Analysis of neurotransmitter release mechanisms by photolysis of caged Ca2+ in an autaptic neuron culture system
Analysis of neurotransmitter release mechanisms by photolysis of caged Ca2+ in an autaptic neuron culture system
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DOI：
10.1038/nprot.2012.074
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发表时间：
2012-07-01
期刊：
影响因子：
14.8
通讯作者：
Rhee, Jeong-Seop
中科院分区：
文献类型：
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作者：
Burgalossi, Andrea;Jung, SangYong;Rhee, Jeong-Seop
神经递质的释放是由膜去极化、Ca 2+内流和释放机制的Ca 2+感应触发的，导致突触囊泡（SV）与质膜融合。相互连接是一个复杂的膜循环，其中囊泡被拴在释放位点，引发，融合和回收。由于这些过程中有许多是钙离子依赖性的，并且同时发生，因此很难在实验上对它们进行剖析。这个问题可以部分地规避通过控制突触的Ca 2+浓度通过UV光解笼中的Ca 2+。我们开发了一种培养方案的基础上产生的小神经胶质细胞岛上的单个神经元，这是足够小，被紫外线闪光灯覆盖的Ca 2 + uncaging在小突触。神经元装载有对光不稳定的Ca 2+螯合剂硝基苯基-EGTA和Ca 2+指示剂，并且使用UV闪光来触发Ca 2 +-解除包裹和SV融合。该协议需要三个星期才能完成，并提供了前所未有的见解发射机释放的机制。
Neurotransmitter release is triggered by membrane depolarization, Ca2+ influx and Ca2+ sensing by the release machinery, causing synaptic vesicle (SV) fusion with the plasma membrane. Interlinked is a complex membrane cycle in which vesicles are tethered to the release site, primed, fused and recycled. As many of these processes are Ca2+ dependent and simultaneously occurring, it is difficult to dissect them experimentally. This problem can be partially circumvented by controlling synaptic Ca2+ concentrations via UV photolysis of caged Ca2+. We developed a culture protocol for Ca2+ uncaging in small synapses on the basis of the generation of small glia cell islands with single neurons on top, which are sufficiently small to be covered with a UV-light flash. Neurons are loaded with the photolabile Ca2+-chelator nitrophenyl-EGTA and Ca2+ indicators, and a UV flash is used to trigger Ca2+-uncaging and SV fusion. The protocol takes three weeks to complete and provides unprecedented insights into the mechanisms of transmitter release.