Release of the styryl dyes from single synaptic vesicles in hippocampal neurons
Release of the styryl dyes from single synaptic vesicles in hippocampal neurons
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DOI：
10.1523/jneurosci.4518-07.2008
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发表时间：
2008-02-20
影响因子：
5.3
通讯作者：
Almers, Wolfhard
中科院分区：
文献类型：
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作者：
Chen, Xi;Barg, Sebastian;Almers, Wolfhard
在大脑中的小突触前小泡中，突触小泡在释放递质时被认为不会与质膜融合，而是关闭其融合孔并完好无损地存活以供将来使用（亲吻并运行胞吐作用）。这一观点最有力的证据是荧光膜标记 FM1-43 [N-(3-三乙基铵丙基)-4-(4-(二丁基氨基)苯乙烯基)吡啶鎓二溴化物]从单个囊泡中缓慢且不完全地释放。我们研究了在没有神经胶质细胞的盖玻片上生长的海马神经元稀疏培养物中 FM1-43 的释放。这使得突触前波顿能够以有利的信噪比成像。少量染色的纽扣以高时间分辨率成像，而高频电刺激引起胞吐作用。 FM1-43 的释放是量子的，并且以突然的步骤发生，每个步骤代表一个单一的聚变事件。沿着轴突行进的囊泡簇的荧光具有相同量子大小的分布，表明囊泡释放其包含的所有染料。在大多数融合事件中，染料释放的时间常数< 100 ms，并且很少观察到较慢的释放。胞吐作用后，在布顿任一侧的轴突中均未检测到 FM1-43，这表明染料在扩散之前已被释放。我们的结果与高频刺激期间突触小泡与质膜完全融合一致。
In small presynaptic boutons in brain, synaptic vesicles are thought not to merge with the plasma membrane when they release transmitter, but instead to close their fusion pores and survive intact for future use ( kiss-and-run exocytosis). The strongest evidence for this idea is the slow and incomplete release of the fluorescent membrane marker, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide], from single vesicles. We investigated the release of FM1-43 from sparse cultures of hippocampal neurons grown on coverslips with no glia. This allowed presynaptic boutons to be imaged at favorable signal-to-noise ratio. Sparingly stained boutons were imaged at high time resolution, while high-frequency electrical stimulation caused exocytosis. The release of FM1-43 was quantal and occurred in abrupt steps, each representing a single fusion event. The fluorescence of vesicle clusters traveling along axons had a distribution with the same quantal size, indicating that a vesicle releases all the dye it contains. In most fusion events, the time constant of dye release was < 100 ms, and slower release was rarely observed. After exocytosis, no FM1-43 could be detected in the axon to either side of a bouton, indicating that dye was released before it could spread. Our results are consistent with synaptic vesicles fusing fully with the plasma membrane during high-frequency stimulation.