In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli.
In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli.
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DOI：
10.3791/62172
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发表时间：
2021-02-11
期刊：
影响因子：
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通讯作者：
Macpherson LJ
中科院分区：
文献类型：
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作者：
Fowler BE;Macpherson LJ
在过去的十年里，基因编码的钙指示剂(GECI)的进步推动了体内功能成像的革命。利用钙作为神经元活动的指标，这些技术提供了一种实时监测大型神经元群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膝状神经节神经元对施加到活体麻醉小鼠舌头上的味觉刺激的反应。膝状神经节由支配舌前部和腭部的味觉神经元胞体以及支配耳廓的一些体感神经元组成。用GCaMP对单个膝状神经节神经元的味觉诱发反应进行成像，为野生型小鼠这些神经元的调谐曲线提供了重要信息，也为检测遗传操作小鼠的外周味觉错配表型提供了一种方法。在这里，我们演示了暴露膝状神经节的手术程序，GCaMP荧光图像的获取，数据分析的初始步骤，以及故障排除。该技术可用于转基因编码的GCaMP，或AAV介导的GCaMP表达，并可被修改以成像特定的感兴趣基因亚群(即，Cre介导的GCaMP表达)。总体而言，膝状神经节神经元的体内钙成像是监测外周味觉神经元活动的一项强大技术，并为更传统的全神经鼓索记录或味觉行为分析提供补充信息。在这里，我们描述了一种手术暴露活体、麻醉的实验室小鼠膝状神经节的技术，以及如何使用钙成像来测量膝状神经节神经元对口腔中施加的味觉刺激的反应。这项技术显示了神经元群体对味觉刺激的反应，允许用不同的刺激剂进行多次试验。这样就可以深入比较哪些神经元对哪些味觉作出反应。
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. Here we describe a technique to surgically expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use Calcium Imaging to measure the responses of ensembles of geniculate ganglion neurons to taste stimuli applied to the oral cavity. This technique shows the response of populations of neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.