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

项目摘要 描述（由申请人提供）： 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、持续时间和视场施加了严格的约束。开发更亮、更红和双光子（2 P）兼容的电压指示器将大大增加可用于电压成像的大脑结构的数量，并且还将实现定性的新型测量，这可能会对神经科学产生变革。该提案将开发一个家庭的混合蛋白质小分子（化学）电压传感器的基础上一个新的传感机制，光诱导电子转移（PET）。基因编码的PET电压传感器将接受各种生物可利用的HaloTag染料，通过单光子（1 P）或2 P成像报告膜电压。这种方法结合了基因编码蛋白质的精细分子特异性和合成荧光团的上级物理特性。原理证明实验证明了负载有远红生物可利用染料的化学发生电压传感器蛋白（称为HaloVSD）。这些HaloVSD以优异的灵敏度和速度报告了培养的神经元中的阈下电压和尖峰。在目标1中，该团队将进化这种支架，以创建改进的基于远红外PET的化学发生电压传感器。传感器将进行详细的生物物理表征，并将在小鼠体内进行验证。在Aim 2中，该团队将使用1000-1300 nm的激发波长生成2 P兼容电压传感器（HaloVSD-2 P）的调色板，用于可访问的2 P成像。HaloVSD-2 P将是一个模块化平台，可与多种明亮，耐光和生物可利用的染料一起使用。在目标3中，该团队将把HaloVSD与通道视紫红质联合收割机起来，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。由于它们的高亮度，HaloVSD与现有的远红Achaerhodopsin衍生的电压传感器相比需要少约100倍的激发光。这将使荧光背景、光毒性和漂白最小化，并将防止通道视紫红质的假红光激活。这些工具将能够在活体动物中进行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，通过对电路动力学的全光学询问来研究动物模型。因为它们是基因编码的，这些传感器可以很容易地引入到各种模式生物中，并将在健康和疾病的脑回路功能研究中得到广泛应用。

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.