We have identified ([3H]7-OH-DPAT) as a selective probe for the recently cloned dopamine D3 receptor and used it to assess the presence of this receptor and establish its distribution and properties in brain. In transfected Chinese hamster ovary (CHO) cells, it binds to D3 receptors with subnanomolar affinity, whereas its affinity is approximately 100-, 1000-, and 10,000-fold lower at D2, D4, and D, receptors, respectively. Specific [3H]7-OH-DPAT binding sites, with a Kd of0.8 nM and a pharmacology similar to those at reference D3 receptors of CHO cells, were identified in rat brain. D3 receptors differ from D2 receptors in brain by their lower abundance (2 orders of magnitude) and distribution, restricted to a few mainly phylogenetically ancient areas-e.g., paleostriatum and archicerebellum-as evidenced by membrane binding and autoradiography studies. Native D3 receptors in brain are characterized by an unusually high nanomolar affinity for dopamine and a low modulatory influence of guanyl nucleotides on agonist binding. These various features suggest that D3 receptors are involved in a peculiar mode of neurotransmission in a restricted subpopulation of dopamine neurons.Dopamine is an important neurotransmitter in brain, being involved physiologically in the control of cognitive, motor, and endocrine processes and pathologically in Parkinson and possibly mental diseases.Until recently, it was largely believed that its various actions were mediated by two receptor subtypes, termed D1 and D2 (1, 2). Molecular biology approaches have led, however, to the identification and cloning of the genes corresponding not only to these two receptor subtypes (3-7) but also to additional and less expected ones, termed D3 (8), D4 (9), and D5 (10, 11). For the latter receptors, the information so far available derives from molecular biology approaches; their pharmacology and signaling system have only been studied in transfected cells, generally fibroblasts, and their distribution has been indirectly approached by studies of mRNA localization. Nevertheless, these otherwise valuable approaches suffer from some limitations. When expressed in fibroblasts, receptors might find a membrane environment, which could modify their pharmacological specificity, and a repertoire of cellular components, particularly GTP-binding proteins (G proteins), which may differ from that found in neurons. Regarding distribution, mRNA detection reveals the sites of receptor synthesis rather than receptor localization, which may be different. Therefore, there is an obvious interest in studying the native receptor protein in brain, particularly in the case ofthe D3 receptor, whose pharmacology in Chinese hamster ovary (CHO) cells and cerebral localization of its mRNA suggest that it may represent an important target for antipsychotics (8,12,13).Starting with the idea that designing a selective radioactive probe for the D3 receptor would help to settle these issues, we have screened a series of dopamine agonists and thereby identified 7-hydroxy-N,...

A polyclonal antibody was generated using synthetic peptides designed in a specific sequence of the rat D(3) receptor (D(3)R). Using transfected cells expressing recombinant D(3)R, but not D(2) receptor, this antibody labeled 45-80 kDa species in Western blot analysis, immunoprecipitated a soluble fraction of [(125)I]iodosulpride binding, and generated immunofluorescence, mainly in the cytoplasmic perinuclear region of the cells. In rat brain, the distribution of immunoreactivity matched that of D(3)R binding, revealed using [(125)I]R(+)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino] tetralin ([(125)I]7-trans-OH-PIPAT), with dense signals in the islands of Calleja and mammillary bodies, and moderate to low signals in the shell of nucleus accumbens (AccSh), frontoparietal cortex, substantia nigra (SN), ventral tegmental area (VTA) and lobules 9 and 10 of the cerebellum. Very low or no signals could be detected in other rat brain regions, including dorsal striatum, or in D(3)R-deficient mouse brain. Labeling of perikarya of AccSh and SN/VTA appeared with a characteristic punctuate distribution, mostly at the plasma membrane where it was not associated with synaptic boutons, as revealed by synaptophysin immunoreactivity. In SN/VTA, D(3)R immunoreactivity was found on afferent terminals, arising from AccSh, in which destruction of intrinsic neurons by kainate infusions produced a loss of D(3)R binding in both AccSh and SN/VTA. D(3)R-immunoreactivity was also found in all tyrosine hydroxylase (TH)-positive neurons observed in SN, VTA and A8 retrorubral fields, where it could represent D(3) autoreceptors controlling dopamine neuron activities, in agreement with the elevated dopamine extracellular levels in projection areas of these neurons found in D(3)R-deficient mice.

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