Synaptic strength and reliability are determined by the number of vesicles released per action potential and the availability of release-competent vesicles in the readily releasable pool (RRP). Compared with release of a single vesicle (univesicular release), multivesicular release (MVR) would speed up RRP depletion, yet whether the RRP is refilled differently during the two different release modes has not been investigated. Here, we address this question by quantitative optical imaging with an axon-targeting glutamate sensor, iGluSnFRpre. We found that hippocampal synapses preferentially release multiple vesicles per action potential at high extracellular calcium or by paired-pulse stimulation. When MVR prevails, the RRP is recovered very rapidly with a time constant of 430 ms. This rapid recovery is mediated by dynamin-dependent endocytosis followed by direct reuse of retrieved vesicles. Furthermore, our simulation proved that the portion of retrieved vesicles that directly refill the RRP increases dramatically (>70%) in MVR compared with that in univesicular release (<10%). These results suggest that the contribution of rapid and direct recruitment of retrieved vesicle to the RRP changes dynamically with release mode at the level of individual synapses, which suggests a form of presynaptic homeostatic plasticity for reliable synaptic transmission during various synaptic activity.

SIGNIFICANCE STATEMENT The number of vesicles released in response to an action potential and the number of release competent vesicles in the readily releasable pool (RRP) are the fundamental determinants of synaptic efficacy. Despite its functional advantages, releasing multiple vesicles, especially at small synapses, can deplete the RRP after a couple of action potentials. To prevent failure of synaptic transmission, the RRP should be refilled rapidly, yet whether the RRP replenishment process is regulated by the release mode has not been investigated. Here, using quantitative optical glutamate imaging and simulation, we demonstrate that the contribution of the fast refilling mechanism changes with release mode at the level of individual synapses, suggesting a rapid form of presynaptic homeostatic plasticity during various synaptic activity.

突触强度和可靠性取决于每个动作电位释放的囊泡数量以及易释放池（RRP）中具有释放能力的囊泡的可用性。与单个囊泡的释放（单囊泡释放）相比，多囊泡释放（MVR）会加快RRP消耗，但尚未研究在两种不同释放模式下RRP是否被不同地补充。在这里，我们通过使用轴突靶向谷氨酸传感器iGluSnFRpre进行定量光学成像解决了这个问题。我们发现，在高细胞外钙或通过成对脉冲刺激，每个动作电位海马突触优先释放多个囊泡。当使用MVR时，RRP以430毫秒的时间常数非常快速地恢复。这种快速恢复是由动力蛋白依赖的内吞作用介导的，然后直接重新利用回收的囊泡。此外，我们的模拟证明，与非囊泡释放（<10％）相比，MVR中直接补充RRP的回收囊泡部分显着增加（> 70％）。这些结果表明，在单个突触水平上，快速而直接募集的囊泡对RRP的贡献随着释放模式的变化而动态变化，这暗示了突触前稳态可塑性的形式，可在各种突触活动期间可靠地进行突触传递。10％）。这些结果表明，在单个突触水平上，快速而直接募集的囊泡对RRP的贡献随着释放模式的变化而动态变化，这暗示了突触前稳态可塑性的形式，可在各种突触活动期间可靠地进行突触传递。10％）。这些结果表明，在单个突触水平上，快速而直接募集的囊泡对RRP的贡献随着释放模式的变化而动态变化，这暗示了突触前稳态可塑性的形式，可在各种突触活动期间可靠地进行突触传递。

重要性声明响应动作电位而释放的囊泡的数量和易释放库（RRP）中能释放的囊泡的数量是突触功效的基本决定因素。尽管具有功能优势，但释放多个囊泡（尤其是在较小的突触处）可能会在几个动作电位后耗尽RRP。为防止突触传递失败，应快速补充RRP，但尚未研究RRP补充过程是否受释放模式调节。在这里，使用定量光学谷氨酸成像和模拟，我们证明了快速再填充机制的贡献随单个突触水平上的释放模式而变化，表明在各种突触活动期间突触前稳态可塑性的快速形式。