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 the cloned rat D-1A and D-3 dopamine receptor subtypes in order to determine 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 receptor proteins expressed in stably transfected Chinese hamster ovary (CHO) cells. Central nervous system (CNS) areas of the male rat were examined using standard immunofluorescent methods in fresh frozen tissues. This paradigm detected D-1A-like and D-3-like dopamine receptor staining 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 noted in the substantia nigra, not associated with perikarya. Differences in cellular staining patterns and intensity were evident between the D-1A-like and D-3-like dopamine receptor subtypes. Equivalent morphological elements exhibited dopamine receptor expression following incubation using antisera generated against either extracellular or intracellular epitopes of either the D-1A or D-3 native proteins. Dopamine receptor immunoreactivity could not be detected in the cerebellum at equivalent antisera dilutions used to discriminate cellular staining patterns within the forebrain. Fluorescent-labeled latex microspheres were infused into the substantia nigra terminal fields to retrogradely identify the cell bodies of the striatonigral projection system. This paradigm showed that 80% of striatonigral neurons expressed D-1A-like receptors, while 65% demonstrated D-3-like dopamine receptor staining. This distribution for the D-1A-like and D-3-like receptor subtypes suggests that overlap may occur in the expression of the receptors in the striatonigral neuron population. Our previous results localizing cellular 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.