Tricoire Ludovic, Lambolez Bertrand
Neurobiologie des processus adaptatifs, UMR7102, Université Pierre et Marie Curie, Paris, France.
Methods Mol Biol. 2014;1098:33-45. doi: 10.1007/978-1-62703-718-1_3.
Genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca(2+) indicators is widely used to record transient intracellular Ca(2+) increases associated with bioelectrical activity. The natural bioluminescent Ca(2+) sensor aequorin has been historically the first Ca(2+) indicator used to address biological questions. Aequorin imaging offers several advantages over fluorescent reporters: it is virtually devoid of background signal; it does not require light excitation and interferes little with intracellular processes. Genetically encoded sensors such as aequorin are commonly used in dissociated cultured cells; however it becomes more challenging to express them in differentiated intact specimen such as brain tissue. Here we describe a method to express a GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices using recombinant Sindbis virus. This technique allows expressing GA in several hundreds of neurons on the same slice and to perform the bioluminescence recording of Ca(2+) transients in single neurons or multiple neurons simultaneously.
基因编码指示剂是利用荧光或生物发光成像技术实时研究细胞内信号级联反应的宝贵工具。钙（Ca2+）指示剂成像被广泛用于记录与生物电活动相关的细胞内钙（Ca2+）瞬变增加。天然生物发光钙（Ca2+）传感器水母发光蛋白历来是第一个用于解决生物学问题的钙（Ca2+）指示剂。与荧光报告基因相比，水母发光蛋白成像具有几个优点：它几乎没有背景信号；它不需要光激发，对细胞内过程干扰很小。基因编码传感器如水母发光蛋白通常用于解离的培养细胞；然而，在分化的完整标本如脑组织中表达它们变得更具挑战性。在这里，我们描述了一种使用重组辛德毕斯病毒在新皮质急性切片的锥体细胞中表达绿色荧光蛋白-水母发光蛋白（GA）融合蛋白的方法。这项技术允许在同一切片上的数百个神经元中表达GA，并同时对单个神经元或多个神经元中的钙（Ca2+）瞬变进行生物发光记录。
