Two-photon all-optical electrophysiology in behaving mice

项目总结/摘要 行为小鼠的双光子全光学电生理学神经元通过电信号进行通信，因此在行为动物的大脑中同时记录数十或数百个点的膜电位的能力将是神经科学的变革能力。本计画旨在发展先进的工具-分子报告器与显微镜，以进行基因定位的全光学行为小鼠电生理研究。具体而言，我们建议共同开发双光子（2 P）可激发的遗传编码电压指示器（GEVI）和一种新型的2 P电压成像显微镜。一个重要的组成部分将是制定在体内使用这些工具的协议，并将这些工具传播给神经科学界。 第一个目标是开发与2 P激发相容的膜电压的改进的分子报告。我们提出了一套详细的光谱研究，以了解微生物视紫红质为基础的GEVI如何与2 P激发相互作用。然后，我们建议从不同的天然存在的微生物视紫红质中筛选视蛋白支架，以提高2 P电压敏感性，然后通过靶向突变的高通量筛选来提高所选支架的2 P电压指示特性。这项工作的成果将是新的2 P可激发GEVI，具有更好的亮度，光稳定性和电压灵敏度。 即使使用最好的GEVI，信号也只能与用于测量的光学系统一样好。由于动作电位持续时间短（1 ms）、信号小（1 - 10%）以及有用信号局限于纳米厚的细胞膜，体内2 P电压成像提出了严格的技术要求。在我们的第二个目标中，我们提出了一种新的高速扫描方法，可以在不到1 ms的时间内访问多达512个点，比其他扫描系统快一个数量级。 第三个目标是利用这些工具实现新的定性测量。我们将开发（1）体内功能连接映射，（2）在感觉和光遗传学输入下测量微电路动力学，以及（3）映射树突整合和单个神经元内动作电位的反向传播的协议。 体内电压成像的发展历来是一个挑战，因为蛋白质工程，仪器和数据分析问题交织在一起。本提案描述了一种综合方法，将体内电压成像变成神经科学的主流工具。

PROJECT SUMMARY/ABSTRACT Two-photon all-optical electrophysiology in behaving mice Neurons communicate through electrical signals, so the ability to record membrane potential from dozens or hundreds of points simultaneously within the brain of a behaving animal would be a transformative capability for neuroscience. This proposal is to develop advanced tools-molecular reporters and microscopes for genetically targeted all-optical electrophysiology in behaving mice. Specifically, we propose to co-develop two-photon (2P)-excitable genetically encoded voltage indicators (GEVIs) and a new type of 2P voltage-imaging microscope. An important component will be to develop protocols for using these tools in vivo and to disseminate the tools to the neuroscience community. The first aim is to develop improved molecular reporters of membrane voltage, which are compatible with 2P excitation. We propose a set of detailed spectroscopic studies to understand how microbial rhodopsin-based GEVIs interact with 2P excitation. We then propose to screen opsin scaffolds from diverse naturally occurring microbial rhodopsins for improved 2P voltage sensitivity, followed by a high-throughput screen of targeted mutations to improve 2P voltage indicating properties of selected scaffolds. The output of this effort will be new 2P-excitable GEVIs with improved brightness, photostability, and voltage sensitivity. Even with the best GEVI imaginable, the signals will only be as good as the optical system used for measurement. 2P voltage imaging in vivo presents stringent technical demands due to the short duration of action potentials (1 ms), the small signals (1 – 10%), and the confinement of useful signals to the nanometers- thick cell membrane. In our second aim, we propose a new approach to high-speed scanning which can visit up to 512 points in less than 1 ms, an order of magnitude faster than other scanning systems. The third aim is to use the tools to enable qualitatively new types of measurements. We will develop protocols for (1) functional connectivity mapping in vivo, (2) measurements of microcircuit dynamics under sensory and optogenetic inputs, and (3) mapping dendritic integration and back-propagation of action potentials within individual neurons. The development of in vivo voltage imaging has historically been a challenge because the protein engineering, instrumentation, and data analysis problems are intertwined. The present proposal describes an integrated approach to turn in vivo voltage imaging into a mainstream tool for neuroscience.