Chemigenetic voltage indicators for far-red and two-photon imaging in vivo

用于体内远红和双光子成像的化学遗传学电压指示器

• 批准号: 10731843
• 负责人: Ahmed Abdelfattah
• 金额: $ 216.49万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10731843
• 关键词: Action Potentials; Amino Acids; Animal Model; Animals; Behavior; Biological Availability; Brain; Cells; Color; Couples; Dependence; Development; Disease; Dyes; Electron Transport; Electronics; Electrons; Electrophysiology (science); Event; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Genetic; Genetic Engineering; Health; Image; Individual; Investigation; Language; Light; Maps; Measurement; Membrane; Membrane Potentials; Molecular; Molecular Conformation; Motion; Mus; Nervous System; Neurons; Neurosciences; Optics; Penetration; Photons; Phototoxicity; Process; Property; Protein Engineering; Proteins; Reporter; Reporting; Research; Resolution; Sensory; Signal Transduction; Slice; Specificity; Speed; Structure; System; Techniques; Tertiary Protein Structure; Testing; Time; Tryptophan; Validation; Variant; Work; cell type; design; experimental study; fluorophore; hybrid protein; improved; in vivo; in vivo evaluation; in vivo fluorescence imaging; in vivo two-photon imaging; millisecond; model organism; neural circuit; novel; optogenetics; patch clamp; postsynaptic; prevent; prototype; response; scaffold; sensor; small molecule; spatiotemporal; tool; transmission process; two-photon; voltage

PROJECT SUMMARY DESCRIPTION (provided by applicant): Changes in membrane potential are the fundamental language of the nervous system, but these voltage signals are not directly visible. Existing membrane voltage sensors impose severe constraints on the depth, duration, and field of view of in vivo voltage imaging. The development of brighter, redder, and two-photon (2P) compatible voltage indicators would dramatically increase the number of brain structures accessible to voltage imaging and would also enable qualitatively new types of measurements which could be transformative for neuroscience. This proposal will develop a family of hybrid protein-small molecule (chemogenetic) voltage sensors based on a new sensing mechanism, photoinduced electron transfer (PET). Genetically encoded PET voltage sensors will accept diverse bioavailable HaloTag dyes to report membrane voltage via one-photon (1P) or 2P imaging. This approach combines the exquisite molecular specificity of genetically encoded proteins with the superior photophysical properties of synthetic fluorophores. Proof-of-principle experiments demonstrated chemogenetic voltage sensor proteins (termed HaloVSDs) loaded with a far-red bioavailable dye. These HaloVSDs reported subthreshold voltages and spikes in cultured neurons with excellent sensitivity and speed. In Aim 1, the team will evolve this scaffold to create improved far-red PET-based chemogenetic voltage sensors. The sensors will undergo detailed photophysical characterization and will be validated in mice in vivo. In Aim 2, the team will generate a palette of 2P-compatible voltage sensors (HaloVSD-2P) for accessible 2P imaging using 1000–1300 nm excitation wavelengths. HaloVSD-2P will be a modular platform that can be used with multiple bright, photostable, and bioavailable dyes. In Aim 3, the team will combine the HaloVSDs with channelrhodopsins for a bidirectional optical neuro-electronic interface, i.e., all-optical electrophysiology. These tools will be used to construct functional connectivity maps in vivo. Due to their high brightness, HaloVSDs require ~100-fold less excitation light compared to existing far-red Achaerhodopsin- derived voltage sensors. This will minimize fluorescence background, phototoxicity, and bleaching, and will prevent spurious red-light activation of channelrhodopsins. These tools will enable robust crosstalk-free all- optical electrophysiology experiments in live animals. HaloVSDs will provide neuroscientists with unprecedented means of investigating animal models with all-optical interrogation of circuit dynamics. Because they are genetically encoded, these sensors can be easily introduced to various model organisms and will be of broad use in studies of brain circuit function in health and disease.

项目概要 描述（申请人提供）：膜电位的变化是神经系统的基本语言，但这些电压信号并不直接可见。现有的膜电压传感器对体内电压成像的深度、持续时间和视野施加了严格的限制。更亮、更红和双光子 (2P) 兼容电压指示器的开发将极大地增加可进行电压成像的大脑结构的数量，并且还将实现新的定性测量类型，这可能会给神经科学带来变革。该提案将开发一系列基于新传感机制光诱导电子转移（PET）的混合蛋白质-小分子（化学遗传学）电压传感器。基因编码的 PET 电压传感器将接受多种生物可利用的 HaloTag 染料，通过单光子 (1P) 或 2P 成像报告膜电压。这种方法将基因编码蛋白质的精致分子特异性与合成荧光团的卓越光物理特性结合起来。原理验证实验证明了化学遗传学电压传感器蛋白（称为 HaloVSD）负载有远红生物可利用染料。这些 HaloVSD 报告了培养神经元中的阈下电压和峰值，具有出色的灵敏度和速度。在目标 1 中，该团队将改进这种支架，以创建改进的基于 PET 的远红化学发生电压传感器。这些传感器将进行详细的光物理表征，并将在小鼠体内进行验证。在目标 2 中，该团队将生成一系列 2P 兼容电压传感器 (HaloVSD-2P)，以便使用 1000–1300 nm 激发波长进行 2P 成像。 HaloVSD-2P 将是一个模块化平台，可与多种明亮、光稳定和生物可利用的染料一起使用。在目标 3 中，该团队将 HaloVSD 与视紫红质通道结合起来，形成双向光学神经电子接口，即全光学电生理学。这些工具将用于构建体内功能连接图。由于其高亮度，HaloVSD 所需的激发光比现有的远红 Achaerhodopsin 电压传感器少约 100 倍。这将最大限度地减少荧光背景、光毒性和漂白，并防止通道视紫红质的虚假红光激活。这些工具将在活体动物中实现稳健的无串扰全光学电生理学实验。 HaloVSD 将为神经科学家提供前所未有的手段，通过全光学询问电路动力学来研究动物模型。由于它们是经过基因编码的，因此这些传感器可以很容易地引入到各种模型生物体中，并将​​广泛用于健康和疾病中脑回路功能的研究。