Background: Synapse regions in the brain are difficult to isolate and study; resealed nerve terminals (synaptosomes) are a widely used in vitro system for the study of neurotransmission, but nonsynaptosomal elements in the homogenate complicate data interpretation. With the goal of quantitative analysis of pathways leading to synapse loss in neurodegenerative disease, we have developed a method that allows focus on the intact synaptosomes within a crude synaptosomal preparation by gating the largest particles based on forward angle light scatter (FSC).
Methods: Crude synaptosomal fractions (P-2) were prepared and labeled with a viability dye (calcein AM), a presynaptic marker (SNAP-25), and a postsynaptic marker (PSD-95). Forward scatter gates based on size standards were drawn to identify the large population (1.4-4.5 microm), and the enrichment of each marker was quantified in preparations from fresh rat homogenates and from cryopreserved human cortex.
Results: Gating on forward scatter resulted in an increase that was highly significant (P < 0.001) for all three markers examined. The calcein-AM-positive fraction in the large synaptosomes was 98% +/- 0.8, and 75% +/- 9.8 for rat and human, respectively. Of large particles, 90% +/- 2.7 in rat and 82% +/- 2.6 in human were positive for SNAP-25, indicating a relatively pure population of intact synaptosomes. A total of 76% +/- 2.9 of the large particles were positive for PSD-95 in rat. This compared to 36% +/- 3.0 in human tissue, and indicates that both presynaptic and postsynaptic elements may be analyzed with this methodology.
Conclusions: Most nonsynaptosomal elements can be excluded and the intact subpopulation of interest within the P-2 can be identified based on size. Size-based gating analysis provides a simple and cost-effective method to monitor fluorescence changes in synapse regions.
Copyright 2004 Wiley-Liss, Inc.