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; 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.

项目摘要/摘要 在行为小鼠神经元中通过电信号进行通信的两光子全光学生理学，因此仅在行为动物的大脑中，从数十个或数百个点记录膜电位的能力就是神经科学的变革能力。该建议是为行为小鼠的遗传靶向全光学生理学开发先进的工具分子记者和显微镜。具体而言，我们建议共同开发两光子（2p） - 可驱动的一般编码电压指示器（GEVIS）和一种新型的2P电压成像显微镜。一个重要的组成部分是开发用于在体内使用这些工具的协议，并将工具传播到神经科学社区。第一个目的是开发改善膜电压的分子记者，与2p兴奋相兼容。我们提出了一组详细的光谱研究，以了解基于微生物蛋白的GEVIS如何与2P兴奋相互作用。然后，我们建议从天然存在的微生物紫红蛋白中筛选Opsin支架，以提高2p电压敏感性，然后进行靶向突变的高通量屏幕，以改善2P电压，以表明所选支架的性质。这项工作的输出将是新的2p可取消的GEVIS，具有提高的亮度，光稳定性和电压灵敏度。即使拥有最佳的GEVI可以想象，信号也只能与用于测量的光学系统一样好。 2P电压成像在体内提出了严格的技术需求，这是由于动作电位短的时间（1 ms），小信号（1-10％）以及将有用信号限制在纳米厚的细胞膜上。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，该方法可以在不到1 ms的情况下访问512点，比其他扫描系统快的速度更快。第三个目的是使用工具来实现定性的新型测量。我们将开发（1）在体内的功能连通性映射的方案，（2）在感觉和光遗传输入下的微电路动力学测量，以及（3）映射树突状集成和单个神经元内动作电位的后传播。从历史上看，体内电压成像的开发一直是一个挑战，因为蛋白质工程，仪器和数据分析问题是交织在一起的。本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。