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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 dopamine 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.

As a result of alternative splicing, the D2 gene of the dopamine receptor family exists in two isoforms. The D2 long is characterized by the insertion of 29 amino acids in the third cytoplasmic loop, which is absent in the short isoform. We have produced subtype-specific antibodies against both the D2 short and D2 long isoforms and found a unique compartmentalization between these two isoforms in the primate brain. The D2 short predominates in the cell bodies and projection axons of the dopaminergic cell groups of the mesencephalon and hypothalamus, whereas the D2 long is more strongly expressed by neurons in the striatum and nucleus accumbens, structures targeted by dopaminergic fibers. These results show that the splice variants of the dopamine D2 receptor are differentially distributed and possess distinct functions. The strategic localization of the D2 short isoform in dopaminergic cell bodies and axons strongly suggests that this isoform is the likely dopamine autoreceptor, whereas the D2 long isoform is primarily a postsynaptic receptor.Among all the neurotransmitter receptors cloned to date, the D2 subtype of dopamine receptors has been the one most closely linked to the positive symptoms of schizophrenia (1, 2) and often implicated in extrapyramidal side effects (3, 4). Neuroanatomical (5, 6) and physiological (7,8) studies indicate that this receptor has both autoreceptor and postsynaptic functions. Further, recent evidence of loss of autoreceptor function in D2-deficient mice (8) but sparing of this function in D3 mutant mice (9) strongly suggests that the D2 subtype is the only autoreceptor within the dopamine system. However, the D2 receptor exists in two isoforms (10, 11), and it is not known if both isoforms contribute to autoreceptor function. With the use of recently developed subtype-specific antibodies to dopamine D2 short (D2S) and D2 long (D2L) isoforms, we have found prevalent labeling of the D2S isoform in the cell bodies and axons of brain stem dopamine neurons, suggesting that this isoform is the autoreceptor of the dopamine system. METHODSPreparation of Antibodies. The D2S peptide TPLKDAAR and D2L peptide SNGSFPVNRRRM corresponding to residues 238-245 and 259-270 (10, 11), respectively, were derived from the third cytoplasmic loop of the receptor. The D2S peptide was arranged by adopting four amino acids from each side of the insertion site to differentiate between D2S and D2L isoforms. A similar procedure has previously been used for the preparation of antibodies to ␥-aminobutyric acid (GABA) receptors (12). The peptides were coupled to keyhole limpet hemocyanin (KLH) protein. Peptide-KLH conjugate (100 g) emulsified in complete Freund's adjuvant was injected into rabbits for antibody development. Affinity purification of the antisera was as described elsewhere (12). In brief, peptide (5 mg) was coupled to 1 g of activated thiopropyl-Sepharose 6B (Pharmacia LKB). Phosphate-buffered saline (PBS) diluted antiserum (1:5) was circulated through the column. After washing with PBS, th...

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