Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system

Following the fusion of synaptic vesicles with the presynaptic plasma membr ane of nerve terminals by the process of exocytosis, synaptic-vesicle compo nents are recycled to replenish the vesicle pool. Here we use a pl l-sensit ive green fluorescent protein to measure the residence time of VAMP, a vesi cle-associated SNARE protein important for membrane fusion, on the surfaces of synaptic terminals of hippocampal neurons following exocytosis. The tim e course of VAMP retrieval depends linearly on the amount of VAMP that is a dded to the plasma membrane, with retrieval occurring between about 4 secon ds and 90 seconds after exocytosis, and newly internalized vesicles are rap idly acidified. These data are well described by a model in which endocytos is appears to be saturable, but proceeds with an initial maximum velocity o f about one vesicle per second. We also find that, following exocytosis, a portion of the newly inserted VAMP appears on the surface of the axon.