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

我们研究了在突触前神经末梢激活钙通道后，细胞内钙离子和质膜附近（<150 nm）突触小泡的动力学。荧光探针的图像由配备有高速图像增强CCD相机的渐逝显微镜捕获。从金鱼视网膜分离的开型双极细胞用填充有Ca指示剂Fluo-4FF的贴片移液管进行全细胞电压钳位。我们测量了钙电流、胞吐相关的膜电容变化以及轴突末端荧光强度的时空变化。钙电流的激活诱导了荧光强度的斑片状增加。这些明亮的补丁不诱发细胞外钴离子的存在下，因此确定为钙域。在激活Ca电流时，Ca结构域中心的荧光强度迅速增加，但在其外围区域（约1.5 μ m）延迟。距离中心500 nm）。然而，这种延迟不足以解释胞吐作用的即时和晚期成分之间的延迟。这种差异可能归因于来自成像系统的伪影或位于Ca结构域中心区域的突触囊泡与位于其周边区域的突触囊泡之间的Ca敏感性差异。为了研究突触小泡的动力学，在胞吞过程中将FM 1 -43插入突触小泡的膜中。消逝显微镜显示多个亮点。这些斑点大多数是由单个突触囊泡发出的荧光。我们可以区分胞吐的囊泡和那些从质膜上移开的囊泡。前者是在钙电流激活过程中观察到的，而后者是自发发生的，与钙电流无关。

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.