培养的大鼠海马神经元胞内Ca2＋和 NO 双标记方法研究

以培养 8 ～ 10 天的大鼠海马神经元为对象,选择 Calcium Orange AM 和 DAF-FM diacetate 为 Ca2＋和一氧化氮 (NO) 的荧光指示剂,建立了基于激光扫描共聚焦显微技术的细胞内 Ca2＋和 NO 双标记检测方法 . 此方法对 Ca2＋和 NO 进行分步染色,然后应用激光扫描共聚焦显微镜 (LSCM) 的双轨迹 (Two Track) 模式,通过快速切换激光实现对细胞内 Ca2＋和 NO 的同时检测 . 实验结果显示,两种染料之间无串扰现象;在 N- 甲基 -D- 天冬氨酸 (NMDA) 刺激下,海马神经元胞内 Ca2＋快速升高,随后达到平台期并有波动, NO 则稳定持续升高,这些变化过程与单标记的结果一致;双标记层切序列图像显示细胞内 Ca2＋和 NO 都较集中分布于细胞中部,但在细节上两者的分布存在差异 . 此双标记方法能同时检测培养的海马神经元胞内 Ca2＋和 NO ,为研究神经元胞内 Ca2＋和 NO 的相互调控作用提供了一种新的手段 .

Simultaneous Detection of Ca2+ and Nitric Oxide in Cultured Hippocampal Neurons Using Double-label Method

Choosing 8~10 days cultured hippocampal neurons of SD rat, using Calcium Orange AM and DAF-FM diacetate as the fluorescent indicators of intracellular Ca2+ and nitric oxide(NO), simultaneous detection of intracellular Ca2+ and NO was proposed by double-label method on laser scanning confocal microscope (LSCM). The dyeing process includes two steps and "Two Track" mode of LSCM is applied to realize simultaneous detection of intracellular Ca2+ and NO through quickly switching excitation wavelengths. The experiment results show that there is no cross talk between two dyes and the double-label method can reveal the changes of intracellular Ca2+ and NO concentrations under the stimulation of N-methyl-D-aspartate (NMDA), quite consistent with the results of respective single-label experiments. The analysis of slicing image sequences of double-labeled neurons shows that both Ca2+ and NO are mainly located in the center area of cell bodies, while their distribution details are different. The results suggest that the double-label method can simultaneously detect the intracellular Ca2+ and NO in cultured hippocampal neurons and thus provide an approach to investigate the roles of Ca2+ and NO in neurons as well as the interaction between them.