Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human cortex.
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.
Gylys
Karen H
KH
UCLA School of Nursing and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90095, USA. kgylys@sonnet.ucla.edu
Fein
Jeffrey A
JA
Yang
Fusheng
F
Cole
Gregory M
GM
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015415:Biomarkers; D001923:Brain Chemistry; D002540:Cerebral Cortex / Q000737:chemistry / Q000378:metabolism; D000076122:Disks Large Homolog 4 Protein; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis; D019636:Neurodegenerative Diseases / Q000378:metabolism / Q000473:pathology; D051381:Rats; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000737:chemistry* / Q000378:metabolism
D015415:Biomarkers; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C494208:SNAP25 protein, human; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; C033652:postsynaptic density proteins