Proc. Nati. Acad. Sci. USAVol. 86, pp. 6412-6416, August 1989Neurobiology

Dopamine D2 receptors in the cerebral cortex: Distribution andpharmacological characterization with [3Hlraclopride

(raclopride binding/neostriatum/rat/monkey)

MICHAEL S. LIDOW*t, PATRICIA S. GOLDMAN-RAKIC*, PASKO RAKIC*, AND ROBERT B. INNIS*fSection of *Neuroanatomy and tDepartment of Psychiatry, Yale University, School of Medicine, New Haven, CT 06510

Contributed by Pasko Rakic, May 17, 1989

ABSTRACT An apparent involvement of dopamine in theregulation of cognitive functions and the recognition of awidespread dopaminergic innervation of the cortex have fo-cused attention on the identity of cortical dopamine receptors.However, only the presence and distribution of dopamine DIreceptors in the cortex have been well documented. Compa-rable information on cortical D2 sites is lacking. We report herethe results of binding studies in the cortex and neostriatum ofrat and monkey using the D2 selective antagonist [3H]raclo-pride. In both structures [3H]raclopride bound in a sodium-dependent and saturable manner to a single population of siteswith pharmacological profiles of dopamine D2 receptors. D2sites were present in all regions of the cortex, although theirdensity was much lower than in the neostriatum. The densityof these sites in both monkey and, to a lesser extent, rat cortexdisplayed a rostral-caudal gradient with highest concentra-tions in the prefrontal and lowest concentrations in the occipitalcortex, corresponding to dopamine levels in these areas. Thus,the present study establishes the presence and widespreaddistribution of dopamine D2 receptors in the cortex.

The dopaminergic innervation ofthe cerebral cortex has beenwell characterized (1-4), and dopamine receptor-mediatedeffects have also been demonstrated in the cortex (5, 6).However, much less is known about the receptors thatmediate these effects. It has been only recently that theavailability of a Dl-specific antagonist, SCH 23390, has madeit possible to describe the pharmacology and distribution ofdopamine D1 sites in both rodents (7, 8) and primate cortex(9, 10). On the other hand, dopamine D2 sites in the cortexhave not been fully documented, and, indeed, their veryexistence in this structure is controversial. Although D2receptors in the cortex have been reported in several studies(7-9, 11-15), an equal number of studies have obtainednegative results (10, 16-21). Most studies on cortical D2 siteshave used either [3H]spiperone, which binds multiple recep-tor sites (16, 22, 23) or [3H]sulpiride, which has very highnonspecific binding in the cortex (11). The few studies thatused [125I]iodosulpiride, a highly selective D2 antagonist, didnot perform saturation analyses of the binding, thus leavingdensity of the receptors unspecified (24-26).

In light ofthe hypothesis that the D2 receptor is a major siteof therapeutic action for neuroleptics (27), it is of interest toobtain quantitative data on D2 binding sites in various regionsof the cortex as well as to extend this study to the primate.Within the past year the availability of a new substitutedbenzamide, [3H]raclopride, another highly selective D2 an-tagonist (28), has provided the opportunity to reexamine D2binding sites in the cerebral cortex and to compare these sitesin primates and rodents. We also have compared the corticalD2 sites with those in the neostriatum.

MATERIALS AND METHODSTissue was obtained from three adult rhesus monkeys(Macaca mulatta): one male and two females. The animalswere anesthesized with sodium pentobarbital (40 mg/kg) andperfused with ice-cold phosphate-buffered saline followed by0.1% paraformaldehyde (9; 15). The cerebral cortex andneostriatum were rapidly removed and immersed in isopen-tane at -700C for 5 min before storing at -800C until use. Forcompetition studies tissue was pooled from several neocor-tical areas. However, saturation studies were conducted ondifferent cortical areas separately. Rodent brains were ob-tained from male Sprague-Dawley rats (200-400 g; CharlesRiver Breeding Laboratories). Animals were decapitated andthe cerebral cortex and neostriatum were removed for im-mediate assay. All competition studies using rat cortex camefrom the prefrontal cortex. Different cortical areas wereassayed separately for the saturation analysis.The tissue was homogenized in ice-cold 50 mM Tris'HCl

buffer (pH 7.4) with a Brinkman Polytron PT10. Homoge-nates were centrifuged twice at 20,000 x g for 10 min with anintermediate rehomogenization in fresh buffer. The finalpellets were resuspended in incubation buffer containing 50mM Tris-HCI (pH 7.4 at 230C), 120 mM NaCl, 5 mM KCI, 2mM CaCI2, 1 mM MgCl2, and 0.1% ascorbic acid. The effectof Na' was tested in 50 mM Tris HCl buffer (pH 7.4 at 230C)containing 0.1% ascorbic acid to which different concentra-tions of NaCl were added; effects of K+, Ca2+, and Mg2+were tested in 50 mM Tris'HCl buffer (pH 7.4 at 230C)containing 120 mM NaCl and 0.1% ascorbic acid to whichdifferent concentrations ofKCI, CaCl2, or MgCl2 were added.Two hundred microliters of tissue homogenate was incubatedwith 25 ml of [3H]raclopride and 25 ml of displacer or buffer.The final dilution of cortical tissue was 1:25 and of neostriataltissue was 1:125. Standard incubation was for 30 min at 230C.The effect of incubation temperature was studied at 00, 230,and 370C with incubation time ranging from 15 to 120 min.The incubation was terminated by rapid filtration and threewashings with 5 ml ofice-cold 50mM Tris HCl buffer (pH 7.4)through Whatman GF/B filters by using a Brandel M-242 cellharvester. The radioactivity trapped in the filters was mea-sured in a Packard 3320 liquid scintillation counter. Satura-tion studies used 0.1-6 nM [3H]raclopride, and displacementstudies used 1.0 nM [3H]raclopride. Specific binding wasdefined as the difference in binding with and without 1.0 AmM(+)-butaclomol. Labeling of tissue with 1.0 nM [3H]sulpiridewas conducted as described for [3H]raclopride. Binding with1.0 nM [3H]spiperone was performed in the presence of 0.3gM ketanserin. The Pierce BCA protein assay was used todetermine protein concentration in the tissue. Data wereanalyzed by a nonlinear curve-fitting computer program

Abbreviation: 5-HT, 5-hydroxytryptamine.tTo whom reprint requests should be addressed at: Yale University,School of Medicine, Section of Neuroanatomy, C303 Sterling Hallof Medicine, 333 Cedar Street, New Haven, CT 06510.

6412

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.

Proc. Natl. Acad. Sci. USA 86 (1989) 6413

Z. 200

150 10 3

0 100-

TIME (min)

FIG. 1. Effect of temperature on the time course of specific[3H]raclopride binding in monkey cortex. Error bars represent ±SEM of three experiments.

(EDBA/LIGAND, Elsevier-Biosoft, Cambridge, U.K.) and re-ported as mean ± SEM.

Radioligands were obtained from New England Nuclear.Drugs were either purchased from Research Biochemicals(Natick, MA) or were gifts from Astra Lakemedel AB,Sodertalje, Sweden (raclopride), Smith Kline & French(chlorpromazine), Sandoz (clozapine), Lilly (LY 171555), orMiles (ipsapirone).

RESULTSEffect of Temperature and Buffer Composition on [3H]-

Raclopride Binding. Maximal specific binding was reachedwithin 30-min incubation at 230C and was greater in magni-tude and more stable over 2 hr than incubation at 00C or 370C(Fig. 1). NaCl in the Tris HCl incubation buffer tended toincrease specific binding, with a maximal effect at 120 mMNaCI (Fig. 2). KCI at 0-15 mM had no influence on totalspecific binding, measured in the presence of 120 mM NaCl(data not shown). The divalent cations Mg2' and Ca2+showed a small depressant effect on specific binding, with-25% decrease at 8 mM CaCl2 or MgCl2. Nevertheless, 2 mMCaCl2 and 1 mM MgCl2 were retained in the incubation bufferfor the displacement of [3H]raclopnde binding by D2 agonists,a process that requires divalent cations (29).

Saturation Analysis of [311]Raclopride Binding. Saturationstudies showed that [3H]raclopride binding to rat and monkey

o200

100-

Eff100 o Na7 oo ec ofCd a+ and Cdg C C

0I.2. Efc f aCz +adM2 on the~spcfic[H]

in0 s arsEi0 a0 3&,CI0ME C

E

Effect of Na+ Effect of Ca++ and Mg++

FIG. 2. Effect of Na', Ca2' and Mg2+ on the specific [3HI-raclopride binding in monkey cortex. The effect of Na+ was testedin 50 mM Tris-HCl buffer (pH 7.4 at 230C) -containing 0.1% ascorbicacid to which different concentrations of NaCl were added. Theeffects of Ca2+ and Mg2+ were tested in 50 mM Tris HCl buffer (pH7.4 at 23°C) containing 120 mM NaCI and 0.1% ascorbic acid to whichdifferent concentrations of CaCl2 or MgC12 were added. The columnlabeled 120 mM NaCl in the first data set is the same as the baselinefor the second data set and is labeled No CaCI2 and MgCl2. Error barsrepresent + SEM determined from three replications.

neostriatum had a very low proportion of nonspecific bindingover a wide range of radioligand concentrations (Fig. 3a). Thespecific binding in this structure was saturable with Bma, = 220± 19 fmol/mg of protein (rat) and 180 ± 9 fmol/mg of protein(monkey). Furthermore, Scatchard plots of [3H]raclopridebinding were consistent with a single binding site with Kd = 2.5± 0.2 nM and 1.3 ± 0.2 nM in rat and monkey, respectively.As in the neostriatum, the specific binding of [3H]raclopride inrat and monkey cerebral cortex was saturable and of highaffinity (Fig. 3b), and Scatchard plots similarly indicated thepresence of a single binding site. However, the number of[3Hlraclopride-binding sites in the cortex was considerablylower than that in the neostriatum: 50-75 times lower in rat and75-300 times lower in monkey (Fig. 3). Also, saturation studiesin the cortex, in contrast to the neostriatum, showed that[3H]raclopride had a substantial proportion of nonspecificbinding at most concentrations (Fig. 3b). In fact, specificbinding exceeded nonspecific binding only within a limitedconcentration range of the ligand (-0.1-2.5 nM).

Competition Experiments. [3H]Raclopride-binding sites inneostriatum and cortex showed virtually identical pharma-cological profiles in both species (Table 1). In all tissues only

z ] svBOUND 0 w 0 D $ BOUND(fmol/mg prot) 0 ; (fmol/mg prot)

C 150- 6-

Ro n nonspecific

uL E 100 _ | °40~oo~~~~~~~50~~~~~~~~~~~~~~~~

0 1 2 3 4 5 6 0 1 2 3 4 5 6[3H]RACLOPRIDE FREE (nM) [3H]RACLOPRIDE FREE (nM)

FIG. 3. Saturation binding of [3Hlraclopride in monkey neostriatum (a) and cortex (temporal lobe) (b). [3H]Raclopride binding was measuredat equilibrium conditions (30 min at 23°C), with specific binding defined as excess over blanks containing 1 AM (+)-butaclamol. (Insets)Corresponding Scatchard plots of the saturation binding data. Each value represents the average of three experiments. These Scatchard plotsyielded Kd = 1.3 ± 0.2 nM and Bmax = 180 ± 9 fmol/mg of protein (neostriatum) and Kd = 1.0 + 0.2 and Bmax = 2.3 - 0.1 fmol/mg of protein(cortex).

Neurobiology: Lidow et al.

a

Proc. Natl. Acad. Sci. USA 86 (1989)

Table 1. Drug displacement of [3H]raclopride binding sites in neostriatum and cortex of monkeyand rat

Ki, nMMonkey Rat

Compounds Striatum Cortex Striatum CortexD2 antagonists

Spiperone 0.010 0.007 0.002 0.002Haloperidol 1.0 1.4 1.4 1.7(+)-Butaclomol 2.3 1.9 3.2 1.1Raclopride 1.3 1.1 2.5 2.3Fluphenazine 3.4 7.2 1.2 1.0Chlorpromazine 4.8 4.0 1.9 5.1Pimozide 6.1 10.1 1.1 1.0(-)-Sulpiride 38 13 31 27Clozapine 190 400 130 400(+)-Sulpiride 440 610 700 540(-)-Butaclomol >10000 >10000 >10000 >10000

D2 agonists(-)-N-1-propyl- 0.4 (h) 0.6 (h) 0.6 (h) 0.5 (h)

norapomorphine 30.2 (1) 26.4 (1) 23.3 (1) 22.5 (1)LY 171555 3.6 (h) 4.7 (h) 3.7 (h) 4.1 (h)

3700 (1) 2700 (1) 3500 (1) 4200 (1)Dopamine 3.5 (h) 4.7 (h) 3.8 (h) 4.5 (h)

4300 (1) 3800 (1) 5000 (1) 3500 (1)(+)-N-1-propyl-norapomorphine 620 570 780 650

Other receptorsSCH 23390 (D1) 350 400 430 530SKF 83566 (D1) 480 420 1000 1200Serotonin (S1) 5900 7600 >10000 >10000Ipsapirone (SlA) 390 480 820 940Ketanserin (S2) 230 240 250 310Mepyramine

(histamine) 6800 6500 >10000 >10000(-)-Propranolol ((3) 7100 6400 >10000 >10000Prazosin (al) >10000 >10000 >10000 >10000Clonidine (a2) >10000 >10000 >10000 >10000Atropine (M) >10000 >10000 >10000 >10000Bicuculine(GABAA) >10000 >10000 >10000 >10000

The data represent the mean Ki values of three experiments. The pseudo-Hill coefficients of alldisplacing agents were close to 1.0 except for D2 agonists, which had values of 0.6-0.8. For theseligands K; values are given for both the high (h)- and low (1)-affinity sites. Also in parentheses are theconcerned receptor types: D, dopamine; S, serotonin; f3, j3-adrenergic; a, a-adrenergic; M, muscarinic;GABA, y-aminobutyric acid.

D2-selective ligands showed high affinity for [3Hlraclopridebinding. Cortical and striatal binding sites demonstratedsimilar stereoselectivity, with active isomers [(-)-sulpiride,(+)-butaclomol, and (-)-N-1-propylnorapomorphine] show-ing much higher affinity for these sites than their inactiveenantiomers [(+)-sulpiride, (-)-butaclomol, and (+)-N-1-propylnorapomorphine]. Drugs selective for other receptorsshowed relatively low affinity for the [3H]raclopride bindingsites. All ligands used in this study displaced [3H]raclopridebinding with pseudo-Hill coefficients close to 1.0, with theexception of D2 agonists, which demonstrated pseudo-Hillcoefficients ranging from 0.6 to 0.8. Such low pseudo-Hillcoefficients are characteristic for inhibition of D2 antagonistbinding by agonists (30).

Areal Distribution of [3H]Raclopride Binding in the Cortex.The distribution of [3H]raclopride binding sites in monkeycortex showed a general rostral-caudal gradient with nearlyfive times higher density of sites in prefrontal and temporalregions than in the occipital region (Fig. 4). Motor, soma-tosensory, and posterior parietal cortical regions containedintermediate concentrations. The rat cortex showed moreeven distribution of [3H]raclopride binding, although a shal-

low rostral-caudal gradient was present, with -50% moresites in prefrontal compared with occipital cortex (Fig. 4Inset). [3H]Raclopride binding had similar Kd values in allcortical regions of both species (Fig. 4).

Displacement of [3H]Sulpfride and [3H]Spiperone by Raclo-pride. To determine whether differences exist between pop-ulations of D2 receptors labeled by [3H]raclopride and otherneuroleptics, we studied the ability of raclopride to competefor D2 binding sites with [3H]sulpiride (a substituted benza-mide) and [3H]spiperone (a spirodecanone neuroleptic). Thebinding with [3H]spiperone duplicated common practice,using 0.3 AM ketanserin in the incubation buffer to preventbinding to 5-hydroxytryptamine type 2 (5-HT2) sites (7-10,16). The raclopride displacement of [3H]sulpiride binding inneostriatum and cortex of both species showed simple com-petition curves with pseudo-Hill coefficients close to 1.0 (Fig.5). Raclopride displacement of [3H]spiperone binding in theneostriatum also resulted in a simple competition curve (Fig.5). In contrast, raclopride displacement of [3H]spiperonebinding in the cortex was shallow, with a pseudo-Hill coef-ficient of -0.6. Computer analysis of the displacement curveis consistent with two binding sites: high affinity (Ki = 1.0

6414 Neurobiology: Lidow et al.

Proc. Natl. Acad. Sci. USA 86 (1989) 6415

_t6CRR

MONKEY04

C

Kd(nM)1.30.31.00.21.10.2O.90 2... .

0 2

FI. 4 C. E

~~ ~ ~ ~ ~ ~ ~ ~ At N piNE

~~~~~~~~~~~~4V

0

frm moke (mi6itga)adrt(Ist.B n dvle

E ~ ~ 0W

E~0

Kd (nM) 1.3±0.3 2 1.1±0.2 0.9±0.2 1.0±0.2 1.0±0.2

FIG. 4. Regional [3H]raclopride binding in the neocortical areas

from monkey (main histogram) and rat (Inset). Bma, and Kd values

are expressed as mean ± SEM determined in three experiments.

nM) accounting for -37% of binding and low affinity (Ki =

6 /zM) representing the remaining 63% of the sites. Todetermine whether these low-affinity sites represent a2-adrenergic and/or 5-HT1A serotonergic receptors, binding of[3H]spiperone to cortical membranes (in the presence of 0.3tkM ketanserin) was displaced with idazoxan (a2-adrenergicantagonist) and ipsapirone (5-HT1A agonist). The resultsshow that displacement of [3H]spiperone binding by bothligands contain high- and low-affinity components (Table 2).The high-affinity sites for idazoxan represented 39% andthose for ipsapirone represented 38% of the specific [3H]-spiperone binding in the cortex.

DISCUSSIONSpecificity of [3lHlRaclopride Binding. Our results support

earlier findings (21, 27) that [3H]raclopride is a potent andselective D2 ligand, which can be displaced from its bindingsites only by D2-selective drugs. We found that nonspecific

100

1 striatum

FoST _ X F

11 10 9 8 7 6 5 11 10 9 8 7 6 5-log [RACLOPRIDE] (M)

FIG. S. Raclopride displacement of [3H]sulpiride and [3H]-spiperone binding (in the presence of 0.3 ,uM ketanserin) to monkeyneostriatum and cortex. Data represent the average of three exper-iments. Raclopride displaced [3H]sulpiride with pseudo-Hill coeffi-cients 0.98 (neostriatum) and 0.99 (cortex). Ki in the neostriatumequals 1.3 nM and K. in the cortex equals 1.2 nM. In the- neosetriatumraclopride displaced [3H]spiperone with pseudo-Hill coefficient 0.85;Ki equals 3.0 nM. In the cortex, raclopride displaced [3Hlspiperonewith pseudo-Hill coefficient 0.6. Ki values can be seen in Table 2.

Table 2. Pseudo-Hill coefficients, Ki values, and percentages forhigh- and low-affinity sites obtained by computer analysis of thedisplacement of [3H]spiperone binding (in the presence of 0.3A&M ketanserin) to monkey cortex with D2 dopaminergic-,a-adrenergic-, and 5-HT1A serotonergic-specific ligands

High- Low-affinityLigands Pseudo-Hill affinity sites sites

(receptor sites) coefficient K;, nM % K1, nM %Raclopride (D2) 0.6 1 37 6000 63Idazohan (a2) 0.7 10 39 5000 61Ipsapirone (5-HT1A) 0.6 2 38 3000 62Data represent mean values determined in three experiments.

binding of [3H]raclopride in the neostriatum is very low.However, in the cortex the range of [3H]raclopride concen-trations at which the specific binding was higher than non-specific binding was extremely limited. Thus, the use of lowconcentrations (<3 nM) of this radioligand is essential forsuccessful labeling of D2 sites in the cortex. This finding mayexplain the failure of previous attempts to use relatively highconcentrations of [3H]raclopride for the autoradiographicstudy of cortical D2 receptors (21). Our results indicate that,like other substituted benzamides, [3H]raclopride shows so-dium-dependent binding (11, 31, 32), whereas the divalentcations Ca2' and Mg2' have inhibitory effect (32). Contraryto the recent suggestion that substituted benzamides shouldhave very low nonspecific binding when incubation is done at0°C (12), we like others (33) found that incubation at 23°C for30 min resulted in the maximal stable [3H]raclopride bindingto both striatal and cortical tissue.

Cortical Dopamine D2 Receptors. The major finding of thisstudy is that both rodent and primate cortices contain D2dopamine receptor binding sites, which appear virtuallyidentical to those in the neostriatum, although the formersites are present in considerably lower concentration. Thelow density of D2 binding sites is consistent with the lowconcentration of dopamine in the cortex as compared withthat in the neostriatum. Thus, dopamine concentration in therat cortex is 130- to 220-fold lower than in rat neostriatum (2,34) and 130- to 1700-fold lower in monkey cortex than inmonkey neostriatum (1). The D2 receptors in monkey cortexshow a strong rostral-caudal density gradient that closelyparallels the concentration gradient of dopamine itself (1). Asimilar, but less dramatic, gradient was seen in the ratcortex-a finding consistent with the shallow gradient ofdopamine in the rodent cortex (2).Apparent Single Population of Dopamine D2 Receptors.

Recent electrophysiological studies in the rat have suggestedthat cortical and striatal D2 receptors have different pharma-cological properties (35, 36). However, homogenate bindingstudies with [3H]sulpiride (16) and [1251]iodosulpiride (25) andthe present results with [3H]raclopride have not detectedsuch differences. The reasons for these disparities betweenelectrophysiological and binding studies are unclear butcould be due to a unique subpopulation of cortical D2 sitesthat binding studies with substituted benzamides failed todetect. Indeed, it has been suggested that the substitutedbenzamides bind only to a subpopulation ofD2 receptors andthat other radioligands label the entire population of D2 sites(29, 37, 38). In our study, we found that, while both [3H]-spiperone (in the presence of ketanserin) and raclopridecompete for the same population of receptor sites in theneostriatum, raclopride shows a high affinity for only asubpopulation of receptors labeled by [3H]spiperone in thecortex. However, [3H]spiperone is known to bind multiplereceptor sites (16, 22, 23), and while our assay conditionsprevented binding to 5-HT2 sites (16), the low-affinity sitesdetected with raclopride may represent a2-adrenergic and/or5-HTIA receptors (22, 23). Indeed, in competition studies

Neurobiology: Lidow et al.

Proc. Natl. Acad. Sci. USA 86 (1989)

with idazoxan and ipsapirone, we found that more thantwo-thirds ofthe [3H]spiperone binding was to these receptorsites. Thus, in the neostriatum, the density of D2 receptors ishigh enough to make nondopaminergic binding of [3H]-spiperone comparatively insignificant. In contrast, the lowdensity of D2 receptors in the cortex together with a sub-stantial concentration of other high-affinity sites for [3H]-spiperone significantly increases the proportion of nondopam-inergic binding and makes [3H]spiperone a poor label forcortical D2 receptors.

Functional Significance. A widespread distribution of do-pamine D2 receptors in the cerebral cortex is of considerableclinical significance because this may be the site for antipsy-chotic effects of neuroleptics (27). Thus, our findings shouldbring attention to the cortex as a possible site of dysfunctionin diseases like schizophrenia. Recently, dopamine afferentshave been seen to form synapses with dendritic spines ofcortical pyramidal cells, placing dopaminergic receptors in astrategic position to directly affect the activity of efferents toother cortical and subcortical areas (P.S.G.-R., C. Leranth,M. S. Williams, N. Mons, and M. Geffard, unpublishedresults). Thus, even a small number ofD2 receptors may havea great influence on cortical functions. Also, the low densityof D2 receptors in the cortex makes any alteration in theirnumber of biochemical properties especially profound.

We are thankful to Drs. D. W. Gallager and I. Creese for theirvaluable comments. This work was supported by grants from theU.S. Public Health Service.

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M"16 Neurobiology: Lidow et al.