Preferred Sites of Exocytosis and Endocytosis Colocalize during High- But Not Lower-Frequency Stimulation in Mouse Motor Nerve Terminals
Preferred Sites of Exocytosis and Endocytosis Colocalize during High- But Not Lower-Frequency Stimulation in Mouse Motor Nerve Terminals
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DOI：
10.1523/jneurosci.4646-09.2009
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发表时间：
2009-12-02
影响因子：
5.3
通讯作者：
Betz, William J.
中科院分区：
文献类型：
--
作者：
Gaffield, Michael A.;Tabares, Lucia;Betz, William J.
运动神经末梢胞吐和内吞作用的空间关系已被探索，结果不一，大多是固定的制剂，没有关于每个胞吐部位利用的直接信息。我们试图利用synaptopHluorin （spH）和FM4-64实时测定这些空间特性。早些时候我们发现，神经刺激引起spH荧光热点的出现，这标志着胞吐的首选部位。在这里，我们发现苯乙烯染料FM4-64存在下的神经刺激唤起了FM4-64荧光的热点。它们的大小、密度和出现速度与spH热点相似，但它们在刺激后的消失速度要慢得多（t（1/2）类似于9分钟，而spH热点类似于10秒），与FM4-64斑点一致，这些斑点表明新生囊泡的大量内吞和随后缓慢的细胞内弥散。两种荧光团的同时成像显示spH和FM4-64点的强共定位，但仅在高（100 Hz）刺激下。在40 Hz的刺激下，胞外点和内吞点没有共定位。我们的结果与假设一致，即在高频刺激期间，内吞作用的热点可能以大量摄取的形式发生在或非常接近高度活跃的胞外部位。
The spatial relationship of exocytosis and endocytosis in motor nerve terminals has been explored, with varied results, mostly in fixed preparations and without direct information on the utilization of each exocytic site. We sought to determine these spatial properties in real time using synaptopHluorin (spH) and FM4-64. Earlier we showed that nerve stimulation elicits the appearance of spH fluorescence hot spots, which mark preferred sites of exocytosis. Here we show that nerve stimulation in the presence of the styryl dye FM4-64 evokes hot spots of FM4-64 fluorescence. Their size, density, and rate of appearance are similar to the spH hot spots, but their rate of disappearance after stimulation was much slower (t(1/2) similar to 9 min vs similar to 10 s for spH hot spots), consistent with FM4-64 spots identifying bulk endocytosis and subsequent slow intracellular dispersion of nascent vesicles. Simultaneous imaging of both fluorophores revealed a strong colocalization of spH and FM4-64 spots, but only during high (100 Hz) stimulation. At 40 Hz stimulation, exocytic and endocytic spots did not colocalize. Our results are consistent with the hypothesis that hot spots of endocytosis, possibly in the form of bulk uptake, occur at or very near highly active exocytic sites during high-frequency stimulation.