Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve...

A study was made to determine the disposition of vesicle-associated proteins (syntaxin, SV2, SNAP-25) and calcium channels with respect to the spatial extent of spontaneous and evoked quantal release within regions of amphibian motor-nerve terminal branches delineated by FM1-43 stained vesicle clusters (blobs). Discrete concentrations of vesicles revealed approximately 2 microm apart along the length of terminal branches through FM1-43 staining were identical in size and spacing to those identified along terminal branches with SV2 antibody (AbSV2). Fluorescent antibodies to syntaxin 1 (AbS), SNAP-25 (AbS25) and the calcium channel alpha1B subunit (Abalpha1B) were found in relatively high concentrations coincident with the AbSV2 blobs. Three extracellular recording electrodes were placed in the vicinity of individual FM1-43 blobs, and an algorithm was used to determine the spatial origin of miniature endplate potentials (MEPPs) and EPPs together with their relative amplitudes. MEPPs and EPPs originated throughout the region stained by FM1-43 but not elsewhere; amplitude-frequency distributions of MEPPs and EPPs were similar for all FM1-43 blobs with average coefficients of variation of no less than 0.28. A linear relationship existed between the size of an FM1-43 blob, measured as the integrated extent of FM1-43 staining of a blob, and the frequency of MEPPs as well as the probability of EPPs from the blob. There was a proximo-distal gradient in the size of FM1-43 blobs along the length of single terminal branches, suggesting a gradient in release probability along the branches. The frequency distribution of the distances between blobs was approximately Gaussian, whereas the frequency distribution of the size of blobs was highly skewed and was best fitted with a gamma distribution. It is concluded that there are correlations among the extent of labeling of SNAP-25, syntaxin and calcium channels at a release site, the store of vesicles to be found there, and the probability of spontaneous and evoked quantal release.