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

点击翻译按钮获取中文摘要

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.