Cellular distribution of the rat D4 dopamine receptor protein in the CNS using anti-receptor antisera

Abstract

A polyclonal antiserum was generated against a unique peptide fragment in the rat D4 dopamine (DA) receptor. The titer was monitored using solid-phase ELISA and once it was established, specificity was assessed using Chinese Hamster Ovary (CHO) cells, stably transfected with the full-length cDNA for the rat D4 DA receptor. Immunofluorescent staining produced by incubation with the anti-D4 DA receptor antiserum was selective for D4 DA receptor-transfected CHO cells, and was expressed at their cell membranes and cytoplasm. Attenuated staining for D4 DA receptor protein was visible in untransfected, K1 CHO cells, and in D2 or D3 DA receptor-transfected CHO cells. The regional and cellular CNS distribution patterns for the D4 DA receptor subtype were examined, and illustrated significant protein levels within the frontal (FCx) and parietal cortices. Lesser amounts of receptor protein staining occurred in the thalamus, globus pallidus, hippocampus, cerebellar vermis, and very low expression was detected in the striatum (CPu). D4 DA receptor protein staining was correlated with the cellular expression of its mRNA transcripts in these same brain regions using concurrent fluorescent analyses. The homologous coincidence in staining patterns for the D4 DA receptor transcripts and encoded proteins in identified neurons of the FCx and CPu showed variations in receptor expression in these identified basal ganglia pathways.