Department of Biology, The University of Texas at San Antonio.
Department of Biology, The University of Texas at San Antonio;
J Vis Exp. 2021 Feb 11(168). doi: 10.3791/62172.
Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.
在过去的十年中，基因编码钙指示剂（GECIs）的进步推动了体内功能成像的革命。这些技术将钙作为神经元活动的替代物，提供了一种实时监测大神经元集合中单个细胞对各种刺激的反应的方法。我们和其他人应用这些技术来成像单个神经节神经元对活体麻醉小鼠舌头上施加的味觉刺激的反应。神经节由支配前舌和 palate 的味觉神经元的细胞体以及支配耳朵 pinna 的一些躯体感觉神经元组成。用 GCaMP 对单个神经节神经元的味觉诱发反应进行成像，为了解这些神经元在野生型小鼠中的调谐特性提供了重要信息，也为检测遗传操作小鼠中的外周味觉误连接表型提供了一种方法。在这里，我们展示了暴露神经节、GCaMP 荧光图像采集、数据分析的初始步骤以及故障排除的手术过程。该技术可用于转染的 GCaMP，或 AAV 介导的 GCaMP 表达，并可进行修改以成像特定的感兴趣的遗传亚群（即 Cre 介导的 GCaMP 表达）。总之，神经节神经元的体内钙成像技术是监测外周味觉神经元活性的有力工具，为更传统的全神经鼓索记录或味觉行为测定提供了互补信息。
