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Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion

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journal contribution
posted on 2019-02-21, 10:05 authored by V Marra, JJ Burden, F Crawford, K Staras
Fast activity-driven turnover of neurotransmitter-filled vesicles at presynaptic terminals is a crucial step in information transfer in the CNS. Characterization of the relationship between the nanoscale organization of synaptic vesicles and their functional properties during transmission is currently of interest. Here we outline a procedure for ultrastructural investigation of functional vesicles in synapses from native mammalian brain tissue. FM dye is injected into the target region of a brain slice and upstream axons are electrically activated to stimulate vesicle turnover and dye uptake. In the presence of diaminobenzidine (DAB), photoactivation of dye-filled vesicles yields an osmiophilic precipitate that is visible in electron micrographs. When combined with serial-section electron microscopy, fundamental ultrastructure-function relationships of presynaptic terminals in native circuits are revealed. We outline the utility of this protocol for the 3D reconstruction of a recycling vesicle pool in CA3-CA1 synapses from an acute hippocampal slice and for the characterization of its anatomically defined docked pool. This protocol requires 6-7 d.

Funding

This work was supported by Wellcome Trust (WT084357MF), Biotechnology and Biological Sciences Research Council (BBSRC) (BB/K019015/1), Medical Research Council (MRC) (MR/K004999/1) and European Union (EU) (FP7-308943) grants to K.S.

History

Citation

Nat Protoc, 2014, 9 (6), pp. 1337-1347

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Neuroscience, Psychology and Behaviour

Version

  • AM (Accepted Manuscript)

Published in

Nat Protoc

Publisher

Nature Research

eissn

1750-2799

Copyright date

2014

Available date

2019-02-21

Publisher version

https://www.nature.com/articles/nprot.2014.088

Language

en

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