Objective: Presynaptic voltage-gated Ca(2+) channels mediate rapid Ca(2+) influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduced as a fluorescence probe by Betz and his colleagues in 1992, and has been used to monitor exocytosis, endocytosis and endosomal traffic in a variety of cell types. The present study aims to investigate the feasibility of applying the FM 1-43 dye in the functional analysis of calcium channel-mediated exocytosis in synaptic terminals.
Methods: The hippocampi were isolated from embryos of pregnant rats, and hippocampal neurons were then transfected with Ds-Red conjugated plasmid. The neurons were then loaded with 8 micromol/L FM 1-43 and 47 mmol/L KCl for 90 s after transfection. After that, 90 mmol/L KCl was employed to induce FM dye destaining, which was recorded by FM imaging system.
Results: The neuron synaptic terminals of rat hippocampus could be effectively stained by the FM 1-43 dye. Besides, the destaining of the labeled neuron terminals was in accordance with the transmitter release, which could be blocked by the application of nifedipine (inhibitor for L-type calcium channel).
Conclusion: The FM imaging technique is an advanced and effective method for analyzing synaptic vesicle exocytosis and neurotransmitter release, and can be applied in various synaptic functional studies.
目的: 突触前膜上的钙离子通道介导钙离子内流, 从而实现突触小泡的胞吐以及神经递质释放。1992年Betz等首次发现FM 1-43染料可以作为一种标记荧光用于科学研究。
目前: FM 1-43染料已被广泛用于检测各种细胞类型的胞吞和胞吐过程。本研究旨在探讨将FM 1-43染料用于检测突触前膜钙离子通道介导的神经递质释放的可行性。
方法: 取孕鼠进行原代大鼠神经元的培养, 利用带有Ds-Red 标记的质粒进行转染, 48 小时后经8 μmol/L 的FM 1-43染料和47 mmol/L的KCl孵育90秒, 然后用90 mmol/L的KCl再次孵育, 其间用FM 1-43成像技术记录代表突触的亮点荧光衰减的过程。
结果: FM 1-43染料能有效标记大鼠海马神经元突触末端, 刺激后被标记部位的荧光强度衰减的过程与神经递质的释放过程相一致, 并且这个过程可以被L型钙离子通道抑制剂硝苯地平抑制。
结论: FM 1-43成像技术能够检测钙离子介导的神经递质的释放过程, 是一种非常先进并切实有效的检测神经元突触末端神经递质释放过程的手段和方法。