Flow cytometric analysis of rat striatal nerve terminals

Methods were developed for the analysis and isolation of striatal nerve terminals (synaptosomes) using fluorescence-activated cell sorting (FACS). Comparison of the light-scattering properties of synaptosomal and mitochondrial fractions indicated that particles in the synaptosomal fraction were generally larger and more sensitive to hypotonic lysis, consistent with results obtained by other methods of analysis. FACS analysis using indirect immunofluorescence techniques indicated that approximately 84% of the synaptosomal fraction was labeled by monoclonal antibody (mAb) A2B5 and thus appeared to be of neuronal origin. After permeabilization, between 5 and 10% of the particles were labeled by a mAb to glial fibrillary acidic protein, suggesting that they were derived from astrocytes. A fluorescent voltage-sensitive dye (VSD) was used to distinguish intact synaptosomes from free mitochondria (only the former maintain a membrane potential under the present experimental conditions). Approximately 83% of the synaptosomal fraction exhibited increased fluorescence after incubation with the VSD; furthermore, the fluorescence signal decreased in response to depolarizing agents (elevated potassium and veratridine). A portion of the mitochondrial fraction responded similarly, consistent with the presence of contaminating synaptosomes. Analysis of synaptosomal labeling by 11 fluorescein-conjugated plant lectins indicated that striatal nerve terminals differ significantly in their cell surface glycoconjugates. Subpopulations of synaptosomes defined on the basis of lectin binding were collected by FACS onto filters and probed with a mAb to tyrosine hydroxylase (TH) using Western blot techniques. While subpopulations exhibited different amounts of TH immunoreactivity, none of the lectins appeared to recognize TH-positive (i.e., dopaminergic) synaptosomes exclusively. These findings demonstrate that synaptosomes can be characterized and isolated for further study based on FACS analysis of properties such as size, membrane potential, and the presence of intracellular or cell surface molecules.