Two-photon all-optical electrophysiology in behaving mice

行为小鼠的双光子全光电生理学

• 批准号: 10401180
• 负责人: Adam Ezra Cohen
• 金额: $ 219.09万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10401180
• 关键词: Action Potentials; Address; Animals; Back; Biological Sciences; Brain; Cell membrane; Cells; Communities; Computer software; Data Analyses; Development; Disease; Electrophysiology (science); Engineering; Fluorescence; Fluorescence Resonance Energy Transfer; Genetic; Health; Image; Individual; Letters; Licensing; Mainstreaming; Measurement; Measures; Membrane; Membrane Potentials; Methods; Microbial Rhodopsins; Microscope; Microscopy; Molecular; Mus; Mutation; Neurons; Neurosciences; Opsin; Optics; Organism; Output; Photons; Plasmids; Property; Protein Engineering; Protocols documentation; Reporter; Resolution; Scanning; Sensory; Signal Transduction; Speed; System; Technology; Thick; Time; Variant; Visit; base; bioimaging; chromophore; design; drug discovery; high throughput screening; improved; in vivo; in vivo imaging; instrumentation; microscopic imaging; nanometer; novel strategies; open data; optogenetics; research and development; scaffold; spectroscopic survey; tool; two-photon; voltage; voltage sensitive dye

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.

项目概要/摘要 行为小鼠的双光子全光电生理学神经元通过电信号进行通信，因此同时记录行为动物大脑内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。该提案旨在开发先进的工具——分子报告仪和显微镜，用于行为小鼠的基因靶向全光学电生理学。具体来说，我们建议共同开发双光子（2P）可激发基因编码电压指示器（GEVI）和新型2P电压成像显微镜。一个重要的组成部分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。 第一个目标是开发改进的膜电压分子报告器，其与 2P 激发兼容。我们提出了一系列详细的光谱研究，以了解基于微生物视紫红质的 GEVI 如何与 2P 激发相互作用。然后，我们建议从多种天然存在的微生物视紫红质中筛选视蛋白支架，以提高 2P 电压敏感性，然后进行靶向突变的高通量筛选，以提高 2P 电压，指示所选支架的特性。这项工作的成果将是新型 2P 激发 GEVI，其亮度、光稳定性和电压灵敏度均得到改善。 即使使用可以想象的最好的 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%) 以及有用信号被限制在纳米厚的细胞膜内，体内 2P 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 毫秒的时间内访问多达 512 个点，比其他扫描系统快一个数量级。 第三个目标是使用这些工具来实现新型的定性测量。我们将开发以下协议：（1）体内功能连接映射，（2）感觉和光遗传学输入下微电路动力学的测量，以及（3）映射单个神经元内动作电位的树突整合和反向传播。 体内电压成像的发展历来都是一个挑战，因为蛋白质工程、仪器和数据分析问题相互交织。目前的提案描述了一种将体内电压成像转变为神经科学主流工具的综合方法。

项目成果

Statistically unbiased prediction enables accurate denoising of voltage imaging data.

统计上无偏见的预测可以准确地降低电压成像数据。

• DOI: 10.1038/s41592-023-02005-8
• 发表时间: 2023-10
• 期刊: NATURE METHODS
• 影响因子: 48
• 作者: Eom, Minho;Han, Seungjae;Park, Pojeong;Kim, Gyuri;Cho, Eun-Seo;Sim, Jueun;Lee, Kang-Han;Kim, Seonghoon;Tian, He;Boehm, Urs L.;Lowet, Eric;Tseng, Hua-an;Choi, Jieun;Lucia, Stephani Edwina;Ryu, Seung Hyun;Rozsa, Marton;Chang, Sunghoe;Kim, Pilhan;Han, Xue;Piatkevich, Kiryl D.;Choi, Myunghwan;Kim, Cheol-Hee;Cohen, Adam E.;Chang, Jae-Byum;Yoon, Young-Gyu
• 通讯作者: Yoon, Young-Gyu