Using the Fluorescent Styryl Dye FM1-43 to Visualize Synaptic Vesicles Exocytosis and Endocytosis in Motor Nerve Terminals

The styryl dye FM1-43 is a powerful tool to track exocytosis, endocytosis and recycling of secretory granules or vesicles. Due to its unique structure, dye molecules reversibly partition into the outer leaflet of surface membrane without permeating due to two cationic charges located in their headgroup. When a secretory cell is stimulated to evoke exocytosis, FM1-43 molecules that were inserted in the membrane are internalized during compensatory endocytosis and newly formed secretory granules or vesicles become stained with dye (staining/endocytosis). If stained secretory granules or vesicles undergo exocytosis in dye-free medium, due to concentration gradient, FM1-43 molecules dissociate from the membrane and loose fluorescence (destaining/exocytosis). Using a fluorescence microscope attached to a CCD camera or a confocal, it is possible to follow secretion in live cell or tissue preparations and in this chapter, we will make a description of FM1-43 staining and destaining protocol using the neuromuscular junction as experimental model. This technique has allowed answering important questions concerning synaptic vesicle recycling, which is a key step for neuronal communication. In addition, FM1-43 has proven to be an excellent tool for investigating membrane internalization and endosome recycling in a variety of cell types.