Imaging synaptic vesicle exocytosis and endocytosis with FM dyes

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.

Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells

“…To determine whether aggregates were internalized by endocytosis, cells were concomitantly labeled with the fixable membrane FM 4-64FX dye that reveals endocytic vesicles (22) and inoculated with mutant SOD1 aggregates for various periods of time. Five minutes after inoculation, aggregates were clearly colocalized with the endocytic membranes ( Fig.…”

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

“…An alternative approach is to combine a functional measure of vesicle pools with subsequent ultrastructural investigation. One of the most widelyused methods exploits FM-dyes, fluorescent reporters that readily bind to lipid membrane and are taken up into recycling vesicles during endocytosis to provide a functional readout of vesicle turnover [21][22][23] . One dye variant, FM1-43FX, also efficiently drives the polymerization of diaminobenzidine (DAB) when photoactivated, leading to the formation of an osmiophilic precipitate [24][25][26][27][28][29][30][31] .…”

Synaptic Glutamate Release Is Modulated by the Na+-Driven Cl−/HCO3−Exchanger Slc4a8

“…For analysis of presynaptic vesicle release in WT and KO hippocampal neurons, synaptic vesicles were labeled with a fluorescent FM dye. Vesicle exocytosis was investigated upon electrical field stimulation by quantification of fluorescence intensity decline (Gaffield and Betz, 2006).…”