Differential Regional and Cellular Distribution of Dopamine D2-Like Receptors: An Immunocytochemical Study of Subtype-Specific Antibodies in Rat and Human Brain ZAFAR U. KHAN, * ANTONIA GUTIE ´ RREZ, ROSA MARTI ´ N, ANTONIO PEN ˜ AFIEL, ALICIA RIVERA, AND ADELAIDA DE LA CALLE Department of Cell Biology, Faculty of Science, University of Malaga, Teatinos 29071, Malaga, Spain ABSTRACT Dopamine D2-like receptors (D2, D3, and D4) are major targets for action of typical and atypical neuroleptics, commonly used in the treatment of schizophrenia. To understand their individual functional contribution, subtype-selective anti-peptide antibodies were raised against D2, D3, and D4 receptor proteins. The antibodies were shown to be specific on immunoblots of rat brain membranes and immunoprecipitated the solubilized native dopa- mine receptors in an antibody concentration-dependent manner. In addition, they also bind selectively to the respective recombinant D2, D3, and D4 receptor membrane proteins from cDNA transfected cells. Immunolocalization studies show that the D2-like receptor proteins had differential regional and cellular distribution in the cerebral cortex, hippocampus, basal ganglia, cerebellum, and midbrain, thus providing anatomical substrate for area-specific regulation of the dopamine neurotransmission. In cortical neurons, D4 receptor protein was found in both pyramidal and nonpyramidal cells, whereas D2 and D3 seem to be mostly associated with nonpyramidal interneurons. In rat hippocampus, the expression pattern of D2-like receptors (D4D3D2) mirrored that obtained with immunoprecipitation studies. D2 and D4 receptor immunolabeling was observed in the thalamic reticular nucleus, which was negative for the D3 subtype. Species differences were also observed; for example, the D4 subtype receptor is the most highly expressed protein in the rat cortex, whereas it is significantly less in human cortex. Differential patterns of D2, D3, and D4 receptor expression in rat and human brain should shed light on the therapeutic actions of neuroleptic drugs and may lead to the development of more specifically targeted antipsychotic drugs. J. Comp. Neurol. 402:353–371, 1998.  1998 Wiley-Liss, Inc. Indexing terms: D2 subtype; D3 subtype; D4 subtype; immunoblot; immunoprecipitation; immunocytochemistry Dopamine exerts its action through four different well known pathways and modulates motor, cognition, emo- tional, and endocrine functions. The nigrostriatal pathway originates in the substantia nigra pars compacta and projects to the striatum. The striatal structure has been associated with planning, initiation, and coordination in voluntary movement (Gerfen, 1992) and also receives motivational, sensory, and motor inputs arriving by means of afferents from cortical and limbic areas (Graybiel et al., 1994). The loss in dopaminergic neurons of substantia nigra pars compacta and consequently the lack of dopa- mine innervation to the striatum is thought to be respon- sible for the deficits in motor function seen in Parkinson’s disease. The two other pathways of dopamine neurotransmission originate in the ventral tegmental area and constitute the Grant sponsor: Spanish DGICYT; Grant number: PB94–0219-C02; Grant sponsor: Junta de Andalucia; Grant number: CTS-0161; Grant sponsor: European Union BIOMED I; Grant number: BMH1-CT94–1060. *Correspondence to: Zafar U. Khan, M.S., Ph.D., Section of Neurobiology, School of Medicine, Yale University, C303 Sterling Hall of Medicine, 333 Cedar Street, New Haven, CT 06520–8001. Received 29 January 1998; Revised 28 May 1998; Accepted 10 August 1998 THE JOURNAL OF COMPARATIVE NEUROLOGY 402:353–371 (1998)  1998 WILEY-LISS, INC.