Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses.

We have investigated mechanisms in postendocytic processing of synaptic vesicles at hippocampal synapses, using synaptobrevin/vesicle-associated membrane protein (VAMP) tagged with variants of the green fluorescent protein. Following exocytosis, VAMP is retrieved at synaptic and adjoining axonal regions. Retrieved VAMP-containing vesicles return to synaptic vesicle clusters at a rate slower than endocytosis. Vesicles containing a different protein, synaptophysin, recluster at a similar rate, suggesting common vesicular intermediates for the two proteins. Activity prolongs the time taken by endocytosed vesicles to return to synapses. Exogenous calcium buffers slow endocytosis but have no additional effect on the time course of reclustering. In contrast, the protein kinase inhibitor staurosporine does not affect endocytosis but slows reclustering. Finally, since VAMP can move freely on surface membranes, sustained synaptic activity leads to mixing of this vesicular component between adjacent synapses.