title: Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening.
abstract: Small molecules modulating synaptic vesicle endocytosis (SVE) may ultimately be useful for diseases where pathological neurotransmission is implicated. Only a small number of specific SVE modulators have been identified to date. Slow progress is due to the laborious nature of traditional approaches to study SVE, in which nerve terminals are identified and studied in cultured neurons, typically yielding data from 10-20 synapses per experiment. We provide a protocol for a quantitative, high-throughput method for studying SVE in thousands of nerve terminals. Rat forebrain synaptosomes are attached to 96-well microplates and depolarized; SVE is then quantified by uptake of the dye FM4-64, which is imaged by high-content screening. Synaptosomes that have been frozen and stored can be used in place of fresh synaptosomes, reducing the experimental time and animal numbers required. With a supply of frozen synaptosomes, the assay can be performed within a day, including data analysis.
authors: Cell Signalling Unit, Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, Australia.
authors: James A
authors: JA
authors: Daniel
authors: Chandra S
authors: CS
authors: Malladi
authors: Emma
authors: E
authors: Kettle
authors: Adam
authors: A
authors: McCluskey
authors: Phillip J
authors: PJ
authors: Robinson
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000032:analysis / Q000493:pharmacokinetics; D005615:Freezing; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D008297:Male; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D016548:Prosencephalon / Q000166:cytology; D011726:Pyridinium Compounds / Q000032:analysis / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000032:analysis / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013572:Synaptic Vesicles / Q000502:physiology*; D013574:Synaptosomes / Q000378:metabolism*
chemical_list: C092350:FM 4-64; C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds