Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum

Abstract

Fluorescence-activated cell sorting (FACS) was used to identify and isolate permeabilized dopaminergic nerve teminals from rat striatum based on immunofluorescent labeling of tyrosine hydroxylase (TH). Striatal synaptosomes were permeabilized by fixation with modified Zamboni fluid. A highly fluorescent subpopulation of particles was detected by FACS following sequential incubation with a monoclonal antibody to TH (LNC 1) and a fluorescein-conjugated secondary antibody. After correcting for nonsynaptosomal particles present in the synaptosomal fraction, analysis of these data suggested that approximately 12-15% of striatal synaptosomes were dopaminergic, consistent with previous estimates. Specific labeling by LNC 1 was decreased if synaptosomes were prepared from rats that had received intraventricular injections of 6-hydroxydopamine. The decrease in labeling was highly correlated with the extent of the lesion as determined by measurement of striatal dopamine levels, suggesting that LNC 1-labeled synaptosomes were derived from nigrostriatal dopamine terminals. In order to verify that LNC 1-labeled synaptosomes were enriched for TH, equal numbers of labeled and control synaptosomes were isolated by FACS and analyzed by SDS-PAGE. LNC 1-labeled synaptosomes were shown by Western blot techniques to be enriched 6-fold for TH compared with control synaptosomes, suggesting that the labeled population consisted almost entirely of dopaminergic synaptosomes.