Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo.
Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo.
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用于记录果蝇光感受器和间神经元对体内轻刺激的细胞内电压反应的电生理方法。
DOI：
10.3791/54142
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发表时间：
2016-06-19
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影响因子：
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通讯作者：
Rien D
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文献类型：
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作者：
Juusola M;Dau A;Zheng L;Rien D
用传统的锐微电极可以准确地记录昆虫光感受器和视觉中间神经元的电压响应。本文描述的方法使研究者能够测量单个果蝇R1-R6光感受器和大单极细胞（LMCs）对光刺激的长时间（从几分钟到几小时）高质量的细胞内反应。由于记录系统具有低噪声，因此可以用于研究苍蝇眼睛中单个细胞之间的可变性，以及它们的输出如何反映视觉环境的物理特性。我们概述了执行此技术的所有关键步骤。描述了构建适当的电生理记录装置的基本步骤，例如实验设备的设计和选择。我们还解释了如何通过制作合适的（锋利的）记录电极和（钝的）参考电极来为记录做准备。详细介绍了如何将一只完整的苍蝇固定在一个定制的苍蝇架上，在它的眼睛上准备一个小窗口，并通过这个孔插入一个记录电极，以最小的损害。我们解释了如何定位细胞接受野的中心，使所研究的细胞适应黑暗或光明，并记录其对动态光刺激的电压响应。最后，我们描述了稳定正常记录的标准，展示了单个细胞对不同光刺激的特征高质量电压响应，并简要定义了如何量化它们的信号传导性能。这种方法的许多方面在技术上具有挑战性，需要练习和耐心才能掌握。但一旦了解并优化研究者的实验目标，它提供了出色的体内神经生理数据。
Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.