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.

项目摘要 描述（由适用提供）：膜电位的变化是神经系统的基本语言，但是这些电压信号却不直接可见。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重的约束。兼容电压指标的明亮，红色和两光子（2p）的发展将显着增加电压成像可访问的大脑结构的数量，并且还可以实现定性的新型测量，这些测量可能对神经科学具有变革性。该建议将基于新的传感器机制，即光诱导的电子传递（PET），开发一个杂化蛋白质 - 小分子（化学）电压传感器。遗传编码的PET电压传感器将接受潜水员可生物利用的吊带染料染料，以通过单光子（1p）或2p成像报告膜电压。这种方法结合了遗传编码蛋白的等效分子特异性与合成荧光团的优异的光物理特性。原则证明的实验表明化学发生电压传感器蛋白（称为Halovsds）装有远红色的生物利用染料。这些HALOVSD报告了具有出色敏感性和速度的培养神经元中的亚阈值电压和峰值。在AIM 1中，团队将发展这种支架，以创建改进的基于远红色的宠物的化学遗传电压传感器。传感器将经历详细的光物理表征，并将在体内验证。在AIM 2中，该团队将使用1000–1300 nm兴奋的波长生成2P兼容电压传感器（HALOVSD-2P）的调色板。 HALOVSD-2P将是一个模块化平台，可与多个明亮，光稳定和可生物利用染料一起使用。在AIM 3中，该团队将将HalovsD与ChannelRhodopsins结合起来，用于双向光学神经电子界面，即全光学电生理学。这些工具将用于在体内构建功能连接图。由于它们的亮度高，与现有的远红色achaerhopoptin衍生的电压传感器相比，Halovsds的刺激性低约100倍。这将最大程度地减少荧光背景，光毒性和漂白剂，并防止通道旋转蛋白的潮流红光激活。这些工具将实现活动物中强大的无串扰全光生理实验。 HALOVSD将为神经科学家提供前所未有的方法，用于研究动态动态的动物模型。由于它们是遗传编码的，因此可以轻松地将这些传感器引入各种模型生物体中，并且在健康和疾病中的脑电路功能研究中将广泛使用。