Synaptic vesicle transport revealed by electrophysiological and imaging studies

电生理学和成像研究揭示突触小泡运输

• 批准号: 21K06445
• 负责人: MAHAPATRA Satyajit
• 金额: $ 2.58万
• 依托单位: Okinawa Institute of Science and Technology Graduate University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2021
• 资助国家: 日本
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21K06445/
• 关键词: Vesicle transport; The calyx of Held; Hippocampal CA1 synapse; Endocytosis; Release-site clearance; Synaptic depression; Recovery from STD; Physiological condition; SV transport; STD; Synaptic vesicles (SVs); Release site clearance; Calyx of H

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.

为了了解突触小泡 (SV) 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小泡对接，但在生理上使用内吞抑制剂（Dynasore 和 Pitstop 2）后，短期抑制 (STD) 的恢复并未出现任何延迟。这些发现与之前的报告形成鲜明对比（Hosoi 等人，Neuron. 2009）。1) 由于 Dynasore 和 Pitstop-2 抑制内吞蛋白导致突触强度降低近一半，且起效时间超快，为 10 毫秒。这种快速抑制不能用普遍的观点来解释，即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，并且可能涉及一些快速内吞形式。为了测试这一点，在 37°C 和 2.0 mM 钙条件下，使用内吞抑制剂（Dynasore、Dynamin1 PRD 肽和 Pitstop 2），我们发现所有这些阻断剂除了慢速内吞作用外，还可以有效阻断快速内吞作用。2) 接下来，我们测试了阻断活性区支架蛋白 intersectin 的生理学意义，抑制活性区支架蛋白 intersectin 已被证明可以强烈延迟从细胞中恢复的快速时间过程。 Held 花萼处的 STD（Sakaba 等人，PNAS. 2013）。3) 为了抑制 Intersectin 通路，我们分别使用 ML141 和 Latrunculin B 靶向其下游效应子 CDC42 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。这两种阻滞剂对内吞恢复都没有任何影响。 这两种药物都能强烈增强性传播疾病，但不会影响性传播疾病的恢复。

项目成果

Physiological impact of release-site vesicle recruitment on central excitatory transmissions

释放位点囊泡募集对中枢兴奋性传递的生理影响

• 发表时间: 2021
• 作者: Tomioka Ryohei;Takemoto Makoto;Song Wen-Jie;Satyajit Mahapatra and Tomoyuki Takahashi;冨岡良平 周博 宋文杰;Satyajit Mahapatra and Tomoyuki Takahashi
• 通讯作者: Satyajit Mahapatra and Tomoyuki Takahashi

Release-site clearance and rapid vesicle replenishment at rodent central synapses

啮齿动物中央突触的释放位点清除和快速囊泡补充

• 发表时间: 2022
• 作者: Tomioka Ryohei;Takemoto Makoto;Song Wen-Jie;Satyajit Mahapatra and Tomoyuki Takahashi
• 通讯作者: Satyajit Mahapatra and Tomoyuki Takahashi