Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening

Nat Protoc. 2012 Jul 5;7(8):1439-55. doi: 10.1038/nprot.2012.070.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Fluorescent Dyes / analysis
  • Fluorescent Dyes / pharmacokinetics
  • Freezing
  • High-Throughput Screening Assays / instrumentation
  • High-Throughput Screening Assays / methods*
  • Male
  • Molecular Biology / instrumentation
  • Molecular Biology / methods*
  • Prosencephalon / cytology
  • Pyridinium Compounds / analysis
  • Pyridinium Compounds / pharmacokinetics
  • Quaternary Ammonium Compounds / analysis
  • Quaternary Ammonium Compounds / pharmacokinetics
  • Rats
  • Rats, Sprague-Dawley
  • Synaptic Vesicles / physiology*
  • Synaptosomes / metabolism*


  • FM 4-64
  • FM1 43
  • Fluorescent Dyes
  • Pyridinium Compounds
  • Quaternary Ammonium Compounds