The synaptic vesicle cycle is vital for sustained neurotransmitter release. It has been assumed that functional synaptic vesicles are replenished autonomously at individual presynaptic terminals. Here we tested this assumption by using FM dyes in combination with fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured rat hippocampal neurons. After photobleaching, synapses acquired recently recycled FM dye-labeled vesicles originating from nonphotobleached synapses by a process requiring dynamic actin turnover. The imported vesicles entered the functional pool at their host synapses, as revealed by the exocytic release of the dye upon stimulation. FM1-43 photoconversion and ultrastructural analysis confirmed the incorporation of imported vesicles into the presynaptic terminal, where they mixed with the native vesicle pools. Our results demonstrate that synaptic vesicle recycling is not confined to individual presynaptic terminals as is widely believed; rather, a substantial proportion of recycling vesicles are shared constitutively between boutons.

The arrival of an action potential at a synapse triggers neurotransmitter release with a limited probability, p(r). Although p(r) is a fundamental parameter in defining synaptic efficacy, it is not uniform across all synapses, and the mechanisms by which a given synapse sets its basal release probability are unknown. By measuring p(r) at single presynaptic terminals in connected pairs of hippocampal neurons, we show that neighboring synapses on the same dendritic branch have very similar release probabilities, and p(r) is negatively correlated with the number of synapses on the branch. Increasing dendritic depolarization elicits a homeostatic decrease in p(r), and equalizing activity in the dendrite significantly reduces its variability. Our results indicate that local dendritic activity is the major determinant of basal release probability, and we suggest that this feedback regulation might be required to maintain synapses in their operational range.

SummarySynapse-specific vesicle pools have been widely characterized at central terminals. Here, we demonstrate a vesicle pool that is not confined to a synapse but spans multiple terminals. Using fluorescence imaging, correlative electron microscopy, and modeling of vesicle dynamics, we show that some recycling pool vesicles at synapses form part of a larger vesicle “superpool.” The vesicles within this superpool are highly mobile and are rapidly exchanged between terminals (turnover: ∼4% of total pool/min), significantly changing vesicular composition at synapses over time. In acute hippocampal slices we show that the mobile vesicle pool is also a feature of native brain tissue. We also demonstrate that superpool vesicles are available to synapses during stimulation, providing an extension of the classical recycling pool. Experiments using focal BDNF application suggest the involvement of a local TrkB-receptor-dependent mechanism for synapse-specific regulation of presynaptic vesicle pools through control of vesicle release and capture to or from the extrasynaptic pool.