A radioimmunoassay to monitor synaptic activity in hippocampal neurons in vitro

O. Mundigl, C. Verderio, K. Kraszewski, P. De Camilli, M. Matteoli

Research output: Contribution to journalArticlepeer-review

Abstract

Exocytosis of synaptic vesicles (SV) results in the surface exposure of lumenal epitopes of SV proteins. We have recently described the use of antibodies directed against the lumenal N-terminus of synaptotagmin I (Syt(lum)·Abs) to morphologically monitor exo-endocytic recycling of SVs. We report here that a radioimmunoassay based on these anti bodies can be used to quantify levels of synaptic activity in primary neuronal cultures. High density cultures of hippocampal neurons grown in the absence of glia were used for these studies. A significant cell surface pool of synaptotagmin I immunoreactivity was detectable by Syt(lum)·Abs at steady state. The increase in the amount of Syt(lum)·Abs which became cell bound during a 3 min incubation at 37°C over the Ab bound to this cell surface pool, was substantially higher in depolarizing media containing extracellular Ca2+ than in Ca2+-free media. Incubation of the cultures with Syt(lum)·Abs for longer time periods indicated a sustained increase in the rate of SV exocytosis in depolarizing media which lasted for at least 1 h. This increase was completely abolished by pretreating the neurons with tetanus toxin and this block correlated with a disappearance of synaptobrevin immunoreactivity. This radioimmunoassay therefore offers a new way to monitor SV exocytosis of neuronal populations in vitro irrespective of the type of neurotransmitter secreted and of postsynaptic effects.

Original languageEnglish
Pages (from-to)246-256
Number of pages11
JournalEuropean Journal of Cell Biology
Volume66
Issue number3
Publication statusPublished - 1995

Keywords

  • Cell culture
  • Hippocampal neurons
  • Nerve terminal
  • Neurotransmitter
  • Radioimmunoassay
  • Synaptic vesicle

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

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