Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6412-6416, August 1989 Neurobiology Dopamine D2 receptors in the cerebral cortex: Distribution and pharmacological characterization with [3Hlraclopride (raclopride binding/neostriatum/rat/monkey) MICHAEL S. LIDOW*t, PATRICIA S. GOLDMAN-RAKIC*, PASKO RAKIC*, AND ROBERT B. INNIS*f Section 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 the MATERIALS AND METHODS regulation of cognitive functions and the recognition of a widespread dopaminergic innervation of the cortex have fo- Tissue was obtained from three adult rhesus monkeys cused attention on the identity of cortical dopamine receptors. (Macaca mulatta): one male and two females. The animals However, only the presence and distribution of dopamine DI were anesthesized with sodium pentobarbital (40 mg/kg) and receptors in the cortex have been well documented. Compa- perfused with ice-cold phosphate-buffered saline followed by rable information on cortical D2 sites is lacking. We report here 0.1% paraformaldehyde (9; 15). The cerebral cortex and the results of binding studies in the cortex and neostriatum of neostriatum were rapidly removed and immersed in isopen- rat and monkey using the D2 selective antagonist [3H]raclo- tane at -700C for 5 min before storing at -800C until use. For pride. In both structures [3H]raclopride bound in a sodium- competition studies tissue was pooled from several neocor- dependent and saturable manner to a single population of sites tical areas. However, saturation studies were conducted on with pharmacological profiles of dopamine D2 receptors. D2 different cortical areas separately. Rodent brains were ob- sites were present in all regions of the cortex, although their tained from male Sprague-Dawley rats (200-400 g; Charles density was much lower than in the neostriatum. The density River Breeding Laboratories). Animals were decapitated and of these sites in both monkey and, to a lesser extent, rat cortex the cerebral cortex and neostriatum were removed for im- displayed a rostral-caudal gradient with highest concentra- mediate assay. All competition studies using rat cortex came tions in the prefrontal and lowest concentrations in the occipital from the prefrontal cortex. Different cortical areas were cortex, corresponding to dopamine levels in these areas. Thus, assayed separately for the saturation analysis. the present study establishes the presence and widespread The tissue was homogenized in ice-cold 50 mM Tris'HCl distribution of dopamine D2 receptors in the cortex. buffer (pH 7.4) with a Brinkman Polytron PT10. Homoge- nates were centrifuged twice at 20,000 x g for 10 min with an intermediate rehomogenization in fresh buffer. The final The dopaminergic innervation ofthe cerebral cortex has been pellets were resuspended in incubation buffer containing 50 well characterized (1-4), and dopamine receptor-mediated mM Tris-HCI (pH 7.4 at 230C), 120 mM NaCl, 5 mM KCI, 2 effects have also been demonstrated in the cortex (5, 6). mM CaCI2, 1 mM MgCl2, and 0.1% ascorbic acid. The effect However, much less is known about the receptors that of Na' was tested in 50 mM Tris HCl buffer (pH 7.4 at 230C) mediate these effects. It has been only recently that the containing 0.1% ascorbic acid to which different concentra- availability of a Dl-specific antagonist, SCH 23390, has made tions of NaCl were added; effects of K+, Ca2+, and Mg2+ it possible to describe the pharmacology and distribution of were tested in 50 mM Tris'HCl buffer (pH 7.4 at 230C) dopamine D1 sites in both rodents (7, 8) and primate cortex containing 120 mM NaCl and 0.1% ascorbic acid to which (9, 10). On the other hand, dopamine D2 sites in the cortex different concentrations ofKCI, CaCl2, or MgCl2 were added. have not been fully documented, and, indeed, their very Two hundred microliters oftissue homogenate was incubated existence in this structure is controversial. Although D2 with 25 ml of [3H]raclopride and 25 ml of displacer or buffer. receptors in the cortex have been reported in several studies The final dilution of cortical tissue was 1:25 and ofneostriatal (7-9, 11-15), an equal number of studies have obtained tissue was 1:125. Standard incubation was for 30 min at 230C. negative results (10, 16-21). Most studies on cortical D2 sites The effect of incubation temperature was studied at 00, 230, have used either [3H]spiperone, which binds multiple recep- and 370C with incubation time ranging from 15 to 120 min. tor sites (16, 22, 23) or [3H]sulpiride, which has very high The incubation was terminated by rapid filtration and three nonspecific binding in the cortex (11). The few studies that washings with 5 ml ofice-cold 50mM Tris HCl buffer (pH 7.4) used [125I]iodosulpiride, a highly selective D2 antagonist, did through Whatman GF/B filters by using a Brandel M-242 cell not perform saturation analyses of the binding, thus leaving harvester. The radioactivity trapped in the filters was mea- density of the receptors unspecified (24-26). sured in a Packard 3320 liquid scintillation counter. Satura- In light ofthe hypothesis that the D2 receptor is a major site tion studies used 0.1-6 nM [3H]raclopride, and displacement of therapeutic action for neuroleptics (27), it is of interest to studies used 1.0 nM [3H]raclopride. Specific binding was obtain quantitative data on D2 binding sites in various regions defined as the difference in binding with and without 1.0 AmM of the cortex as well as to extend this study to the primate. (+)-butaclomol. Labeling of tissue with 1.0 nM [3H]sulpiride Within the past year the availability of a new substituted was conducted as described for [3H]raclopride. Binding with benzamide, [3H]raclopride, another highly selective D2 an- 1.0 nM [3H]spiperone was performed in the presence of 0.3 tagonist (28), has provided the opportunity to reexamine D2 gM ketanserin. The Pierce BCA protein assay was used to binding sites in the cerebral cortex and to compare these sites determine protein concentration in the tissue. Data were in primates and rodents. We also have compared the cortical analyzed by a nonlinear curve-fitting computer program D2 sites with those in the neostriatum. Abbreviation: 5-HT, 5-hydroxytryptamine. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Yale University, payment. This article must therefore be hereby marked "advertisement" School of Medicine, Section of Neuroanatomy, C303 Sterling Hall in accordance with 18 U.S.C. §1734 solely to indicate this fact. of Medicine, 333 Cedar Street, New Haven, CT 06510. 6412 Downloaded by guest on September 26, 2021 Neurobiology: Lidow et al. Proc. Natl. Acad. Sci. USA 86 (1989) 6413 Z. 200 o200 150 10 3 - 100 0 100- in0 s arsEi0 a Eff100 o7 Naoo ec Cdof a+ and Cdg C C 0 3 E 0I.2. Efc f &,CIaCz +adM20MEon the~spcfic[H]C Effect of Na+ Effect of Ca++ and Mg++ TIME (min) FIG. 2. Effect of Na', Ca2' and Mg2+ on the specific [3HI- raclopride binding in monkey cortex. The effect of Na+ was tested FIG. 1. Effect of temperature on the time course of specific in 50 mM Tris-HCl buffer (pH 7.4 at 230C) -containing 0.1% ascorbic [3H]raclopride binding in monkey cortex. Error bars represent ± acid to which different concentrations of NaCl were added. The SEM of three experiments. effects of Ca2+ and Mg2+ were tested in 50 mM Tris HCl buffer (pH 7.4 at 23°C) containing 120 mM NaCI and 0.1% ascorbic acid to which different concentrations of CaCl2 or MgC12 were added. The column (EDBA/LIGAND, Elsevier-Biosoft, Cambridge, U.K.) and re- labeled 120 mM NaCl in the first data set is the same as the baseline ported as mean ± SEM. for the second data set and is labeled No CaCI2 and MgCl2. Error bars Radioligands were obtained from New England Nuclear. represent + SEM determined from three replications. Drugs were either purchased from Research Biochemicals (Natick, MA) or were gifts from Astra Lakemedel AB, neostriatum had a very low proportion of nonspecific binding Sodertalje, Sweden (raclopride), Smith Kline & French over a wide range of radioligand concentrations (Fig. 3a). The (chlorpromazine), Sandoz (clozapine), Lilly (LY 171555), or specific binding in this structure was saturable with Bma, = 220 Miles (ipsapirone). ± 19 fmol/mg of protein (rat) and 180 ± 9 fmol/mg of protein (monkey). Furthermore, Scatchard plots of [3H]raclopride RESULTS binding were consistent with a single binding site with Kd = 2.5 Effect of Temperature and Buffer Composition on [3H]- ± 0.2 nM and 1.3 ± 0.2 nM in rat and monkey, respectively. Raclopride Binding. Maximal specific binding was reached As in the neostriatum, the specific binding of [3H]raclopride in within 30-min incubation at 230C and was greater in magni- rat and monkey cerebral cortex was saturable and of high tude and more stable over 2 hr than incubation at 00C or 370C affinity (Fig. 3b), and Scatchard plots similarly indicated the (Fig. 1). NaCl in the Tris HCl incubation buffer tended to presence of a single binding site. However, the number of increase specific binding, with a maximal effect at 120 mM [3Hlraclopride-binding sites in the cortex was considerably NaCI (Fig. 2). KCI at 0-15 mM had no influence on total lower than that in the neostriatum: 50-75 times lower in rat and specific binding, measured in the presence of 120 mM NaCl 75-300 times lower in monkey (Fig.