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Imaging synaptic vesicle exocytosis and endocytosis with FM dyes

Nature Protocols volume 1, pages 2916–2921 (2006)Cite this article

Abstract

FM dyes have been used to label and then monitor synaptic vesicles, secretory granules and other endocytic structures in a variety of preparations. Here, we describe the general procedure for using FM dyes to study endosomal trafficking in general, and synaptic vesicle recycling in particular. The dye, dissolved in normal saline solution, is added to a chamber containing the preparation to be labeled. Stimulation evokes exocytosis, and compensatory endocytosis that follows traps FM dye inside the retrieved vesicles. The extracellular dye is then washed from the chamber, and labeled endocytic structures are examined with a fluorescence microscope. Fluorescence intensity provides a direct measure of the labeled vesicle number, a good measure of the amount of exocytosis. If the preparation is stimulated again, without dye in the chamber, dimming of the preparation provides a measure of exocytosis of labeled vesicles. With a synaptic preparation on hand, this protocol requires 1 day.

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Figure 1: FM 1-43 structure.
Figure 2: Typical FM dye experiment.
Figure 3: FM dye-labeled synaptic vesicles in nerve terminals.
Figure 4: Expected destain rates.

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Acknowledgements

This work is supported by grants from MDA and NIH to W.J.B. We thank Steve Fadul for technical assistance, Michael Grybko for discussions on brain slices, Dr Silvio Rizzoli for discussions on phototoxicity, and Dr Joe Johnson and Dr Leah Sheridan for discussions on the text.

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Authors and Affiliations

  1. Neuroscience Program, University of Colorado Medical School, Aurora, 80045, Colorado, USA

    Michael A Gaffield

  2. Department of Physiology & Biophysics, University of Colorado Medical School, Aurora, 80045, Colorado, USA

    William J Betz

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  1. Michael A Gaffield
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Correspondence to William J Betz.

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Supplementary information

Supplementary Video 1

FM 1-43 destain in frog motor nerve terminal. The nerve was stimulated at 30 Hz. One revolution of the clock represents one minute. (AVI 1363 kb)

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Gaffield, M., Betz, W. Imaging synaptic vesicle exocytosis and endocytosis with FM dyes. Nat Protoc 1, 2916–2921 (2006). https://doi.org/10.1038/nprot.2006.476

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