Two types of exocytosis at retinal ribbon synapses and their functions in visual information processing

在感觉神经元的末端可以观察到带有突触小泡光环的突触带，这些突触带以强直的方式释放递质。因此，条带被认为是持续胞吐的先决条件。视网膜双极细胞在其终末有大量的带，并将输出发送到神经节和/或无长突细胞。在本研究中，我们应用全内反射荧光显微镜对全细胞电压钳下的金鱼视网膜Mb1双极细胞终末进行了观察，观察到了钙离子进入的部位、条带和囊泡的融合过程。结果发现，突触前L型钙离子电流的激活引起突触前囊泡的瞬间高速率融合，随后在突触带内发生低速融合，而在突触外则发生延迟性持续融合。蛋白激酶C(PKC)被佛波酯激活，特异性地增加了带状区域外的囊泡融合事件。囊泡融合事件的空间随机性检验表明，囊泡融合不是随机发生的，而是在这两个区域的“热点”发生的。对金鱼MB1双极细胞终末的电子显微镜观察显示，PKC激活选择性地增加了停靠在无带部位的小泡的数量，这些小泡面对具有突触后密度(PSD)的神经元突起。用连续超薄切片重建MB1双极细胞的整个终末，结果表明，具有二个突起的带状突触的数目几乎与具有单个突起的无带状突触的数目相当。这些结果表明，胞吐作用可能发生在有带和无带的活动区。视网膜神经节细胞瞬时和持续光反应的二分法可能部分归因于双极细胞终末的两种类型的活动区。

Synaptic ribbons with a halo of synaptic vesicles are observed at the terminal of sensory neurons that release transmitter tonically. Thus, ribbons are assumed to be prerequisite to sustained exocytosis. Retinal bipolar cells have plenty of ribbons in their terminal and send outputs to ganglion and/or amacrine cells. In the present study, applying total internal reflection fluorescence microscopy to goldfish retinal Mb1 bipolar cell terminals under whole-cell voltage clamp, we visualized Ca^2+ entry sites, ribbons, and vesicle fusion events. We found that main Ca^2+ entry sites were located at ribbons, and that activation of the presynaptic L-type Ca^2+ current induced a transient high-rate vesicle fusion followed by a low-rate fusion inside the ribbon region and a delayed sustained fusion outside the ribbon region. Activation of protein kinase C (PKC) by phorbol ester specifically increased vesicle fusion events outside the ribbon region. Test for spatial randomness of vesicle fusion events revealed that vesicle fusion occurred not randomly but at "hot spots" in both regions. Electron microscopic examination of goldfish Mb1 bipolar cell terminals revealed that PKC activation selectively increased the number of docked vesicles at ribbon-free sites, which faced neuronal processes with the postsynaptic density (PSD). Reconstruction of the whole terminal of an Mb1 bipolar cell from serial ultra-thin sections showed that the number of ribbon-associated synapses with dyad processes was almost comparable to that of ribbon-free synapses with single processes. These results indicate that exocytosis may occur at both ribbon-associated and ribbon-free active zones. Dichotomy of transient and sustained photoresponses in retinal ganglion cells may be partly ascribed to two types of active zones in bipolar cell terminals.