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

项目总结 描述(申请人提供)：膜电位的变化是神经系统的基本语言，但这些电压信号不是直接可见的。现有的膜电压传感器严重限制了活体电压成像的深度、持续时间和视场。更亮、更红、与双光子(2P)兼容的电压指示器的开发将极大地增加电压成像可访问的大脑结构的数量，并将使可能对神经科学产生变革的定性新型测量成为可能。这项提议将开发一系列基于一种新的传感机制--光致电子转移(PET)的蛋白质-小分子(化学发生)混合型电压传感器。基因编码的PET电压传感器将接受各种生物可用HaloTag染料，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质的精致分子专一性和合成荧光团的优越光物理性质。原理验证实验证明了化学发生电压传感器蛋白(称为HaloVSD)负载了一种远红生物可用染料。这些HaloVSD报告了培养神经元中的亚阈值电压和尖峰，具有极好的灵敏度和速度。在目标1中，该团队将进化这种支架，以创造改进的基于远红外线PET的化学产生电压传感器。这些传感器将经过详细的光物理表征，并将在小鼠体内进行验证。在目标2中，该团队将生成一个2P兼容电压传感器(HaloVSD-2P)调色板，用于使用1000-1300 nm激发波长进行可访问的2P成像。HaloVSD-2P将是一个模块化平台，可以与多种明亮、光稳定和生物可用的染料一起使用。在目标3中，该团队将把HaloVSD与通道视紫红质结合起来，形成双向光学神经-电子接口，即全光电生理学。这些工具将用于构建体内的功能连接图。由于其高亮度，HaloVSD需要的激发光比现有的远红无视紫红质电压传感器少约100倍。这将最大限度地减少荧光背景、光毒性和漂白，并将防止通道视紫红素的虚假红光激活。这些工具将在活体动物中进行强大的无串扰全光电生理学实验。HaloVSD将为神经科学家提供前所未有的手段来研究动物模型，并对电路动力学进行全光学询问。由于它们是基因编码的，这些传感器可以很容易地引入各种模式生物，并将在健康和疾病的大脑电路功能研究中广泛使用。

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.