Localization of Dopamine D, Receptor Mrna and D, and D, Receptor Binding in the Rat Brain and Pituitary: an in Situ Hybridization-Receptor Autoradiographic Analysis

The Journal of Neuroscience, August 1990, 70(8): 2587-2800

Localization of Dopamine D, Receptor mRNA and D, and D, Receptor Binding in the Rat Brain and Pituitary: An in situ Hybridization-Receptor Autoradiographic Analysis

Alfred Mansour,’ James H. Meador-Woodruff,i James R. Bunzow,2 Olivier Civelli,* Huda Akil,’ and Stanley J. Watson’ ‘Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109-0720, and *Vellum Institute for Advanced Biomedical Research, The Oregon Health Sciences University, Portland, Oregon 97201

Several lines of evidence suggest the existence of multiple Cumulative evidence suggeststhe existence of at least 2 distinct dopamine receptor subtypes, referred to as D, and D,. The dopaminergic receptor subtypes, referred to as D, and D,. D, present study examines the distribution of these dopamine dopamine (DA) receptors are positively coupled to adenylate binding sites in the rat brain and pituitary in relation to the cyclase (Stoof and Kebabian, 1984), widely distributed in the distribution of D, receptor mRNA using a combination of in CNS (Boyson et al., 1986; Dawson et al., 1986; Dubois et al., vitro receptor autoradiographic and in situ hybridization 1986; Bouthenet et al., 1987; Wamsley et al., 1989), and abun- techniques. 3H-Raclopride and 3H-SCH23390 (in the pres- dant in the parathyroid gland (Attie et al., 1980). D, DA recep- ence of 1 ELM ketanserin) were used to label D, and D, re- tors, on the other hand, are either not coupled to adenylate ceptor binding sites, respectively, while a 495 bp cRNA probe cyclase (Memo et al., 1986) or negatively coupled (Onali et al., synthesized from the Sac I-Bglllfragment of a rat D, receptor 1985) and are densely distributed in the basalganglia (Martres cDNA was used to visualize the D, receptor mRNA. Analysis et al., 1985; Charuchinda et al., 1987; Joyce and Marshall, 1987; of adjacent tissue sections in which receptor autoradiog- Richfield et al., 1987) and pituitary gland (Kohler and Fahlberg, raphy and in situ hybridization had been performed revealed 1985; Pazos et al., 1985; DeSouza, 1986). several brain regions where the D, binding site and corre- Selective lesion studies(Creese et al., 1977; Nagy et al., 1978; sponding mRNA appear to be similarly distributed, including Schwartz et al., 1978; Crossand Waddington, 1981; Joyce and the caudate-putamen, nucleus accumbens, olfactory tuber- Marshall, 1987; Trugman and Wooten, 1987; Filloux et al., cle, globus pallidus, substantia nigra, and ventral tegmental 1988; Porceddu et al., 1986) suggestthat while D, and D, re- area. In the pituitary gland, D, binding sites and mRNA ap- ceptorsare both localized on pre- and postsynaptic membranes, pear to be codistributed with very dense levels in the inter- the D, subtype may function as an “autoreceptor,” modulating mediate lobe and individually labeled cells in the anterior the synthesisand/or releaseof DA. Support for this hypothesis lobe. Brain regions demonstrating a lack of correspondence has come from findings demonstrating that D, receptorscan be between the distribution of the D, binding site and D, receptor localized on DA-containing cells (Reisineet al., 1979) and that mRNA include the olfactory bulb, neocortex, paleocortex, application of selective D, agonistsproduces a decreasein cell hippocampus, and zona incerta. Several hypotheses are dis- firing (White and Wang, 1983) DA release,and synthesis(Stoof cussed to explain the lack of correspondence in certain brain et al., 1982; Brown et al., 1985). Similar changesin DA release regions; these include the localization of receptor binding and turnover have not been observed with selective D, receptor sites on both fibers and cell bodies and receptor transport. agonists(Stoof et al., 1982; Brown et al., 1985; Clark and Gal- These studies provide a better understanding of the ana- loway, 1985). tomical distribution of the D, receptor and serve as a frame- The rat D, receptor has been recently cloned (Bunzow et al., work for future regulatory and anatomical mapping studies. 1988) and is structurally similar to membersof the family of G By focusing on specific brain regions, such as the nigro- protein-coupled receptors that include the o(-and @-adrenergic striatal system, hippocampus, and olfactory bulb, they pro- receptors, the muscarinic receptors and rhodopsin. Northern vide insights into D, receptor synthesis, transport, and in- blot analysissuggests that the mRNA coding for the D, receptor sertion into cell membranes. showsa similar distribution to the D, sitesreported with ligand binding, with high levels observed in the striatum and pituitary. Further, transfection of the D, receptor DNA into cells normally not demonstrating DA receptors results in the expression of Received Nov. 6, 1989; revised Mar. 14, 1990; accepted Mar. 15, 1990. specific D, receptor binding. We are grateful to Elizabeth Cox for her expert secretarial assistance and for Given these findings, we have recently examined the distri- grants from the National Institute for Mental Health (MH4225 I, MH15794, bution of the D, receptor mRNA using in situ hybridization MH456 14), National Alliance for the Mentally Ill, Lucille P. Markey Charitable Trust, Theophile Raphael, National Alliance for Research on Schizophrenia and (Meador-Woodruff et al., 1989). D, receptor mRNA was vi- Depression, and Eli Lilly (APA/Lilly Psychiatric Fellowship). sualized in DA projections fields, such as the caudate-putamen, Correspondence should be addressed to Alfred Mansour, Ph.D., Mental Health Research Institute, University of Michigan, 205 Washtenaw Place, Ann Arbor, nucleus accumbens,and olfactory tubercle, as well as in dopa- MI 48 109-0720. mine-containing cell groups such as the substantia nigra (SN), Copyright 0 1990 Society for Neuroscience 0270-6474/90/082587-14$03.00/O ventral tegmental area (VTA), and zona incerta. Such a distri- 2588 Mansour et al. - Localization of Dopamine D, Receptor Binding bution would suggest that the D, receptor has both a pre- and labeled with jH-SCH23390 were given four 4-min washes. All slides postsynaptic localization and the receptors visualized in the SN were then quickly dipped in 250 ml distilled water (4°C) and dried with a portable hair dryer set to “cool.” Nonspecific binding was evaluated and VTA may serve as autoreceptors. Consistent with this hy- by treating a parallel set of slides with the same concentrations of tri- pothesis, unilateral 6-hydroxydopamine lesions of the DA neu- tiated ligand with a 1 PM final concentration of an unlabeled competitor: rons in the medial forebrain bundle produce a complete ipsi- spiperone to displace 3H-raclopride and SCH23390 to displace 3H- lateral loss of D, receptor mRNA in the SN and VTA and a SCH23390. compensatory increase in the D, receptor mRNA of the dener- Saturation studies. Prior to preparing tissue for receptor autoradiography, saturation experiments were performed on slide-mounted brain vated striatum (Mansour et al., 1990). sections to determine the binding kinetics of 3H-raclopride and )H- The purpose of the present set of studies is to compare the SCH23390. Forebrain sections were incubated with a minimum of 8 distribution of D, receptor mRNA in the brain and pituitary to concentrations of either 3H-raclopride (15.0-O. 12 nM) or ‘H-SCH23390 D, and D, binding sites using a combination of in situ hybrid- (7.6-0.06 nM) and washed and dried as described earlier. The binding was quantified by placing brain sections in scintillation vials containing ization and in vitro receptor autoradiographic techniques. These 10 ml of scintillant and vigorously shaking for 30 min in a metabolic anatomical studies provide a more precise analysis of the dis- shaker. Each data point is an average of 2 brain sections. Saturation tribution of D, receptor mRNA and dopaminergic ligand bind- experiments were performed at least twice and graphed as Scatchard ing sites than is possible with Northern analysis and homogenate plots. Kd and B,,, values were determined with the LIGAND program binding. Moreover, while the transfection studies (Bunzow et developed by Munson and Rodbard (1980). Competition studies. To characterize the binding sites labeled by 3H- al., 1988) discussed previously are essential for identifying a D, raclopride and ‘H-SCH23390, competition studies were performed with receptor, studies combining in situ hybridization and receptor slide-mounted brain sections at concentrations 3 times the Kd value for autoradiography are necessary in supporting, validating, and each ligand. These concentrations correspond to the ones used in sub- extending these findings to the CNS. sequent autoradiographic mapping studies and represent a 75% receptor occupancy for each ligand. Competition studies were performed with a Several quantitative studies are available describing the dis- series of dopaminergic compounds [haloperidol, chlorpromazine, spi- tribution of D, and D, binding sites in the brain (e.g., Bouthenet perone, (+) and (-) butaclamol, droperidol, raclopride, and SCH233901, et al., 1987; Charuchinda et al., 1987; Wamsley et al., 1989). It as well as nondopaminergic drugs (propranolol, clonidine, mianserin, is not the focus of the present study to replicate these findings, and bremazocine). The brain sections were incubated, washed, and but to examine in serial sections the distribution of the mRNA dried, and the binding was quantified as described earlier. Autoradiographic mapping. After being brought to room temperature, encoding for the D, receptor in relation to the DA binding sites slide-mounted sections were placed in incubation chambers and incu- to gain insights into regions of possible receptor synthesis, trans- bated with either ‘H-raclopride (5.7 nM) or jHSCH23390 (4.4 nM). port, and eventual insertion into neuronal membranes. The li- These concentrations correspond approximately to 3 times the Kd value gands chosen for these comparisons were 3H-raclopride and 3H- for each ligand, producing an equivalent receptor occupancy. Following a 90 min incubation, the slides were washed and dried, as described SCH23390,2 DA antagonists highly selective for the D, (Kohler above, and apposed to tritium-sensitive Hyperfilm (Amersham) for 24 et al., 1985) and D, (Iorio et al., 1983) sites, respectively. weeks (3H-SCH23390) or 4-8 weeks (SH-raclopride). The Hyperfilm Given the complexity of such a study, this report focuses on was exposed at room temperature, developed in Kodak D-19 (4 min, regions of the CNS whose anatomical circuitry is better under- 19°C) agitated in 2% acetic acid (30 set), fixed in Kodak Rapidfix (5 stood, such as the olfactory bulb, hippocampus, and the basal min), and washed under running water (30 min). Anatomical structures were determined using Nissl-stained sections in conjunction with the ganglia, with minimal discussion of other regions of the rat atlas of Paxinos and Watson (1986). forebrain and midbrain. The pituitary gland is also examined In situ hybridization. Slides adjacent to those used for autoradio- in detail, as this tissue contains a high density of D, receptors, graphic mapping of D, and D, receptor binding sites were directly re- and differences in receptor binding and mRNA distribution are moved from storage at -80°C and placed into 4% formaldehyde for 60 min (22°C) prior to being processed for in situ hybridization (Sherman unlikely to be due to the localization of D, binding sites on et al., 1986; Watson et al., 1987). Following three 5-min rinses in PBS, fibers. In addition to these anatomical considerations, several pH 7.4, sections were treated with proteinase K (1 Kg/ml in 100 mM in situ hybridization and receptor binding controls were per- Tris, pH 8.0, 50 mM EDTA) for 10 min at 37°C. Slides were then rinsed formed to ensureselectivity of hybridization and ligand binding. in water, followed by 0.1 M triethanolamine, pH 8.0, and treated with a mixture of 0.1 M triethanolamine. uH 8.0. and acetic anhvdride (400: 1, vol/vol) with stirring for 10 min: The sections were then rinsed in Materials and Methods 2 x SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.2) for 5 min, Tissue preparation and incubation medium for receptor binding studies. dehydrated through graded alcohols, and allowed to air dry. Adult male Sprague-Dawley rats (Charles River, 200-250 gm) were Brain sections were hybridized with 35S-UTP-labeled riboprobes gen- sacrificed by decapitation, and their brains and pituitaries were quickly erated to the 495 Sac I-Bgl ZZ fragment of a rat D, receptor (Bunzow removed. Brains were frozen in liquid isopentane (-30°C) for 30 set, et al., 1988). cRNA probes were diluted in hybridization buffer (75% while the pituitaries were frozen on crushed dry ice in Lipshaw M-l formamide, 10% dextran sulfate, 3 x SSC, 50 mM Na,PO,, pH 7.4, 1 x - embedding matrix. Frozen tissues were sectioned on a Bright cryostat Denhardt’s, 0.1 mg/ml yeast tRNA, 0.1 m&ml sonicated, denatured (20 pm), thaw-mounted on precleaned and subbed microscope slides, salmon sperm DNA, 10 mM dithiothrietol) to result in a final concen- and stored at -80°C. Immediately prior to using the tissue, the slide- tration of 2 x 10” dpm/30 ~1. Volumes of 30 and 50 ~1 of diluted probe mounted sections are gradually brought to room temperature and were applied to coronal and horizontal sections, respectively. incubated (90 min, 22°C) with 200400 ~1 of either the D,-selective After hybridization (overnight, 55”(Z), the slides were rinsed in 2 x antagonist ‘H-raclopride (83.4 Ci/mmol, New England Nuclear) or the SSC (5 min) and treated with RNase A (200 pg/rnl in 100 mM Tris, pH D,-selective antagonist ‘H-SCH23390 (7 1.3 Ci/mmol, New England 8.0, and 0.5 M NaCl) for 30 min at 37°C. Subsequently, sections were Nuclear) in 50 mM Tris buffer, pH 7.5 at 25°C containing 0.1% ascorbic rinsed in 2 x SSC for 10 min (22”C), 1 x SSC for 10 min (22”Q 0.5 acid, 120 mM NaCl, 5 mM KCl, and 1 mM MgCl,. As SCH23390 has x SSC at 55°C for 60 min, 0.5 x SSC at room temperature for 10 min, been reported to bind serotinergic sites (5HT,), 1 PM ketanserin, a and finally dehydrated in graded alcohols and air-dried. Sections were selective 5-HT, antagonist was added to all )H-SCH23390 binding stud- then either exposed to Kodak XAR-5 X-Ray film for l-3 d and de- ies. veloped, or dipped in Kodak NTB-2 emulsion and stored at 4°C for 6- Following a 90 min incubation period, the slides were drained, washed 17 d prior to development. in 4 consecutive 250 ml, 50 mM Tris, pH 7.6 at 4°C washes containing In situ hybridization controls. To ensure the specificity of the in situ 0.1% ascorbic acid, 120 mM NaCl, 5 mM KCl, and 1 mM MgCl,. Slides hybridization signal, several control studies were performed. (1) The incubated with 3H-raclopride were given four 2-min washes, while those 495 bp cRNA probe used in the present study corresponds to the pu- The Journal of Neuroscience, August 1990, lo(e) 2589

n [3H] RACLOPRIDE (o-o) Table 1. Ki concentrations (nM) of various compounds competing for the D, and D, receptor sites labeled with “H-SCH23390 (4.6 nM) and Bmax = 20.01 fmolimg 3H-raclopride (6.6 nM), respectively Kd = 1.7 nM

[3H] SCH23390 (H) 3H-Raclopride ‘H-SCH2339@ 11 Bmax = 86.0 fmolimg (+)Butaclamol 1.19 227.41 04 (-)Butaclamol >2506.0 >2463.0 0 9 I- Droperidol 3.41 >2463.0 ii 7 Raclopride 5.34 >2463.0 Spiperone 5.35 >2463.0 5 Chlorpromazine 5.74 1037.72 Haloperidol 16.10 >2463.0 3 SCH23390 1157.58 2.58 Mianserin 1991.0 >2463.0 Clonidine >2506.0 >2463.0 I I I I I I I I I I Bremazocine >2506.0 >2463.0 10 20 30 40 50 60 70 80 90 100 Propranolol >2506.0 >2463.0 fmol/mg u ‘H-SCH23390 binding was done in the presence of 1 PM ketanserin. Figure 1. Scatchard plots of jH-raclopride and ‘H-SCH23390 binding in the rat forebrain. Note that each ligand appears to bind a single population of receptor sites and there are approximately 4 times as many D, sites (86 fmol/mg) as D, receptors (20 fmol/mg). 3H-Raclopride binding was readily displaced by D, antagonists, including droperidol, spiperone, raclopride, chlorpromazine, and haloperidol. The binding was stereoselective with (+)butaclamol potently competing for the 3H-raclopride site, and (-)bu- tative third cytosolic loop and the sixth and seventh transmembrane domains ofthe D, receptor. Given the homology ofthe sixth and seventh taclamol failing to displace it even at micromolar concentra- transmembrane domains to other G-protein coupled receptors, such a tions. SCH23390, a D, antagonist, and mianserin, a 5-HT, probe may potentially hybridize these receptors in addition to the D, antagonist, were unable to effectively compete at the site labeled receptor. To examine this issue, we performed in situ hybridization with by 3H-raclopride. Similarly, clonidine (a,-adrenergic agonist), a subcloned 205 bp fragment (EcoRI-Xho II) of the 495 bp clone that codes exclusively for the third cytosolic loop. A series of adjacent brain bremazocine (opioid) and propranolol ((Y- and fi-adrenergic an- sections were hybridized either with the 205 bp or the 495 bp cRNA tagonist) failed to displace the binding at this site. Taken to- probes employing the same in situ conditions described above. (2) For gether, these results suggest that 3H-raclopride binds selectively RNase control, a series of paired, adjacent sections was divided into 2 to D, dopaminergic receptor sites. sets: One slide from each pair was treated as described earlier for in situ hybridization, and the remaining slides from each pair were fixed in 3H-SCH23390 in the presence of 1 PM ketanserin appears to 4% formaldehyde and rinsed in PBS, but prior to treatment with pro- selectively label D, receptor sites. Of the compounds tested, teinase K, were incubated with RNase A (200 &ml) for 30 min at only SCH23390 and (+)butaclamol displaced 3H-SCH23390 37°C. These slides were then processed as described in the in situ hy- (Table 1). Compounds such as (-)butaclamol, haloperidol, dro- bridization protocol. (3) For “sense’‘-strand control, another series of peridol, raclopride, spiperone, mianserin, clonidine, bremazo- paired, adjacent sections was divided into 2 sets: One set was treated according to the in situ hybridization protocol, and the second set was tine, and propranolol failed to compete for the sites labeled by treated identically, except that the cRNA used in the hybridization ‘H-SCH23390. Chlorpromazine did displace 3H-SCH23390, but mixture was %S-UTP-labeled “sense’‘-strand RNA. required micromolar concentrations. The relative difference in potency between (+)butaclamol and its negative enantiomer Results suggests that the 3H-SCH23390 binding is stereoselective. Saturation studies 3H-raclopride and 3H-SCH23390 demonstrate saturable bind- Anatomical distribution ing, and the results best fit a single-site model using the LIGAND Consistent with the saturation results, autoradiographic studies program. As can be seen from Figure 1, 3H-raclopride and 3H- suggest that D, binding sites are more densely distributed than SCH23390 bound to a single population of sites with apparent D, sites in most of the rat CNS. In addition, D, receptors are affinity constants of 1.7 and 1.5 nM, respectively. The mean Kd more widely distributed and are observed at all levels of the values across multiple saturation studies are 2.2 nM for 3H- neuraxis, while D, receptor sites are densely distributed pri- raclopride and 1.55 nM for 3H-SCH23390. While the affinities marily in the basal ganglia, olfactory bulb, and pituitary. By of these ligands for their respective binding sites appear similar, comparison, the distribution of D, receptor mRNA more closely the relative abundance of each receptor varied markedly. In the corresponds to the D, binding sites labeled with 3H-raclopride forebrain slices used in the present study, there were 4 times as than the D, sites demonstrated with 3H-SCH23390. many D, sites (86.0 fmol/mg) as D, sites (20.0 fmol/mg) per The following is a qualitative comparison of the distribution milligram of tissue. of the D, receptor mRNA in relation to the dopaminergic ligand binding sites. The level of mRNA or receptor binding in cau- Competition studies date-putamen is used for comparison with other brain regions To characterize the pharmacological properties of each ligand, and pituitary. The descriptions are also within a ligand or mRNA a series of competition studies was conducted. As can be seen distribution, so that an area described as dense for D, binding from Table 1, 3H-raclopride and 3H-SCH23390 appear to se- sites, for example, may be equivalent to an area of moderate lectively label D, and D, dopamine receptor sites, respectively. D, receptor binding on the basis of receptor number. The em- 2590 Mansour et al. * Loqalization of Dopamine D, Receptor Binding

sites are restricted to the olfactory nerve layer (ON) and glomerular cell layer (GL) where they are denselydistributed (Fig. 2). By comparison, D, receptor mRNA can be visualized in the olfactory nucleus, glomerular, and internal granular cell layers. Within the glomerular layer, D, mRNA is restricted to the periglomerular cells whoseprocesses extend into the glomerula. In contrast, D, sitesare not prominent in the olfactory bulb, with a light amount of binding observed in the glomerular, plexiform, and internal granular layers. Higher levels of D, receptors are seenin the olfactory nucleus, where no D, binding sites are observed. Within the cortical fields, D, receptor binding is densestin the entorhinal cortex. Layer I of entorhinal cortex showsfairly dense D, binding, with no observable binding in layer II and moderate amounts in layer III. Only a very light density of 3H- raclopride binding is seenin the cingulate, frontal, parietal, and temporal cortices, being restricted primarily to deeperlayers. A light density of D, receptors can also be visualized in the piriform cortex. By comparison, denselevels of D, receptor mRNA are found in the superficial aspectsof layer I and in layers II- III of the frontal, parietal, and temporal cortex. Deeper layers (V, VI) of cingulate, frontal, parietal, and temporal cortex show moderate to low levels of D, receptor mRNA. Within paleocortical regions, entorhinal cortex (layers II-III) and the most superficialcells of layer I demonstratedense levels of D, receptor mRNA. Similarly, piriform cortex showsa high density of D, receptor mRNA. In contrast to D, binding, D, receptor sitesare more abundant and widely distributed throughout neo- and paleocortex. Moderate densitiesof D, receptor sitesare seenin deeplayers of cingulate, frontal, parietal, and temporal cortices, with light diffuse labeling observed in more superficial layers. Within the paleocortex, moderate amounts of D, binding are seenin the piriform cortex, with D, binding restricted to layer I of entorhinal cortex. Of the telencephalic regionsexamined, the caudate-putamen contains the highest density of D, receptor mRNA and D, and D, receptor binding sites. The distribution of D, binding sites appearsto be heterogeneouswith this structure, with the highest densitiesin the dorsomedial tips and the dorso- and ventrolat- era1aspects of this nucleus(Fig. 3). D, receptor binding extends into the ventral striatum and is particularly densein the rostra1 part of the nucleus accumbens,as well as in the olfactory tubercle. Somewhat reduced levels of 3H-raclopride binding are seenin the caudal two-thirds of the nucleusaccumbens, where the binding appearsto be lessdense than in the caudate-putamen Figure 2. Dark-fieldautoradiograms comparing D, and D, receptor (Fig. 3). bindingto the distributionof D, mRNA in horizontal sectionsof ol- D, receptor sitesare also densely distributed in the caudate- factory bulb. D, receptor binding is observed in the olfactory nerve layer putamen, with a similar medial-lateral receptor gradient. The (OhJ) and the glomerular layer (GL). D, receptor mRNA, by comparison, can be visualized in the periglomerular cells of GL, olfactory nucleus dopaminergic receptors differ in the nucleusaccumbens, how- (OFN) and the internalgranular layers. D, sitesare predominant only ever, where D, sites are densethroughout this nucleus, with in the olfactory nucleus. particularly high levels observed in the shell of the accumbens (Fig. 3). D, receptor binding extends ventrally and is dense phasis of this study is not to quantify receptor mRNA and throughout the olfactory tubercle. binding levels, but to compare their anatomical distributions. In contrast to the heterogeneousD, ligand distribution, D, Detailed quantitative distribution studiesof D, and D, receptors receptor mRNA is uniformly distributed in the caudate-puta- have been published elsewhere(Boyson et al., 1986; Dawson et men (Fig. 3). D, receptor mRNA levels are also densein the al., 1986;Bouthenet et al., 1987; Charuchindaet al., 1987;Joyce nucleusaccumbens, where the levels appear equivalent to those and Marshall, 1987). seenin the caudate-putamen, making the 2 structures appear as a single unit. More ventrally, the olfactory tubercle contains Telencephalon one of the highest concentrations of D, receptor mRNA. With its preciselaminations, the olfactory bulb is a regionwhere Within the septum,the lateral and medial nuclei demonstrate D, and D, receptor sitescan be easily differentiated. D, binding a light density of D, binding. D, receptor mRNA showsa similar The Journal of Neuroscience, August 1990, fO(8) 2591

Figure 3. Dark-field autoradiograms of D, receptor binding and D, receptor mRNA and ligand binding. Note the high levels and good correspondence of D, receptor mRNA and dopamine receptor binding in the caudate-putamen (CPU), nucleus accumbens (ACB), and olfactorv tubercle (OTU). There av- pears to be a lack ‘of correspondence between the distributions of D, binding sites and D, receptor mRNA in the superficial layers of cortex (CTX). distribution, with a relatively low level of D, receptor mRNA mRNA (Fig. 4). The level of D, ligand binding in this region is found in these nuclei. D, binding sitescan also be localized in fairly light, appearing densestin the lateral portion of the nu- the septum, but these sites are predominantly in the lateral cleus. By comparison, cells containing D, receptor mRNA ap- nucleus. pear widely distributed throughout the globus pallidus and ap- The globus pallidus, a major elSerenttarget of the caudate- pear densely labeled under these hybridization conditions. As putamen, showsD, and D, ligand binding, as well asD, receptor can be appreciated from Figure 4, given the high amounts of 2592 Mansour et al. l Localization of Dopamine D, Receptor Binding

Figure 4. Dark-field autoradiograms of D, and D, receptor binding as compared to D, receptor mRNA localization. D, receptor mRNA and D, and D, binding sites can be visualized in the caudate-putamen (CPU), globus pallidus (GP), and olfactory tubercle (OTU). Note that single-cell resolution can be attained in the globus pallidus in the visualization of D, receptor mRNA. The Journal of Neuroscience, August 1990, IO@) 2593

D, receptor mRNA and the wide distribution of these positive cells, individually labeled pallidal cells can be resolved even at this low magnification. Cells showing D, receptor mRNA appear more numerous in the dorsal portion of the globus pallidus and become more widespread ventrally. D, receptor mRNA can also be visualized in the ventral pallidum, but the apparent density of labeled cells is not as great as observed in the globus pallidus. Moderate densities of D, binding sites can also be observed in the globus pallidus, with dense levels seen in the ventral pallidum. The amygdala, a region where D, and D, receptor sites can be differentiated, demonstrates little or no D, ligand binding in most of the amygdaloid nuclei, with only a light labeling in the medial nucleus. In contrast, D, receptor sites are densely distributed throughout most of the amygdala, including the cortical, lateral, and basalateral nuclei, with moderate densities seen in the medial nucleus. By comparison, light levels of D, receptor mRNA can be visualized in the lateral and basolateral nuclei, with somewhat higher levels observed in the medial nucleus. The hippocampal formation with its laminated structure is an excellent tissue for comparing receptor binding and mRNA distributions. Within the hippocampus, D, receptor binding is restricted to the stratum lacunosum moleculare and subiculum, where a light to moderate density of binding is observed (Fig. 5). D, receptor mRNA, on the other hand, is seen in the pyramidal cell layer (CA 1, CA2, CA3) and in the granular cells of the dentate gyrus. For comparison, moderate densities of D, receptors are observed in the dentate gyrus, with low densities in the stratum moleculare and oriens.

Diencephalon Compared to the telencephalon, far fewer regions of the diencephalon demonstrate D, receptor binding or mRNA. Within the thalamus, the medial and lateral habenula and the zona incerta have particularly high amounts of D, receptor mRNA (Fig. 6). By comparison, D, and D, binding can be seen in the lateral habenula, but does not appear to be present in the medial habenula and zona incerta. In the hypothalamus, D, receptor mRNA is restricted to the anterior and lateral hypothalamic areas, lateral mammillary nucleus, and the paraventricular and ventromedial nuclei, where moderate to low levels are observed. D, receptor binding appears diffuse and light throughout most of the hypothalamus, including the lateral, dorsomedial, ventromedial, and arcuate nuclei. The lateral mammillary nucleus appears to be the ex- ception, with densely localized D, binding sites and mRNA (Fig. 7). D, binding sites are lightly distributed throughout most of the hypothalamus, including the lateral, ventromedial, and arcuate nuclei, with only the suprachiasmatic nucleus demonstrating a high density of D, sites.

Mesencephalon Of the mesencephalic structures, the substantia nigra (SN) and ventral tegmental area (VTA) have the highest densities of dopaminergic receptor binding and D, receptor mRNA. D, ligand Figure 5. Dark-field autoradiograms of D, and D, receptor binding as binding is predominantly in the pars compacta, with light la- compared to D, receptor mRNA in the hippocampus. D, receptor mRNA beling in the pars reticulata (Fig. 7). Consistent with this local- can be visualized in the pyramidal cell layer (CAl, CA2, CA3) of the ization, D, receptor mRNA is restricted to the cells of the pars hippocampal formation and in the granular cells of the dentate gyrus (DG). D, receptor binding, on the other hand, is restricted to the stratum compacta, with large cells in the pars reticulata occasionally lacunosum moleculare (S’LM), while D, binding is observed in the den- labeled. D, receptor binding and mRNA appear to show a sim- tate gyrus and in stratum moleculare and oriens. Other abbreviations: ilar distribution in the VTA, where a moderate density of D, cc, corpus callosum; LHb, lateral habenula. 2594 Mansour et al. l Localization of Dopamine D, Receptor Binding

Figure 6. Dark-field autoradiogram of a D, receptor mRNA at the level of the diencephalon. Note the high levels of D, receptor mRNA in the zona incerta (ZI), caudate-putamen (CPU), and cortex (CTX).

binding and high levels of D, receptor mRNA can be observed. In contrast, D, receptor binding sites are localized predomi- Discussion nantly in the pars reticulata of the substantia nigra, with mod- In agreement with previous receptor autoradiographic studies erate densitiesextending into the VTA. Horizontal sectionsil- (Boysonet al., 1986; Dawsonet al., 1986; Bouthenetet al., 1987; lustrating the distributionsof D, and D, receptorsin relation to D, Charuchinda et al., 1987; Richfield et al., 1987; Wamsley et al., receptormRNA in the basalganglia are presentedin Figure 8. 1989), D, and D, receptor sitesare differentially distributed in More rostrally in the quadragemini, moderate amounts of D, the CNS and pituitary. D, receptors show a widespread distri- receptor mRNA can be visualized in the inferior colliculus, bution, with binding observed in the basalganglia, neocortical while little, if any, D, receptor mRNA can be seenin the superior and paleocortical regions, amygdala, hippocampus, thalamus, colliculus. In contrast, moderate levels of D, binding and high and the neural lobe of the pituitary. In contrast, the distribution densitiesof D, sitesare present in the superficial gray layer of of D, binding sitesappears restricted primarily to the olfactory the superior colliculus. This relationship is somewhatreversed bulb, basal ganglia, and the intermediate lobe of the pituitary. in the inferior colliculus, where moderate amounts of D, recep- Given thesedistinct receptor binding patterns, the distribution tors are present and only light densitiesof D, sitesare found. of D, receptor mRNA generally correspondsto the D, receptor Other mesencephalicregions, such as the periquaductal gray binding sites labeled by 3H-raclopride. These findings support area, raphe nuclei, and interpeduncular nucleus show light to previous dopamine receptor binding studiesin cells transfected moderate levels of D, receptor mRNA, and D, and D, binding. with a full length DNA coding for the D, receptor (Bunzow et al., 1988) and extend thesefindings to the CNS. Pituitary gland Good correspondencebetween the distributions of D, recep- D, receptor binding is very densein the intermediate lobe of tor mRNA and ligand binding can be seenin the caudate-pu- the pituitary, with light labeling in the anterior lobe (Fig. 9). D, tamen, nucleus accumbens,olfactory tubercle, globus pallidus, receptor mRNA showsa corresponding distribution with high lateral mammillary nucleus, substantia nigra, ventral tegmental levels of mRNA observed in the intermediate lobe and indi- area, and pituitary gland. Despite such concordance,subtle dif- vidually labeled cells in the anterior lobe. In contrast, D, re- ferencescan be seenin the precise localization of the mRNA ceptors are restricted to the neural lobe, where there is a light and binding site even at this level of analysis. For example, density of binding sites. within the caudate-putamen D, receptor binding is heteroge- neously distributed, being densest in the lateral extent of the In situ controls nucleus, while the distribution of D, receptor mRNA appears Comparison of sectionshybridized with the 205 bp EcoRI-Xho relatively homogeneousand doesnot demonstrate a mediolater- II probe, which is directed exclusively to the third cytosolic loop, al gradient. This difference may be due to the localization of D, to the longer 495 bp probe, demonstrated that the 2 cRNA binding sites on cortical projections to the caudate-putamen probes labeled the same brain structures (Fig. lo), suggesting (e.g., Schwartz et al., 1978), which would affect the D, ligand the 495 bp probe does not cross-hybridize to other G-protein binding distribution, but not the mRNA localization within the coupled receptors. As can be seen from Figure 10, given the striatum. Similar subtle differences can be seenin the globus higher specificactivity of the 495 bp probe, the quality of the in pallidus, where D, receptor binding is lightly and diffusely dis- situ signal is improved over the shorter 205 bp cRNA probe. tributed, while in situ studies demonstrate distinctly labeled No specific hybridization was observed in any of the brain pallidal cells. Here too, the difference may be more apparent areasidentified following either RNasepretreatment or “sense”- than real, as the binding sitesare likely on pallidal fibers and strand hybridization. Direct comparisonsof brain sectionswith terminals, while the D, receptor mRNA is localized to the cell and without RNase pretreatment, or following “sense” and “an- bodies of widely distributed pallidal neurons. tisense” hybridization, are illustrated in Figure 11. While D, receptor binding sites can be observed in many of The Journal of Neuroscience, August 1990, fO(8) 2595.

Figure 7. Dark-fieldautoradiograms of D, and D, receptorbinding as compared to D, receptormRNA at 2 levelsof the substantianigra. A good correspondencebetween the localizationof D, bindingsites and mRNA canbe seenin the substantianigra, pars compacta (SNc), ventral tegmental area(PTA), and lateralmammillary nucleus (~34). In contrast,D, receptorsites can be observedin the substantianigra, pars reticulata (SW), with little or no bindingseen in the lateralmammillary nucleus. Other abbreviations:J; fornix; jii, fasciculusretroflexus; PAG, periaquaductalgray. the samestructures that also expressD, receptors(e.g., caudate- lobes. A final example is the lateral mammillary nucleus,where putamen, nucleus accumbens,globus pallidus, substantianigra, D, receptors are fairly dense and little or no D, receptors are &nd pituitary), their precise distribution often varies markedly observed. In other regions where there is a high density of both from the D, receptor mRNA and binding sites. For example, D, and D, receptor sites, such as the caudate-putamen, nucleus in the substantia nigra, D, receptor binding is localized in the accumbens,and olfactory tubercle, determining whether the D, pars reticulata, while D, receptor mRNA and binding is ob- receptor mRNA distribution correspondsbetter to a D, or D, served primarily in the pars compacta. Similarly, in the pitu- receptor pattern is difficult. itary, D, sitesare restricted to the neural lobe, while D, receptor Despite the overall good correspondencebetween the distri- mRNA and binding are seenin the intermediate and anterior bution of D, receptor mRNA and D, ligand binding, there are 2596 Mansour et al. l Localization of Dopamine D, Receptor Binding

Figure 8. Dark-field autoradiogram of horizontal rat brain sections showing the distributions of D, receptor mRNA and D, and D, receptor binding. Note the good correspondence in the localization of D, binding Figure 9. Dark-field autoradiograms of D, and D, receptor binding in sites and mRNA in the caudate-putamen (CPU), nucleus accumbens the rat pituitary as compared to the distribution of D, receptor mRNA. (AC@, and substantia nigra (STQ. Regions where there is a lack of Note the high levels of D, receptor mRNA and ligand binding in the correspondence between D, binding and mRNA include cortex (CTX), intermediate lobe and the light labeling in the anterior lobe (AL). D, hippocampus, and zona incerta (ZI). binding sites, in contrast, are restricted to the neural lobe (NL). The Journal of Neuroscience, August 1990, 70(a) 2597

Figure 10. Comparisonof adjacent telencephalicand mesencephalicsec- tions hybridized to either the 495 bp probe(A and B) or the 205 bp probe (C andD). The dark-fieldimages dem- onstratethat the samebrain regions are labeledwith eachprobe, suggesting that the longer495 bp probeis not cross- hybridizing with other 7 transmem- branereceptors. Note also that, because of its higherspecific activity, the 495 bp probeproduces a better quality in situ signal.Abbreviations; CPU, caudate-putamen;SN, substantianigra; SP, septum. a number of brain areas that demonstrate a lack of correspon- undesired labeling of other receptor mRNAs or binding sites. dence. These include the neocortex, zona incerta, olfactory bulb, However, the in situ hybridization controls of RNase pretreat- and hippocampus. While it is at present difficult to determine ment and “sense-strand” labeling would arguethat there is spe- the precise reason for this discrepancy, several possible expla- cific hybridization. Further, in situ hybridization with the 205 nations may be applicable depending on the anatomical region bp cRNA probe that has no sequence identity to any other involved. cloned receptor results in the samemRNA distribution as ob- One possible explanation for a lack of correspondence in some served with the longer 495 bp probe. We have usedthe 495 bp brain regions between receptor binding and mRNA distribu- for this mapping study becauseof the higher specific activity tions may be inherent technical limitations. The visualization that can be achieved, thereby enhancing the sensitivity and of D, receptor mRNA with S5-labeled riboprobes may be a quality of the in situ procedure. more sensitive means of detecting dopaminergic receptors than With regardsto the receptor binding data, 3H-raclopride and is possible with receptor autoradiography with 3H-labeled li- 3H-SCH23390have beenreported to be highly selective ligands gands. In addition, differential quenching of the 2 isotopes may for the D, and D, receptor sites,respectively (Iorio et al., 1983; contribute subtle differences in grain distributions. Billard et al., 1984; Kohler et al., 1985), and the competition Other technical problems may be methodological, such as the results generatedunder the autoradiographic conditions usedin 2598 Mansour et al. l Localization of Dopamine D, Receptor Binding

Figure II. In situ controls. A and B compare adjacent slides that were either treated with RNase A (30 min, 37°C) (II) or not treated (A) prior to in situ hybridization. C and D compare a second set of adjacent sections hybridized with either “antisense” (c) or “sense” (0) cRNA probes. Note that neither RNase pretreatment nor hybridization with a “sense’‘-strand cRNA produced a specific signal. Abbreviations: AC& nucleus accumbens; CPU, caudate-putamen; CTX, cortex; HPC, hippocampus; HYP, hypothalamus; THL, thalamus.

the present study are in full agreement with these conclusions. ing, suggestingthat the D, receptor is translated and transported 3H-Raclopride is stereoselectively displacedby D, antagonists to sitesdistant from the point of transcription. The zona incerta, [e.g., (+)butaclamol, haloperidol, spiperone], while compounds for example, projects to the lateral septum and hypothalamus, such as (-)butaclamol, clonidine, bremazocine, or propranolol where D, receptor binding can be observed. fail to displace this ligand. Similarly, binding sites labeled by The olfactory bulb and hippocampus provide the converse’ 3H-SCH23390are only displacedby unlabeled SCH23390, and example of the presenceof D, receptor binding and no D, re- at higher concentrations, (+)butaclamol and chlorpromazine. ceptor mRNA. In the olfactory bulb, D, receptor binding is While technical limitations may contribute to a lack of cor- densein the olfactory nerve layer (ON) and glomerular layer respondence,they cannot explain the marked differencesin D, (GL), while D, receptor mRNA is found in the periglomerular receptor binding and mRNA distributions observed in some cells of GL and in the internal granular layer. The lack of D, brain regions. Discordance in the distributions may be due to receptor mRNA in the ON is becausethis layer is composedof presenceof D, receptor mRNA and no receptor binding or the denselypacked unmyelinated axons originating from the olfac- converse of D, receptor binding and no D, receptor mRNA. tory receptors. Similarly, the presenceof D, receptor mRNA in The zona incerta and neocortex are examplesof regions which the periglomerular cells and D, ligand binding in the glomerular demonstrate receptor mRNA and little or no D, receptor bind- cells would suggestthat D, receptors may be synthesized in the The Journal of Neuroscience, August 1990, 70(8) 2599

periglomerular cells and transported down their dendritic probe similar to the other G-protein coupled receptors that have jections (Halasz and Shepherd, 1983) to the glomerular cells. been cloned (e.g., muscarinic, cy- and P-adrenergic), one might A similar pattern may be observed in the hippocampus, where expect that there may be several receptor subtypes that encode D, receptor mRNA is localized in the pyramidal cell layer of for pharmacologically similar receptor binding sites, making the hippocampal formation and the granular cells of the dentate future studies relating D, receptor mRNA(s) and D, binding gyrus, while D, receptor binding is restricted to the stratum sites even more challenging. lacunosum moleculare. The pyramidal cells of the hippocampus and granular cells of the dentate gyrus are oriented with their apical dendrites synapsing in the stratum lacunosum moleculare, so that the D, binding protein may be synthesized in these References cells and transported to this dendritic field. This scenario would Aiso M, Potter WZ, Saavedra JM (1987) Axonal transport of dopa- imply that there is a mechanism involved to direct the transport mine D, receptors in the rat brain. Brain Res 426:392-396. of the nascent receptor from the site of translation to the point Attie MF, Brown EM, Gardner DG, Spiegel AM, Aurbach GD (1980) of its subsequent insertion in the membrane. While such an Characterization of dopamine-responsive adenylate cyclase of bovine parathyroid cells and its relationship to parathyroid hormone secre- explanation is appealing, it cannot be determined at present tion. Endocrinology 107:1776-1781. whether the D, receptor binding observed in the stratum la- Billard W, Ruperto V, Crosby G, Iorio LC, Bamett A (1984) Char- cunosum moleculare is not from an extrahippocampal projec- acterization of the binding of [3H] SCH23390, a selective D, receptor tion, such as the entorhinal cortex. antagonist ligand, in rat striatum. Life Sci 351885-1893. Recent studies (Dal Toso et al., 1989; Giros et al., 1989; Bouthenet M-L, Martres MP, Sales N, Schwartz JC (1987) A detailed mapping of dopamine D, receptors in rat central nervous system by Monsma et al., 1989) have identified a second D, receptor that autoradiorrraohv with 11251110dosuIaride. Neuroscience 20: 117-l 55. is identical to the receptor cloned by Bunzow et al. 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