br Visualizing single synaptic vesicle exocytosis at CNS syn

Visualizing single synaptic vesicle A-71623 at CNS synapses We visualized the dynamics of individual synaptic vesicles as they fuse and immediately before fusion at the mammalian conventional synapses for the first time (Midorikawa and Sakaba, 2015). We use a calyx of Held terminal, a large presynaptic terminal in the auditory pathway at the brainstem, as a preparation (Forsythe, 1994). Normally neuronal presynaptic terminals are so small and stuck tightly to the postsynaptic side of the synapse, which makes it difficult to isolate the presynaptic terminal and let the active zones face and adhere to the glass coverslip. A calyx of Held terminal is large enough to amenable whole-cell clamp recording, and thus has been studied extensively as a model CNS synapse by electrophysiology (Borst and Sakmann, 1996; Takahashi et al., 1996; Schneggenburger et al., 1999; Wu and Borst, 1999). We managed to dissociate the terminals and attach them to coverslips, thus overcame this technical issue. When a terminal was observed under TIRFM illumination, FM-labeled synaptic vesicles were visualized as bright spots, and owing to the sparse labeling and thin illumination, most of spots appeared as isolated spots. These spots had the diffraction limited size (∼200 nm), which supported the idea that these were single synaptic vesicles. The activity of the calyx terminal was controlled by presynaptic voltage clamp recording, and the net amount of exocytosis was measured by capacitance measurement technique simultaneously with TIRFM imaging. When a 100-ms depolarization was applied while the FM-labeled synaptic vesicles were visualized under the TIRFM, some of the vesicles disappeared during the depolarization. The disappearance of the spots often accompanied with spreading of the dye into the surrounding plasma membrane by lateral diffusion. These disappearance events were highly synchronized to the stimulation, which strongly indicated that these events reflected fusion. The synchrony can be evaluated in tens of milliseconds order (frame rate of image acquisitions) owing to the precise control of stimulation periods by voltage clamp. There, it was shown that only those vesicles, which had been stably tethered within the evanescent field at the plasma membrane before the stimulus, can be released during and up to 1 s after a pool-depleting stimulus. Surprisingly, newcomers – vesicles, which are first appeared after the step-like stimulus – are not released within a second of exposure to high [Ca2+]. This was surprising, since vesicle tethering, docking, and priming are generally believed to be rate-limited by tethering (for review, Neher and Sakaba, 2008; Hallermann and Silver, 2013). Also, newcomers are suggested to be released rapidly in some types of synapses (Griesinger et al., 2005; Chen et al., 2013). In addition to disappearance events, there are appearance events which appear during the recording and stay thereafter. These events indicate the approaching and subsequent anchoring of the vesicles into the evanescent field, thus should represent tethering of newly approached vesicles nearby the plasma membrane. By comparing the time course of fusion events and tethering events, it was suggested that tethering of new vesicles could refill the empty release sites with a time constant of ∼4 s. This is also surprising since the replenishment of the readily releasable vesicles (RRVs) at the calyx of Held terminal was estimated to be ∼300 ms by the electrophysiological experiments (Neher and Sakaba, 2008; Hallermann and Silver, 2013). Given that a depolarization elicit fusions of only a small fraction of already-tethered vesicles in TIRFM imaging, the discrepancy suggests that the replenishment time course was determined by the step after the tethering, i.e. priming. The rate of tethering vesicles was not affected by changing the intracellular calcium buffer condition from 0.5 EGTA to 5 mM EGTA, thus the tethering rate was not sensitive to intracellular free calcium concentrations.