Calcium Imaging of Anopheles coluzzii Mosquito Antennae Expressing the Calcium Indicator GCaMP6f.-猫眼课题宝

表达钙指示剂 GCaMP6f 的按蚊触角的钙成像。

DOI：

10.1101/pdb.prot107918

发表时间：

2022

期刊：

Cold Spring Harbor protocols

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

Afify,Ali;Potter,ChristopherJ

钙成像是一种用于测量响应刺激的功能性神经元活动的技术。它多年来一直用于研究昆虫（即蜜蜂、果蝇和飞蛾）中气味引起的反应，最近被引入蚊子中。传统上，蚊子的钙成像是使用非特异性钙指示剂染料进行的，以检查整个昆虫大脑区域的神经元反应，但基因编码钙指示剂（GECI）的发展促进了在特定组织上进行功能性钙成像的能力。例如，通过在嗅觉神经元中特异性表达 GECI，可以检查外周器官中这些神经元的气味诱导反应。蚊子触角的钙成像进一步提供了一种在单个实验中同时可视化多个触角神经元活动的有利方法。在本协议中，我们描述了一种钙成像方法，用于研究表达钙指示剂 GCaMP6f 的按蚊天线中的气味诱发反应。该方法需要成像设备（复合显微镜、光源和相机）、气味剂输送系统和图像采集软件。蚊子的准备工作很简单，但需要练习以尽量减少成像过程中蚊子的移动。可以使用斐济软件对录制的视频进行分析，以生成气味诱发反应的热图和活动轨迹。该协议经过一些修改后也可用于研究其他外周器官（如唇瓣、触肢和跗节）。

Calcium imaging is a technique used to measure functional neuronal activities in response to stimuli. It has been used for years to study odorant-induced responses in insects (ie, honeybees, Drosophila, and moths) and was recently introduced into mosquitoes. Traditionally, calcium imaging in mosquitoes was performed using nonspecific calcium indicator dyes to examine neuronal responses in whole insect brain regions, but the development of genetically encoded calcium indicators (GECIs) has facilitated the ability to perform functional calcium imaging on specific tissues. For example, by specifically expressing a GECI in olfactory neurons, the odor-induced responses of these neurons in peripheral organs can be examined. Calcium imaging of mosquito antennae further provides an advantageous method for simultaneously visualizing the activity of several antennal neurons in a single experiment. In this protocol, we describe a calcium imaging method to study odor-evoked responses in Anopheles coluzzii antennae expressing the calcium indicator GCaMP6f. This method requires imaging equipment (compound microscope, light sources, and camera), an odorant delivery system, and image acquisition software. The mosquito preparation is straightforward but requires practice to minimize mosquito movement during imaging. Recorded videos can be analyzed using Fiji software to generate heatmaps and activity traces for odorant-evoked responses. This protocol can also be used, with some modifications, to study other peripheral organs (such as labella, palps, and tarsi).