Proc. NatI. Acad. Sci. USA Vol. 91, pp. 11271-11275, November 1994 Pharmacology Localization of dopamine D3 receptors to mesolimbic and D2 receptors to mesostriatal regions of human forebrain (nucleus accumbens/nucleus basalis/amygdala/ventral tegnental area/pallidum) ANGELA M. MURRAY*, HAN L. Ryoot, EUGENIA GuREVICH, AND JEFFREY N. JOYCEO Departments of Psychiatry and Pharmacology, University of Pennsylvania, Philadelphia, PA 19104-6141 Communicated by William T. Greenough, July 19, 1994 ABSTRACT We characterized the binding of [125I~epide- transfected Chinese hamster ovary (CHO) cells (9, 10). In the pride to dopamine D2-like and D3-like receptors in tissue presence of GTP, binding of agonists to the D2 receptor sections of human striatum. The competition for binding of shows a rightward displacement in the competition curve. [125I]epidepride by domperidone, quinpirole, and 7-hydroxy- The data indicate there is a complete conversion to the N,N-di(l-propyl)-2-aminotetralin (7-OH-DPAT) was best fit low-affinity site ofthe D2 receptor. In contrast, binding to the by assuming one site in the caudate but two sites in nucleus human D3 receptor shows a minor GTP-induced shift (9, 10). accumbens. Guanosine 5'-[3,v-imidoltriphosphate showed a These properties allow for increased selectivity for the dis- large modulatory influence in agonist inhibition of [12(I]epide- placement ofnonselective radioligands by the agonists 7-OH- pride binding in caudate but not in nucleus accumbens. The DPAT and quinpirole. In this paper we have used the binding of [125I~epidepride in the presence of 7-OH-DPAT selective displacement of [1251]epidepride from D3 receptors (1000-fold selective for D3-like versus D2-like sites) and dom- with 7-OH-DPAT and partial displacement from D2 receptors peridone (20-fold selective for D2-like versus D3-like sites) was with domperidone to quantify the distribution of [1251]epide- used to quantify the numbers of D2-like and D3-like receptors pride-bound D2-like and D3-like receptors in the basal ganglia in areas ofhuman brain. The distribution ofD2-like and D3-like and limbic regions of human brain. receptors was largely nonoverlapping. Binding of [125I~epide- pride to D3-like receptors was negligible in the dorsal striatum but was concentrated in islands of dense binding in the nucleus MATERIALS AND METHODS accumbens and ventral putamen that aligned with acetylcho- inetic Experiments. Tissue was obtained post mortem linesterase-poor striosomes. Binding to D3-like receptors was from 24 normal individuals (mean age ± SD, 68 + 14 yr; also enriched in the internal globus pallidus, ventral pallidum, postmortem interval of 9.2 + 6 hr; 17 males, 7 females). For septum, islands of Calleja, nucleus basalis, amygdalostriatal all kinetic experiments 20-gm-thick slide-mounted human transition nucleus of the amygdala, central nucleus of the tissue sections of striatum (containing caudate, putamen, and amygdala, and ventral tegmental area. Binding of [12I5epide- nucleus accumbens) or inferior temporal lobe were preincu- pride to D2 but not D3 receptors was detected in cortex and bated for 30 min in ice-cold TBS (50 mM Tris-HCl/120 mM hippocampus. NaCl, pH 7.4) and then for 25 min in TBS at room temper- ature. The preincubation step was utilized to remove the There are two superfamilies of dopamine receptors, desig- high-affinity binding of dopamine to the D3 receptor (2). To nated Dj-like and D2-like (1). This nomenclature is based on establish optimal conditions for the binding of [125I]epide- their structural and amino acid similarities, as well as their pride, sections of striatum were incubated in TBS and the binding profiles. The D2-like family contains D2, D3, and D4 a2-adrenergic antagonist idazoxan (100 nM) containing 50 pM subtypes, which differ with respect to their distribution in rat [1251]epidepride at 22°C, 30°C, or 37°C for various intervals brain (2-4). Much interest has been focused on the D3 (30-300 min). Results are the means of triplicate determina- receptor because of its high association with "limbic" com- tions for four brains. Following establishment of optimal ponents of the rat brain (e.g., ventral striatum) and high conditions for binding, competition experiments with 18 affinity for dopamine (2, 5). In fact, it has been proposed that differing concentrations of domperidone (0.01-1000 nM), the D3 receptor is the "limbic" dopamine receptor through 7-OH-DPAT (0.1-10,000 nM), and quinpirole (1-100,000 nM) which antipsychotics could act to modify psychosis (5). and one concentration of raclopride (300 nM) for ['25I]epide- However, extension of this hypothesis to the human is pride binding were carried out in the presence and absence of limited by the lack of a detailed mapping of the distribution guanosine 5'-[(3,y-imido]triphosphate (p[NH]ppG; 100 ,M). of the D3 receptor in this species. Such mapping can be Results are the means of triplicate determinations for four accomplished by selectively displacing radioligands nonse- brains, expressed as the percentage of total specific binding lective for D2 and D3 receptors from each receptor site (6). In measured in the absence of any displacing drug. Saturation preliminary studies we utilized that approach to detect the isotherms were established by conducting saturation binding presence of D2-like and D3-like receptors in striatal tissue of [1251]epidepride to striatum and inferior temporal cortex sections with [1251]epidepride (7). The potency of substituted benzamide derivatives similar to [125I]epidepride is only Abbreviations: 7-OH-DPAT, 7-hydroxy-N,N-di(l-propyl)-2-amino- at the tetralin; p(NH]ppG, guanosine 5'-[3,.-iimido]triphosphate; AChE, marginally lower D3 receptor than at the D2 receptor acetylcholinesterase; GPi, globus pallidus internal; GPe, globus (8). Further, drugs such as domperidone, quinpirole, and pallidus external. 7-hydroxy-N,N-di(1-propyl)-2-aminotetralin (7-OH-DPAT) *Present address: Division of Intramural Research Programs, Na- show 32- to 100-fold selectivity for the human D2 or D3 tional Institute of Mental Health Neuropsychiatric Research Hos- receptor labeled with [1251]iodosulpride in membranes of pital, Washington, DC 20032. tPresent address: Department of Neurobiology and Anatomy, Uni- versity of Rochester, Rochester, NY. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Psychiatry, 127 Clinical Research Building, 415 Currie Boulevard, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Philadelphia, PA 19104-6141. 11271 Downloaded by guest on September 30, 2021 11272 Pharmacology: Murray et al. Proc. Natl. Acad Sci. USA 91 (1994) with increasing concentrations of ['251]epidepride (3-500 pM) Caudate in triplicate from four individuals. In all experiments, sec- tions were rinsed, dried, and laid against film for 18 hr (striatum) or 7 days (inferior temporal cortex). Nonspecific binding was determined in the presence of 5 ILM (+)- butaclamol. Competition curves were analyzed by comput- erized nonlinear regression, using either a one-site or a two-site cooperative model to estimate IC50 values and the proportion of sites (11). Saturation curves were analyzed by computer nonlinear regression, using a one-site and two-site cooperative model to obtain equilibrium dissociation con- stants (Kd) and maximal density of receptors (Bmax). Mapping Experiment. The 20-Ium-thick slide-mounted hu- 100000 man tissue sections were preincubated as before and then incubated in TBS and idazoxan (100 nM) at 300C containing 50 pM [125I]epidepride and p[NH]ppG (100 ttM) for 100 min. Adjacent sections were incubated similarly but contained 50 nM 7-OH-DPAT to displace [1251]epidepride binding to D3 co receptors, allowing detection of D2 receptors, or 10 nM '0 domperidone to partially exclude D2 receptors to visualize D3 receptors. Nonspecific binding was determined in the pres- ence of 5 juM (+)-butaclamol. Sections were rinsed, dried, ut and laid against film for the appropriate time. Sections adjacent to those used for autoradiographic experiments were processed for acetylcholinesterase (AChE) histochem- istry or cresyl violet histochemistry for aid in the identifica- tion of regions of interest (12). The maximal density of sites was estimated from the single-concentration mapping exper- iments by using the equation Bma = [B]([L] + Kd)/[L], where [B] represents the amount bound at the concentration of FIG. 1. Inhibition of (1251]epidepride binding by quinpirole, dom- ligand [L] (11). Results are the means of triplicate determi- peridone, or 7-OH-DPAT in tissue sections containing caudate (A) or nations for a minimum offour brains for any region. For these nucleus accumbens (B). Means of four independent experiments are studies, four sections for total binding and two sections for shown. Computer-drawn curves of quinpirole (o, *) inhibition of [1251]epidepride binding in the presence of p[NH]ppG show a large nonspecific binding for each level were analyzed for each rightward shift for caudate and much smaller shift for the nucleus case. An average value for specific binding was obtained for accumbens. It was best fit by assuming one class of sites in the each individual for any region of interest. For the mapping of caudate and two classes of sites in the nucleus accumbens. Displace- D2 and D3 receptor sites the differences between regions were ment of [125I]epidepride binding by domperidone (o, *) was also best tested for significance by analysis ofcovariance (ANCOVA). fit by assuming one class of sites in the caudate and two classes of sites in the nucleus accumbens. Displacement of ['M'I]epidepride binding in the presence of p(NH]ppG by 7-OH-DPAT (A) was best RESULTS fit by assuming two classes of sites in the caudate and nucleus The of accumbens, but the proportion of high-affinity sites was small in Kinetic Experiments.