|dc.description.abstract||Synaptic vesicle recycling is critical for the maintenance and proper function of neurotransmission. Neurotransmission can proceed through action-potential evoked vesicle fusion where, upon depolarization, Ca2+ enters the nerve terminal though voltage-gated channels, interacts with vesicle-associated proteins to promote fusion with the terminal membrane, and causes release of vesicle contents. Neurotransmission can also occur spontaneously in the absence of stimulation, although this process is still Ca2+-dependent. Regardless of the mode of vesicle fusion, the vesicle lipids and protein components must be removed from the terminal membrane; the vesicle must be reconstituted and re-filled with neurotransmitter, so that it may ultimately be reused. Uncoupling the roles of Ca2+ in synaptic vesicle fusion and retrieval has been difficult to date as studies have relied on measurements of bulk synaptic vesicle retrieval. Here, to dissect the role of Ca2+ in these processes, we utilized low signal-to-noise pHluorin-tagged vesicular probes to monitor single synaptic vesicle recycling of both action-potential evoked and spontaneous fusion vesicles in rat hippocampal neurons.
We show that during stimulation, increasing extracellular Ca2+ increases synaptic vesicle fusion probability, but decreases the rate of synaptic vesicle retrieval. This negative regulation of synaptic vesicle retrieval is blocked by the Ca2+ chelation as well as inhibition of calcineurin, a Ca2+-calmodulin-dependent phosphatase. Indeed, the slow time course of aggregate synaptic vesicle retrieval detected during repetitive activity can be explained by a progressive decrease in the rate of synaptic vesicle retrieval during the stimulation train. These results indicate Ca2+ entry during single action potentials slows the pace of subsequent synaptic vesicle recycling. Conversely, we found that synaptic vesicles that undergo spontaneous fusion are retrieved very rapidly and this retrieval time is Ca2+-independent. Interestingly, we found that within a single synaptic bouton, the rate of spontaneous neurotransmission is independent of evoked fusion probability, suggesting there are fundamental regulatory differences between these forms of neurotransmission. Moreover, we found that the glycoprotein Reelin can act presynaptically to enhance spontaneous neurotransmission without affecting evoked neurotransmission by mobilizing a molecularly specific subset of synaptic vesicles. These data illustrate fundamental differences in vesicle recycling between modes of neurotransmission at the single-vesicle level.||en