Synaptic vesicle transport revealed by electrophysiological and imaging studies

为了了解突触囊泡（SV）的转运机制，我们首次尝试通过阻断内吞释放位点清除来阻止囊泡对接，在生理上使用内吞抑制剂（Dynasore和Pitstop 2）后，短期抑制（STD）的恢复没有任何延迟。这些发现与先前的报告（Hoblett等人，Neuron. 1）由于Dynasore和Pitstop-2对内吞蛋白的抑制导致突触强度降低近一半，具有10毫秒的超快起始。这种快速抑制不能用网格蛋白介导的缓慢内吞作用是释放位点清除效应的基础这一共同信念来解释，并且可能涉及一些快速内吞形式。为了测试这一点，在37 ℃和2.0 mM钙中，使用内吞抑制剂（Dynasore、Dynamin 1 PRD肽和Pitstop 2）我们发现所有这些阻断剂除了缓慢的内吞作用之外还有效地阻断快速的内吞作用。2）接下来，我们测试了阻断活性区支架蛋白intersectin的生理意义，已经显示抑制它强烈地延迟了从Held肾盏的STD恢复的快速时程（Sakaba等，PNAS. 3）为了抑制intersectin途径，我们分别通过使用ML 141和Latrunculin B靶向其下游效应子CDC 42和F肌动蛋白。首先，我们测试了这些药物对内吞作用的影响。两种阻断剂对内吞恢复都没有任何影响。 这两种药物都强烈地增强了STD，而不改变STD的恢复。

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.