain, beinginvolved physiologically in the control of cognitive, motor,and endocrine processes and pathologically in Parkinson andpossibly mental diseases.

Until recently, it was largely believed that its variousactions were mediated by two receptor subtypes, termed D1and D2 (1, 2). Molecular biology approaches have led, how-ever, to the identification and cloning of the genes corre-sponding not only to these two receptor subtypes (3-7) butalso to additional and less expected ones, termed D3 (8), D4(9), and D5 (10, 11). For the latter receptors, the informationso far available derives from molecular biology approaches;their pharmacology and signaling system have only beenstudied in transfected cells, generally fibroblasts, and theirdistribution has been indirectly approached by studies ofmRNA localization. Nevertheless, these otherwise valuableapproaches suffer from some limitations. When expressed infibroblasts, receptors might find a membrane environment,which could modify their pharmacological specificity, and arepertoire of cellular components, particularly GTP-bindingproteins (G proteins), which may differ from that found inneurons. Regarding distribution, mRNA detection revealsthe sites of receptor synthesis rather than receptor localiza-tion, which may be different.

Therefore, there is an obvious interest in studying thenative receptor protein in brain, particularly in the case oftheD3 receptor, whose pharmacology in Chinese hamster ovary(CHO) cells and cerebral localization of its mRNA suggestthat it may represent an important target for antipsychotics(8, 12, 13).

Starting with the idea that designing a selective radioactiveprobe for the D3 receptor would help to settle these issues, wehave screened a series of dopamine agonists and therebyidentified 7-hydroxy-N, N-di-n-propyl-2-aminotetralin (7-OH-DPAT) (14-16) as fulfilling this purpose.

MATERIALS AND METHODSBrain Membranes. Male Wistar rats (IFFA Credo, Orldans,

France) weighing 180-200 g were used. They were exposedto an artificial light cycle of 12 h/day and had free access tofood and water. After decapitation, brain tissues were rapidlydissected out, frozen on dry ice, and kept at -70°C until use.Tissues were homogenized with a Polytron (setting 7 for 10sec) in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA andcentrifuged at 2500 x g for 30 sec; the supernatant wascentrifuged at 35,000 x g for 15 min. The pellet was resus-pended and centrifuged again, and this washing procedurewas repeated twice to eliminate endogenous dopamine. Thefinal pellet was resuspended by sonication in a buffer con-taining 50 mM NaHepes, 1 mM EDTA, 50 ,uM 8-hydroxy-quinoleine, 0.005% ascorbic acid, and 0.1% bovine serumalbumin (pH 7.5) (incubation buffer).Membranes from Cell Lines. CHO cell lines expressing rat

D2 or D3 dopamine receptors (CHO-D2 or CHO-D3) havebeen described (12). CHO-D1 were created by transfectingpCD-BS plasmid containing the human D1 receptor gene (6),a generous gift of P. Seeman. COS-7 cells transiently ex-pressing the human D4 receptor were obtained by transfect-ing pCD-PS plasmid, a generous gift of H. H. M. Van Tol,containing the human D4 receptor gene (9). Cells were grownin Dulbecco's modified Eagle's medium containing 10% fetalbovine serum. Cells were harvested by trypsin treatment(0.25%) for 4-5 min and centrifugation at 2000 x g for 5 min.They were homogenized with a Polytron in 10 mM Tris HCl(pH 7.5) containing 1 mM EDTA and were centrifuged at35,000 x g for 15 min. The pellet was then resuspended bysonication in the NaHepes buffer described above.

Abbreviations: 7-OH-DPAT, 7-hydroxy-N,N-di-n-propyl-2-amino-tetralin; p[NH]ppG, 5'-guanylyl imidodiphosphate; G protein, GTP-binding protein.§To whom reprint requests should be addressed.

8155

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

8156 Neurobiology: Ldvesque et al.

A20

60 - D3 D2 D04 D

010

o -11 -10 -9 -8 -7 -6 -5 -4 -3LOG [70H-OPAT] (M)

-I.II

cm

B [3H]70H-OPAT

0 100 200 300 400 500 0 200 400 600 8BOUND (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 theD4 receptor. Results are means from two experiments. (B) Scatchard analysis of [3H]7-OH-DPAT (Left) and [125Iliodosulpride (Right) specificbinding to D2 and D3 receptors in transfected CHO cells. This is a representative example of two such experiments. Note that [3H]7-OH-DPATbinding to D2 receptors was not measurable. (Inset) Chemical structure of 7-OH-DPAT; *, tritium atom.

Binding Assays. Membranes from brain (150-300 pg ofprotein) or cell lines (15-25 ,ug of protein) were added topolypropylene test tubes containing [3H]7-OH-DPAT for theD3 receptor assay, [1251]iodosulpride for D2 and D3 receptors,[3H]spiperone for the D4 receptor, or [3H]SCH23390 for theD1 receptor. Competing drugs were dissolved in incubationbuffer, the final volume being 1 ml, except for [125I]iodosul-pride binding, for which it was 0.4 ml. Tubes were incubatedin triplicate for 1 h at room temperature. The incubationswere stopped by rapid filtration under reduced pressurethrough Whatman GF/C glass filters coated with 0.1% bovineserum albumin (except for the [125I]iodosulpride bindingassay, for which the filters were Whatman GF/B-coated with0.3% polyethyleneimine), followed by three rinses with 3-4ml of ice-cold buffer (50 mM TrisHCl, pH 7.5/120 mMNaCl). Nonspecific binding was measured in the presence of1 ,uM dopamine (for [3H]7-OH-DPAT), 5 ,M (-)-sulpiride(for [1251]iodosulpride), 0.5 ,uM YM-09151-2 (for [3H]spiper-one), and 1 ,uM SCH23390 (for [3H]SCH23390). Radioactiv-ity was counted by y scintigraphy for the 1251 ligand or byscintillation spectrometry, at an efficiency of 41%, for 3Hligands. Saturation curves were analyzed by computer non-linear regression using a one-site cooperative model to obtainequilibrium dissociation constants (Kd) and maximal densityof receptors (Bn.). Competition curves were also analyzedby computerized nonlinear regression using either a one-siteor a two-site cooperative model to estimate IC50 and theproportion of sites (17). The best fit was determined bystatistical analysis between computer calculations. Inhibitionconstants (Kj) were estimated according to the equation Ki =IC50/(1 + S/Kd), where S represents the ligand concentra-tion. Curves were drawn with the INPLOT program (Graph-Pad, San Diego).

Autoradiography. Rats were killed by decapitation; brainswere removed and immediately dipped into liquid monochlo-rodifluoromethane for 5 min. Frozen brains were sectionedwith a Bright-Shandon cryostat and 10-,um sections werethaw-mounted onto precleaned and polylysine-coated glassslides. For [3H]7-OH-DPAT binding, slides were preincu-bated in 50 mM NaHepes (pH 7.5) containing 1 mM EDTAand 0.1% bovine serum albumin for 5 min three times at 22°Cand then incubated in the same buffer containing 0.5 nM[3H]7-OH-DPAT for 1 h. Some slides were incubated in thepresence of 1 ,uM dopamine to estimate nonspecific binding.The slides were then rinsed four times in ice-cold NaHepesbuffer containing 100 mM NaCl. After brief dipping intoice-cold distilled water, the slides were dried and apposed toHyperfilm-3H (Amersham) for 2 months.In Situ Hybridization. Brain sections were fixed in 4%

paraformaldehyde, prehybridized, and hybridized with a

32P-labeled complementary RNA probe specific for the D3receptor (13).

Drugs. 7-OH-DPAT and its precursor 7-hydroxy-N,N-diallylaminotetralin were custom synthesized at the Institut deChimie des Substances Naturelles (Gif-sur-Yvette, France).[3H]7-OH-DPAT was prepared by catalytic reduction, withpure 3H gas, of the double bond located on the N,N-diallylprecursor. The tritiated molecule was then purified by high-performance liquid chromatography through a preparative,uBondapak column in a 0-50%o methanol gradient in watercontaining 0.05% triethanolamine. The specific activity wasestimated to be 158 Ci/mmol (1 Ci = 37 GBq). [M2IJIodosul-pride (2000 Ci/mmol), [N-methyl-3H]SCH23390 (80 Ci/mmol), and [phenyl-4-3H]spiperone (60 Ci/mmol) were pur-chased from Amersham. Dopamine, apomorphine, and 5'-guanylyl imidodiphosphate (p[NHflppG) were purchased fromSigma. (-)-Sulpiride was generously donated by DelagrangeLaboratoires (Paris), haloperidol and domperidone were sup-plied by Janssen Laboratoires (Paris), and SCH23390 wasfrom Schering.

RESULTS

7-OH-DPAT Binding to Ceil Line Membranes. The selec-tivity of nonradioactive 7-OH-DPAT was first assessed incompetition experiments with transfected cell lines express-ing a single dopamine receptor subtype, labeled with anappropriate radioactive ligand. 7-OH-DPAT was much morepotent at the D3 than at the other receptor sites, its Ki valuesbeing 0.78 ± 0.02 nM, 61 ± 2 nM, 650 ± 80 nM, and 5300 ±500 nM at D3, D2, D4, and D1 receptors, respectively (Fig.

5 10 15BOUND (fmol.mg protein-')

20

FIG. 2. Scatchard analysis of[3H]7-OH-DPAT specific binding tomembranes of olfactory tubercle (Olf. Tub.), nucleus accumbens (N.Acc.), and striatum (Str.). [3H]7-OH-DPAT binding in the olfactorytubercle was not affected by 100 jM p[NH]ppG (see Table 2).

CD3

1000

Proc. Natl. Acad. Sci. -USA 89 (1992)

[ 125I]IODOSULPRIDE

Proc. Natl. Acad. Sci. USA 89 (1992) 8157

Table 1. Regional distribution of [3H]7-OH-DPAT binding sitesin rat brain

Binding at 0.7 nM, Bm, fmolfmol per mg of per mg

Region protein of protein Kd, nMOlfactory tubercle 6.6 ± 0.4 (100) 17 ± 1 0.7 ± 0.2Archicerebellum 6.2 ± 0.4 (94) 11 ± 2 0.5 ± 0.1Nucleus accumbens 3.6 ± 0.2 (55) 10 ± 1 1.0 ± 0.2Olfactory bulb 2.9 ± 0.5 (44) 13 ± 2 1.4 ± 0.4Hypothalamus 1.5 ± 0.4 (23)Striatum 1.4 ± 0.3 (21) 5.5 ± 0.4 1.0 ± 0.1Prefrontal cortex 1.0 ± 0.5 (15)Substantia nigra 0.8 ± 0.2 (12)Anterior pituitary 0.8 ± 0.2 (12)Hippocampus 0.3 ± 0.1 (5)

Specific binding was evaluated either at a single [3H]7-OH-DPATconcentration or by analysis of saturation isotherms like those shownin Fig. 2. Values are means ± SEM of three to six independentdeterminations. Values in parentheses represent percentage of ol-factory tubercle binding.

1A). Then, the best conditions to obtain a selective bindingof [3H]7-OH-DPAT to a D3 receptor with negligible bindingto D2 receptors were selected by using the corresponding celllines. In the absence of Mg2+ (and the presence of EDTA),binding to D3 receptor was not affected, whereas specificbinding to D2 receptors was nearly abolished; total binding at0.1-5 nM did not significantly differ from nonspecific bindingin CHO-D2 membranes to which the Bmax of [1251]iodosul-pride was 758 fmol per mg of protein (Fig. 1B). [3H]7-OH-DPAT binding to CHO-D3 membranes occurred monophasi-cally (nH = 0.996) over low nonspecific binding (28% at 0.8nM) with a Kd of 0.67 + 0.03 nM and a Bma. of 403 ± 7 fmolper mg of protein in the same range as the Bn. of [1251]io-dosulpride with CHO-D3 membranes-i.e., 485 + 5 fmol permg ofprotein (Fig. 1B). Upon addition of 100AM p[NH]ppG,in the absence of Mg2+, the Kd value of [3H]7-OH-DPATbecame 0.80 + 0.03 nM; in the presence of 5 mM MgCl2, theKd values were 0.50 + 0.02 and 0.77 + 0.08 nM in the absenceand presence of p[NH]ppG, respectively (data not shown).[3H]7-OH-DPAT and [2'sI]Iodosuipride Binding to Rat

Brain Membranes. A specific saturable [3H]7-OH-DPATbinding was detected in membranes from some brain areas.Saturation experiments with membranes from olfactory tu-bercle, nucleus accumbens, and striatum (Fig. 2) and a fewother areas (data not shown) indicated that the bindingoccurred with a rather similar Kd (Table 1). The specificbinding of [3H]7-OH-DPAT was also evaluated at a singleconcentration close to the Kd (0.7 nM) in additional brain

CEREBELLUM

Table 2. Pharmacology of [3H]7-OH-DPAT binding to ratolfactory tubercle compared to D2 and D3 receptorpharmacology in transfected CHO cells

Ki values, nM

OlfactoryAgent tubercle CHO-D3 CHO-D2

AgonistsDopamine 8 + 1 25 474Dopamine + p[NH]ppG 9 ± 2 27 1705Quinpirole 5 ± 1 5.1 576Apomorphine 14 ± 3 20 247-OH-DPAT 0.7 ± 0.2* 0.78 617-OH-DPAT + p[NH]ppG 0.9 ± 0.4*8-OH-DPAT 328 ± 44

AntagonistsHaloperidol 5 ± 1 10 0.4UH232 9 ± 1 9 40(-)-Sulpiride 12 ± 3 25 9Domperidone 20 ± 3 10 0.3Clozapine 224 ± 99 180 56SCH23390 1581 ± 415 780 720

Values for the olfactory tubercle were obtained by using 0.8 nM[3H]7-OH-DPAT in three or four independent experiments. Valuesfor transfected CHO cells were obtained by using [125I]iodosulprideand are from ref. 8 (or Fig. 1 for 7-OH-DPAT).*Kd value.

areas (Table 1); in olfactory tubercle, it represented 50-60%of total binding, but in striatum and hippocampus it repre-sented only 10-20% and 5-10%o oftotal binding, respectively.The pharmacological properties of [3H]7-OH-DPAT bindingsites were defined by evaluating the Ki values of a series ofdopamine agonists and antagonists with membranes of ol-factory tubercle (Table 2); these values were highly corre-lated (r = 0.996; n = 11; P < 0.001) with those derived froma previous study (8) with CHO-D3, but not with CHO-D2 (r= 0.218; n = 11). As in CHO-D3, the Ki of dopamine and theKd of [3H]7-OH-DPAT were not significantly modified byp[NH]ppG (Table 2). Addition of the guanyl nucleotide in thepresence of 2 mM MgCl2 affected only slightly the dopamineinhibition curve of [125I]iodosulpride binding to membranesof the archicerebellum; the biphasic curve with componentsof K, values of 0.33 ± 0.15 (30% of sites) and 20.2 + 0.1 nMwas transformed into a monophasic curve (Ki = 10.5 ± 1.5nM) (Fig. 3). [125I]Iodosulpride binding in the archicerebel-lum was inhibited by 7-OH-DPAT with a K, of 3.7 ± 0.9 nM;addition ofp[NH]ppG did not modify this Ki value (3.5 + 0.8nM), while clozapine had a Ki of2% ± 29 nM. In comparison,

PITUITARY

0 -12 -11 -10 -9 -8 -7 -6 -5 0 -11 -10 -9 -8 -7 -6 -5LOG [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 mMTris*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 mMNaCl (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.

Neurobiology: Uvesque et al.

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8158 Neurobiology: Ldvesque et al. Po.Nt.Aa.Si S 9(92

the two components of dopamine inhibition of ["25Iliodosul-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± 4.2 (30% of sites) and 1328 ±244 nM in the presence ofp[NH]ppG (Fig. 3).

Autoradiographic Loaiain of [3H17-OH-DPAT BindingSites and D3 Receptor mRNA in Rat Brain. Autoradiogramsindicated a restricted distribution of [3H]7-OH-D)PAT bindingsites, with very high levels on the islands of Calleja and in themolecular layer of lobules 9 and 10 of the cerebellum.Distinct, although lower, labeling was observed in nucleusaccumbens and olfactory bulb, with very low labeling in thestriatum. Binding was completely inhibited in the presence of1 u.M dopamine, leaving a negligible and uniform nonspecificlabeling (data not shown). This distribution of [3H]7-OH-DPAT sites was similar to that of D3 receptor mRNA (Fig. 4).

A B

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Lob.1O

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FIG. 4. Comparison of autoradiographic localization of [3H]7-OH-DPAT specific binding to rat brain sections (A and C) with the

distribution of D3 receptor mRNA measured by in situ hybridization

(B and D). Frontal (A and B) or sagittal (C and D) sections at

approximately similar levels were either incubated with [3H]7-OH-DPAT (0.5 nM) or hybridized with a 32p-labeled complementaryRNA probe. In sections incubated with [3H]7-OH-DPAT and dopa-

mine (1 AM), labeling in nucleus accumbens (Acb), islands of Calleja

(ICj), and cerebellum was completely abolished. Lob. 10, lobule 10;

OR, olfactory bulb; Cpu, caudate putamen; IQJM, islands of Calleja,

major island.

DISCUSSION

The present identification of 7-OH-DPAT as a selective D3

receptor ligand and its consecutive use in tritiated form has

allowed us to establish the occurrence, pharmacological

properties, and regional distribution of the D3 receptor pro-

tein in rat brain.

The compound was selected through screening of various

putative dopamine "autoreceptor-selective" agents, since a

fraction of )3 receptor mRNAs is present within dopaminer-

gic perikarya, suggesting that this receptor, as well as the 1)2

receptor, may play an autoreceptor function (8, 13). Indeed,

7-OH-DPAT had been shown to potently inhibit dopamine

synthesis in vivo (14) and in vitro (15) in slices of rat striatum.

In transfected cell lines expressing a single dopanune

receptor subtype, 7-OH-DPAT displayed subnanomolar af-

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 ensuringa 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-affinityform 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

binding sites correspond selectively to 1)3receptors is shown

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

binding sites was similar in rat olfactory tubercle and in

CHO-)3 cells. Finally, the restricted localization of bindingsites in few dopaminoceptive areas expressing 1)3 receptor

mRNA also supports the selectivity of labeling.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

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

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-

erally correspond to well established, although relatively

minor, dopamine projections, except in archicerebellum

where histochemical (18, 19) but not biochemical approaches

(20) suggest their absence. Nevertheless, these sites in the

cerebellum were previously evidenced by using [1251I]iodosul-pride (21), which binds with subnanomolar affinity to )2 and

1)3receptors and with 10 times lower affinity to 1)4receptors

(data not shown). These features, together with the pharma-

cology of these sites (see Results) as well as with the absence

and low level of 1)2 and 1)4 receptor mRNA, respectively(unpublished observattion), indic-ate- that cerebellar (1f'I5io-dosulpride binding sites correspond to 1)3 receptors. In

substantia nigra. and neostriatum, 1)3 receptor abundance is

extremely low but may largely reflect the expression of 1)3

c

D

Proc. Nad. Acad. Sci. USA 89 (1992)

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Proc. Natl. Acad. Sci. USA 89 (1992) 8159

autoreceptors on perikarya and terminal areas of a subpop-ulation of dopamine neurons.Two major properties of the native D3 receptor protein in

brain could be established through the use of [3H]7-OH-DPAT: its unusually high affinity for the neurotransmitterdopamine and the low modulatory effect of guanyl nucleo-tides on its binding. Both properties, which suggest a peculiarmode of dopaminergic transmission, were previously de-tected in transfected CHO cells but they could have beenrelated to differences in membrane composition or absence ofa cellular component, particularly a G protein, in the trans-formed fibroblast as compared to cerebral neurons (8, 12).The nanomolar affinity ofthe neurotransmitter, quite unusualfor an amine, suggests that dopamine might act, through D3receptors, at some distance from the terminals that release itand/or occupy the D3 receptor for longer periods oftime thanin the case ofother receptor subtypes. Such a quasi-hormonalmode of action might be consistent with the high expressionof the D3 receptor within the core of the islands of Calleja,whereas dopamine axons surround these islands, makingonly few contacts with granule cells (22). In addition, sincethe background dopamine concentration in the extracellularspace of the striatum is 5-20 nM (23), D3 receptors should besubjected to a tonic stimulation.The weak modulatory effect of a guanyl nucleotide on

dopamine binding at native D3 receptors was first evidencedby the absence of significant change in dopamine competitionof [3H]7-OH-DPAT binding to membranes from the olfactorytubercle (Table 2). This observation was made, however, inconditions in which coupling of the G protein was possiblynot favored-i.e., in the absence ofMg2+ (24). Nevertheless,it was confirmed by using membranes of the archicerebellumand [125I]iodosulpride as the ligand. The marginal effect ofp[NH]ppG in this prototypic D3 receptor area contrasts withits well-established effect in pituitary, where the vast major-ity of [125I]iodosulpride binding sites correspond to D2 re-ceptors (Fig. 3). Hence, these data support the view that thetwo native receptor subtypes are coupled to distinct Gproteins or do not interact similarly with a single G protein.This is consistent with observations in transfected CHO cellsshowing that, in contrast with D2, D3 receptors mediate onlymarginal inhibitions of adenylyl cyclase or activation ofarachidonate release (8, 12, 25). In fact, the signaling path-way(s) of D3 receptor remains to be defined. The presentidentification of cerebral areas selectively expressing thisreceptor and 7-OH-DPAT as a potentially selective dopamineD3 receptor agonist should help to solve this problem.

In conclusion, the restricted expression of the D3 receptorin phylogenetically old parts of the brain together with itspeculiar mode of interaction with the neurotransmitter sug-gest that this receptor constitutes a marker for a subset ofneurons whose exact functions, presumably in the control ofemotional, cognitive, and endocrine processes, remain to beestablished.

D.L. is the holder of a postdoctoral fellowship from the NaturalSciences and Engineering Research Council of Canada.

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