An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials.
An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials.
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DOI：
10.1371/journal.pone.0039933
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Nakai J
中科院分区：
文献类型：
--
作者：
Ohkura M;Sasaki T;Kobayashi C;Ikegaya Y;Nakai J
基因编码的钙离子指示剂(GECI)是对特定细胞群体的活动进行成像的强大工具。在这里，我们开发了一种改良的红色荧光Geci，命名为R-CaMP1.07，通过诱变R-GECO1。在HeLa细胞检测中，R-CaMP1.07的荧光反应比R-GECO1强1.5-2倍。在海马锥体神经元中，R-CaMP1.07在50赫兹的帧频率下检测到由单动作电位(AP)触发的钙瞬变的概率为95%，信噪比为7。Ca~(2+)瞬变幅度与AP数目呈线性相关。R-CaMP1.07的表达没有明显改变电生理特性或突触活动模式。R-CaMP1.07和光敏阳离子通道ChR2在锥体神经元中的共表达表明，R-CaMP1.07的激发光几乎不能激活ChR2，因此R-CaMP1.07可用于监测光诱发AP引起的钙瞬变。这些技术进步为监测和操纵单细胞分辨率的神经元活动提供了一种新的策略。
Genetically encoded Ca2+ indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5–2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca2+ transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca2+ transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca2+ transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.
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