Structural basis for the spotted appearance of amphibian neuromuscular junctions stained for synaptic vesicles

We have compared the distribution of vesicles in amphibian motor nerve terminals determined by electron microscopy and by functional labeling with the styryl dye, FM2-10. Our aim was to resolve apparent discrepancies in the literature on the distribution of vesicles determined by the two procedures. Electron photomicrographs of non-serial cross sections of terminal branches were analyzed by stereological procedures to obtain indices of the terminal and vesicle areas. Terminal cross sectional area varied 3-fold on average along terminal branches and was largest particularly when active zone was present in the section. The vesicle area index (a measure of vesicle abundance) was highly correlated with the terminal area index, suggesting that the average density of vesicles is constant throughout the branches. When the data were separated according to whether active zone was present or not in a section, we found a small (26%) but significant increase in the average density of vesicles in active zone compared with non-active zone regions in the terminal. The distribution of spots along terminal branches following vesicle staining with FM2-10, as well as with antibodies to vesicle proteins, suggested that vesicles were distributed in highly concentrated clusters. However, the degree of variation between spot and inter-spot staining intensities found with the FM-dye was similar in magnitude to that for terminal cross sectional area determined from the electron microscopy. We conclude that the spotty pattern of stained vesicles seen with the optical microscope results primarily from vesicle accumulations associated with terminal varicosities.