Voltage Imaging with Genetically Encoded Optical Probes

描述（由申请人提供）：完整神经系统的电活动成像技术对神经元网络如何工作以及神经元集合如何通过复杂的突触相互作用作为紧急过程产生行为的研究具有很大的前景。电压成像主要依赖于光探头的使用，光探头将膜电位的变化转化为可检测的光信号。目前广泛使用的探针有严重的局限性，阻碍了电压成像神经网络研究的进展。本研究为转基因小鼠脑切片活细胞电压成像提供了一种新的方法。该计划将扩展一种称为混合电压传感（hVOS）的方法，该方法使用在细胞中表达的遗传编码荧光蛋白并靶向细胞表面。表达这种蛋白质的细胞用合成化合物二丙胺（DPA）处理，该化合物吸收荧光蛋白发出的光。DPA进入质膜脂双分子层的疏水内部，通过共振能量转移，吸收膜靶蛋白发出的光，从而猝灭其荧光。由于DPA携带负电荷，它在响应膜电位的脂质双分子层内移动。当DPA与膜靶向荧光蛋白之间的距离发生变化时，猝灭量发生变化，因此检测到的荧光可以获得膜电位的信息。该项目的第一个具体目标是对各种荧光蛋白进行关键测试，以找到在hVOS成像中具有最佳性能的荧光蛋白。第二个特定目标将开发一种hVOS方法，该方法使用两个针对质膜相反面的荧光团。这将使研究人员能够从两个hVOS信号中进行膜电位的比率计算。这些探针将使研究人员能够通过生产转基因动物或通过各种其他分子靶向技术来靶向特定类型的细胞。通过这种方式，高性能hVOS探针将成为研究网络行为的有价值的工具，从而为神经科学的许多基础和应用领域的进步做出广泛贡献。公共卫生相关性：该项目将通过为研究人员提供研究脑电活动的通用工具，促进对精神疾病和神经疾病的理解。通过研究特定种类神经元的电活动，研究人员将了解不同种类的细胞如何影响大脑功能。这将使临床科学家能够决定用药物治疗哪种类型的神经元来纠正神经功能异常。

DESCRIPTION (provided by applicant): Techniques for imaging of electrical activity in intact nervous systems hold great promise for the study of how networks of neurons work and how collections of neurons generate behavior as an emergent process through their complex synaptic interactions. Voltage imaging depends critically on the use of optical probes that convert changes in membrane potential into a detectable optical signal. Probes currently in wide use have serious limitations that impede progress in the investigation of neural networks with voltage imaging. This proposal will develop a new and novel approach to voltage imaging in living cells in brain slices from transgenic mice. This plan will extend a method termed hybrid voltage sensing (hVOS), which employs a genetically encoded fluorescent protein expressed in cells and targeted to the cell surface. Cells expressing this protein are treated with the synthetic compound, dipycrylamine (DPA), which absorbs light emitted by the fluorescent protein. DPA enters the hydrophobic interior of the plasma membrane lipid bilayer, and through resonant energy transfer, DPA can absorb light emitted by the membrane targeted protein, and thus quench its fluorescence. Because DPA carries a negative charge, it moves within the lipid bilayer in response to membrane potential. When the distance between DPA and the membrane targeted fluorescent protein changes, the amount of quenching changes so that detected fluorescence yields information about membrane potential. The first Specific Aim of this project is to perform critical tests of various fluorescent proteins to find one with optimal performance in hVOS imaging. The second Specific Aim will develop a method of hVOS that employs two fluorophores targeted to opposite faces of the plasma membrane. This will enable investigators to perform ratiometric calculations of membrane potential from two hVOS signals. These probes will enable investigators to target specific types of cells, either by the production of transgenic animals or by a variety of other molecular targeting techniques. In this way high-performance hVOS probes will serve as valuable tools for investigating network behavior, and thus contribute broadly to advances in many basic and applied areas of neuroscience. PUBLIC HEALTH RELEVANCE: This project will advance understanding of mental illness and neurological diseases by giving investigators general tools for the study of electrical activity in the brain. By studying electrical activity in specific kinds of neurons, researchers will learn how different kinds of cells contribute to brain function. This will allow clinical scientists to decide which types of neurons to target with drugs to correct abnormalities in neural function.