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

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
Karen H GylysGregory M Cole

Abstract

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). 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. 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...Continue Reading

References

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Mar 7, 1998·Journal of Neuroscience Methods·M W JohnsonJ B Watson
Jul 6, 2000·Journal of Neuroscience Research·K H GylysG M Cole
Aug 30, 2002·Neurochemical Research·Karen H GylysGregory M Cole
Oct 26, 2002·Science·Dennis J Selkoe
Oct 23, 2003·Neurochemical Research·Wim E J M GhijsenFernando H Lopes da Silva

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Citations

Sep 29, 2011·Journal of Visualized Experiments : JoVE·Pamela R WestmarkJames S Malter
Jan 1, 2016·The American Journal of Pathology·Tina BilousovaKaren H Gylys
Jan 4, 2012·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Sophie SokolowKaren H Gylys
May 10, 2008·The American Journal of Pathology·Jeffrey A FeinKaren Hoppens Gylys
Dec 8, 2005·Neurobiology of Aging·Karen Hoppens GylysGregory M Cole
Jul 12, 2011·Neurobiology of Aging·Sophie SokolowKaren Hoppens Gylys
Nov 14, 2014·Journal of Neurochemistry·Sophie SokolowKaren H Gylys
Nov 10, 2017·Scientific Reports·Michele M ComerotaGiulio Taglialatela
Jan 9, 2019·Frontiers in Cellular Neuroscience·Valentina LatinaGiuseppina Amadoro
Jun 27, 2019·Scientific Reports·Noemí Pallas-BazarraJesús Avila
Aug 3, 2021·Frontiers in Molecular Neuroscience·Melanie J GrubishaMatthew L MacDonald
Aug 3, 2021·Frontiers in Synaptic Neuroscience·Yesenia Castillo-OcampoJames T Porter

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