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

Nature Protocols volume 9, pages 1337–1347 (2014)Cite this article

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Abstract

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.

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Figure 1: Overview of the experimental protocol.
Figure 2: Schematics of key equipment items.
Figure 3: Labeling functional synapses in acute slice.
Figure 4: Testing activity dependence of synaptic labeling.
Figure 5: Monitoring the progress of photoconversion.
Figure 6: Approach for embedding tissue in EPON.
Figure 7: Ultrastructural readout of functional vesicles.

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Acknowledgements

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.

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Authors and Affiliations

  1. School of Life Sciences, University of Sussex, Brighton, UK

    Vincenzo Marra, Freya Crawford & Kevin Staras

  2. Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK

    Vincenzo Marra

  3. Medical Research Council Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, London, UK

    Jemima J Burden

Authors
  1. Vincenzo Marra
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  2. Jemima J Burden
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Contributions

K.S. and V.M. conceived the method and wrote the paper. F.C. validated the key steps and provided some of the figures. J.J.B. helped develop sample processing methods and carried out serial sectioning.

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Correspondence to Kevin Staras.

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The authors declare no competing financial interests.

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Marra, V., Burden, J., Crawford, F. et al. Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion. Nat Protoc 9, 1337–1347 (2014). https://doi.org/10.1038/nprot.2014.088

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