Flow cytometric analysis of rat striatal nerve terminals

Abstract

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.

Enrichment of presynaptic and postsynaptic markers by size‐based gating analysis of synaptosome preparations from rat and human cortex

“…2D). The continuous nature of the size distribution is in agreement with previous flow cytometry analysis of gradient purified synaptosomes (8), and is consistent with EM observations that the P-2 homogenate contains a heterogenous mixture of membrane fragments and vesicles in addition to synaptosomes (4). Also in agreement with EM observations, the P-2 contains fewer postsynaptic than presynaptic elements: 38% Ϯ 4.6 (n ϭ 3) and 22% Ϯ 2.6 (n ϭ 6) of the total P-2 was positive for PSD-95 in the rat and human, respectively (Fig.…”

“…This compares to 84% for flow cytometric analysis of biochemically purified synaptosomes (8). Light scattering analysis for purified synaptosomes and the P-2 fraction reveal particles ranging from 0.2 to 5 m based on size standards, with the greatest density being small particles less than 1 m in size.…”

“…The error bars in Figure 4 reveal a relatively small interexperiment variability, indicating that, in our hands, the size gate boundaries are consistent from one preparation to the next. Because the population of interest consists of functional synaptic terminals with intact membranes, the calcein AM-positive and SNAP-25-positive fractions in both rat and human preparations indicates that gating analysis results in synaptosome purity equal or superior to density gradient purification (8).…”

Surface-accessible GABA supports tonic and quantal synaptic transmission

“…Subcellular particles present in dissociates of embryonic brain often maintain a functional membrane under our experimental conditions (see Wolf and Kapatos, 1989). About 50% of these particles bound TTFC, sug- Recently plated, (A 2 ) 5-day-old, and (A 3 ) 10-day-old cultures.…”

Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry

“…Intracellular calcium in embryo neurons was determined using Indo-1 (10) and in cerebellar neurons using fluo-3 or Indo-1 (20,27). Moreover, flow cytometry sorting has been used to purify neuronal preparations (12,16,23,28). Therefore, flow cytometry is being used nowadays in neurochemistry and neurophysiology along side other techniques, because it is able to identify various cell parameters rapidly and sensitively (17).…”