Graduate School of Brain Science, Doshisha University, Kyoto 6100394, Japan.
Neuron. 2015 Nov 4;88(3):492-8. doi: 10.1016/j.neuron.2015.09.047.
Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses.
突触小泡与活性区相连，在那里它们被停靠/引发，以便在 Ca(2+)内流时能够迅速融合。为了在中枢神经系统突触前末端直接研究这些步骤，我们在活体分离的 Held 终球顶端使用全内反射荧光（TIRF）显微镜，并测量了刚好在质膜下的单个突触小泡的运动。TIRF 场中的只有一部分囊泡发生胞吐作用。胞吐作用后，新的囊泡（新成员）靠近膜并以几秒钟的时间常数缓慢重新填充释放位点。使用闪光光解均匀升高细胞内 Ca(2+)会引发胞吐爆发，随后是持续成分，分别代表可释放囊泡和囊泡补充的释放。令人惊讶的是，新成员在高 Ca(2+)的情况下不会在一秒内被释放。相反，已经连接的囊泡变得可以释放，并介导补充。我们的结果揭示了传统突触的一个重要特征。
