Department of breast pathology, Tianjin Medical University Cancer Institute and Hospital, Key Laboratory of Breast Cancer Prevention and Therapy of Ministry of Education, Key Laboratory of Cancer Prevention and Therapy of Tianjin, Tianjin 300060, China.
Neurosci Bull. 2009 Aug;25(4):216-20. doi: 10.1007/s12264-009-0507-1.
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.
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.
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).
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.
突触前电压门控钙通道介导钙离子快速内流入突触末梢，触发突触囊泡胞吐和神经递质释放。FM1-43 染料于 1992 年由贝茨及其同事首次作为荧光探针引入，并已被用于监测多种细胞类型的胞吐作用、内吞作用和内体运输。本研究旨在探讨应用 FM1-43 染料分析钙通道介导的突触末梢胞吐作用的功能的可行性。
从孕鼠胚胎中分离出海马组织，并转染 Ds-Red 缀合质粒。转染后，神经元用 8μmol/L FM1-43 和 47mmol/L KCl 负载 90s。之后，用 90mmol/L KCl 诱导 FM 染料褪色，并通过 FM 成像系统进行记录。
大鼠海马神经元的突触末梢可被 FM1-43 染料有效染色。此外，标记神经元末梢的褪色与递质释放一致，可用硝苯地平（L 型钙通道抑制剂）阻断。
FM 成像技术是一种分析突触囊泡胞吐和神经递质释放的先进有效的方法，可应用于各种突触功能研究。
