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Dynamics of Ca ions and synaptic vesicles at the presynaptic membrane region of retinal bipolar cells

视网膜双极细胞突触前膜区Ca离子和突触小泡的动力学

基本信息

批准号：
14380375
负责人：
TACHIBANA Masao
金额：
$ 9.66万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380375/
关键词：
retina synapse Ca ion exocytosis bipolar cell synaptic vesicle Ca current transmitter 伝達物質放出 Caチャネル Caイメージング

项目摘要

We investigated the dynamics of intracellular Ca ions and synaptic vesicles in the vicinity of the plasma membrane (<150 nm) at presynaptic nerve terminals upon activation of Ca channels. Images of fluorescent probes were captured by the evanescent microscope equipped with a high speed, image intensified CCD camera. On-type bipolar cells isolated from the goldfish retina were whole cell voltage clamped with a patch pipette filled with a Ca indicator Fluo-4FF. We measured the Ca current, the membrane capacitance changes associated with exocytosis, and the spatial temporal changes of the fluorescence intensity at the axon terminal, Activation of the Ca current induced patch like increase in the fluorescence intensity. These bright patches were not evoked in the presence of extracellular Co ions, and thus identified as the Ca domains. Upon activation of the Ca current the fluoresc nce intensity was rapidly increased at the center of the Ca domains but delayed at their peripheral region (ca. 500 nm away from the center). However, such delay was not long enough to explain the delay between the immediate and late components of exocytosis. This discrepancy may be ascribed to an artifact derived from the imaging system or to a difference of Ca sensitivitbetween synaptic vesicles located at the central region of the Ca domains and those at their peripheral region. To investigate the dynamics of synaptic vesicles, FM1-43 was inserted into the membrane of synaptic vesicles during endocytosis. Evanescence microscopy revealed multiple bright spots. Most of these spots were fluorescence emitted from single synaptic vesicles. We could differentiate between the exocytosed vesicles and those moved away from the plasma membrane. The former was observed during activation of the Ca current, while the latter happened spontaneously irrespective of the Ca current.

我们研究了 Ca 通道激活后突触前神经末梢质膜 (<150 nm) 附近的细胞内 Ca 离子和突触小泡的动力学。荧光探针的图像由配备高速图像增强 CCD 相机的倏逝显微镜捕获。从金鱼视网膜分离的 On 型双极细胞用装有 Ca 指示剂 Fluo-4FF 的贴片吸管钳住全细胞电压。我们测量了 Ca 电流、与胞吐作用相关的膜电容变化以及轴突末端荧光强度的时空变化，Ca 电流的激活诱导荧光强度的斑块状增加。这些明亮的斑块在细胞外 Co 离子存在的情况下不会被诱发，因此被识别为 Ca 域。激活 Ca 电流后，Ca 域中心的荧光强度迅速增加，但在其外围区域（距中心约 500 nm）延迟。然而，这种延迟的时间不足以解释胞吐作用的即时成分和晚期成分之间的延迟。这种差异可能归因于成像系统产生的伪影或位于 Ca 域中心区域的突触小泡与其周围区域的突触小泡之间的 Ca 敏感性差异。为了研究突触小泡的动力学，在内吞作用期间将 FM1-43 插入突触小泡的膜中。消逝显微镜显示出多个亮点。这些斑点大多数是单个突触小泡发出的荧光。我们可以区分胞吐的囊泡和远离质膜的囊泡。前者是在 Ca 电流激活期间观察到的，而后者是自发发生的，与 Ca 电流无关。