DOPAMINE-RECEPTOR DISTRIBUTION IN THE RAT CNS - ELUCIDATION USING ANTIPEPTIDE ANTISERA DIRECTED AGAINST D-1A AND D-3 SUBTYPES

Anti-peptide antibodies were generated against amino acid sequences of intracellular and extracellular portions of the native proteins for t he cloned rat D-1A and D-3 dopamine receptor subtypes in order to dete rmine the cellular distribution of these specific forms in the brain. These polyclonal antisera exhibited high specific titers, assessed by ELISA and immunofluorescent detection of functional recombinant recept or proteins expressed in stably transfected Chinese hamster ovary (CHO ) cells. Central nervous system (CNS) areas of the male rat were exami ned using standard immunofluorescent methods in fresh frozen tissues. This paradigm detected D-1A-like and D-3-like dopamine receptor staini ng primarily in larger-sized neurons throughout layers 3 and 5 of the cortex, in medium-diameter somata of the striatum, and in the densely packed cells of the olfactory tubercle and hippocampal formation. More attenuated immunoreactivity for both dopamine receptor subtypes was n oted in the substantia nigra, not associated with perikarya. Differenc es in cellular staining patterns and intensity were evident between th e D-1A-like and D-3-like dopamine receptor subtypes. Equivalent morpho logical elements exhibited dopamine receptor expression following incu bation using antisera generated against either extracellular or intrac ellular epitopes of either the D-1A or D-3 native proteins. Dopamine r eceptor immunoreactivity could not be detected in the cerebellum at eq uivalent antisera dilutions used to discriminate cellular staining pat terns within the forebrain. Fluorescent-labeled latex microspheres wer e infused into the substantia nigra terminal fields to retrogradely id entify the cell bodies of the striatonigral projection system. This pa radigm showed that 80% of striatonigral neurons expressed D-1A-like re ceptors, while 65% demonstrated D-3-like dopamine receptor staining. T his distribution for the D-1A-like and D-3-like receptor subtypes sugg ests that overlap may occur in the expression of the receptors in the striatonigral neuron population. Our previous results localizing cellu lar D-3-like receptor expression patterns in this projection system of the rat neostriatum implies that all three of these dopamine receptor subtypes may be co-expressed in this efferent system.