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The spatial relationship of exocytosis and endocytosis in motor nerve terminals has been explored, with varied results, mostly in fixed preparations and without direct information on the utilization of each exocytic site. We sought to determine these spatial properties in real time using synaptopHluorin (spH) and FM4-64. Earlier we showed that nerve stimulation elicits the appearance of spH fluorescence hot spots, which mark preferred sites of exocytosis. Here we show that nerve stimulation in the presence of the styryl dye FM4-64 evokes hot spots of FM4-64 fluorescence. Their size, density, and rate of appearance are similar to the spH hot spots, but their rate of disappearance after stimulation was much slower (t(1/2) approximately 9 min vs approximately 10 s for spH hot spots), consistent with FM4-64 spots identifying bulk endocytosis and subsequent slow intracellular dispersion of nascent vesicles. Simultaneous imaging of both fluorophores revealed a strong colocalization of spH and FM4-64 spots, but only during high (100 Hz) stimulation. At 40 Hz stimulation, exocytic and endocytic spots did not colocalize. Our results are consistent with the hypothesis that hot spots of endocytosis, possibly in the form of bulk uptake, occur at or very near highly active exocytic sites during high-frequency stimulation.

Preferred Sites of Exocytosis and Endocytosis Colocalize during High- But Not Lower-Frequency Stimulation in Mouse Motor Nerve Terminals