FM1-43 DYE ULTRASTRUCTURAL-LOCALIZATION IN AND RELEASE FROM FROG MOTOR-NERVE TERMINALS

Previous work has shown that the fluorescent styryl dye FM1-43 stains nerve terminals in an activity-dependent fashion, This dye appears to label the membranes of recycled synaptic vesicles by being trapped dur ing endocytosis. Stained terminals can subsequently be destained by re peating nerve stimulation in the absence of dye; the destaining eviden tly reflects escape of dye into the bathing medium from membranes of e xocytosing synaptic vesicles, In the present study we tested two key a spects of this interpretation of FM1-43 behavior, namely: (i) that the dye is localized in synaptic vesicles, and (ii) that it is actually r eleased into the bathing medium during destaining. To accomplish this, we first photolyzed the internalized dye in the presence of diaminobe nzidine, This created an electron-dense reaction product that could be visualized in the electron microscope, Reaction product was confined to synaptic vesicles, as predicted. Second, using spectrofluorometry, we quantified the release of dye liberated Into the medium from tubocu rarine-treated nerve-muscle preparations. Nerve stimulation increased the amount of FM1-43 released, and we estimate that normally a stained synaptic vesicle contains a few hundred molecules of the dye. The key to the successful detection of released FM1-43 was to add the micelle -forming detergent holamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which increased FM1-43 quantum yield by more than two orders of magnitude.