Spatial variability in release at the frog neuromuscular junction measuredwith FM1-43

We quantified the spatial variability in release properties at different sy naptic vesicle clusters in frog motor nerve terminals, using a combination of fluorescence and electron microscopy. Individual synaptic vesicle cluste rs labeled with FM1-43 varied more than 10-fold in initial intensity (integ rated FM1-43 fluorescence) and in absolute rate of dye loss during tetanic electrical nerve stimulation. Most of this variability arose because large vesicle clusters spanned more than one presynaptic active zone (inferred fr om postsynaptic acetylcholine receptor stripes labeled with rhodamine-conju gated alpha-bungarotoxin); when the rate of dye loss was normalized to the length of receptor stripe covered, variability from spot to spot was greatl y reduced. In addition, electron microscopic measurements showed that large vesicle clusters (i.e., those spanning multiple active zones) were also th icker, and the increased depth of vesicles led to increased total spot fluo rescence without a corresponding increase in the rate of dye loss during st imulation. These results did not reveal the presence of "hot zones" of secr etory activity.