Synaptic vesicle transport revealed by electrophysiological and imaging studies

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Toward the aim of understanding synaptic vesicles (SVs) transport mechanisms, our first attempt of arresting the vesicle docking through the block of endocytic release site clearance didn’t show any delay in the recovery from short term depression (STD) upon use of endocytosis inhibitors (Dynasore and Pitstop 2) physiologically. These findings are in sharp contrast to the previous report (Hosoi et al., Neuron. 2009).1) Since inhibition of endocytic proteins by Dynasore and Pitstop-2 resulted decrease of synaptic strength by nearly half, with an ultrafast onset of 10 milliseconds. This fast inhibition cannot be explained by the common belief that clathrin mediated slow endocytosis underlies the release site clearance effect, and some fast endocytic form likely is involved. To test this, at 37C and in 2.0 mM calcium, using endocytic inhibitors (Dynasore, Dynamin1 PRD peptide and Pitstop 2) we found all these blockers effectively block fast endocytosis in addition to slow endocytosis.2) Next, we tested the physiological implication of blocking the active zone scaffold protein intersectin, inhibition of which has been shown to strongly delay the fast time course of recovery from STD at the calyx of Held (Sakaba et al., PNAS. 2013).3) To inhibit the intersectin pathway, we targeted its downstream effectors, CDC42 and F actin, by use of ML141 and Latrunculin B, respectively. First, we tested the effect of these drugs on endocytosis.Neither of the blockers had any effect on endocytic recovery. Both drugs strongly enhanced the STD, without altering the recovery from STD.