Ca2+-dependent synaptic vesicle recycling is critical for maintenance of neurotransmission. However, uncoupling the roles of Ca2+ in synaptic vesicle fusion and retrieval has been difficult, as studies probing the role of Ca2+in endocytosis relied on measurements of bulk synaptic vesicle retrieval. Here, to dissect the role of Ca2+ in these processes, we used a low signal-to-noise pHluorin-tagged vesicular probe to monitor single synaptic vesicle recycling in rat hippocampal neurons. We show that Ca2+ increases synaptic vesicle fusion probability in the classical sense, but surprisingly decreases the rate of synaptic vesicle retrieval. This negative regulation of synaptic vesicle retrieval is blocked by the Ca2+chelator, EGTA, as well as FK506, a specific inhibitor of Ca2+-calmodulin-dependent phosphatase calcineurin. The slow time course of aggregate synaptic vesicle retrieval detected during repetitive activity could be explained by a progressive decrease in the rate of synaptic vesicle retrieval during the stimulation train. These results indicate that Ca2+ entry during single action potentials slows the pace of subsequent synaptic vesicle recycling.
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