Voltage Imaging with Genetically Encoded Optical Probes

描述(申请人提供)：完整神经系统的电活动成像技术为研究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产生行为提供了巨大的希望。电压成像在很大程度上依赖于光学探头的使用，这种探头可以将膜电位的变化转换为可检测的光学信号。目前广泛使用的探头具有严重的局限性，阻碍了电压成像神经网络研究的进展。这项提议将开发一种新的、新的方法，在转基因小鼠的脑片中的活细胞中进行电压成像。这项计划将扩展一种名为混合电压传感(HVOS)的方法，该方法使用在细胞中表达的、针对细胞表面的遗传编码的荧光蛋白。表达这种蛋白质的细胞用合成的化合物二吡咯胺(DPA)处理，这种化合物吸收荧光蛋白质发出的光。DPA进入膜脂双层的疏水内部，通过共振能量传递，吸收膜靶蛋白发出的光，从而猝灭其荧光。因为DPA带有负电荷，所以它在脂质双层内移动，以响应膜电位。当DPA和膜靶标荧光蛋白之间的距离改变时，猝灭量发生变化，从而检测到的荧光产生关于膜电位的信息。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一种。第二个具体目标将开发一种利用两个荧光团瞄准质膜相反面的HVOS方法。这将使研究人员能够根据两个HVOS信号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瞄准特定类型的细胞。通过这种方式，高性能的HVOS探测器将成为研究网络行为的有价值的工具，从而广泛地促进神经科学许多基础和应用领域的进步。公共卫生相关性：该项目将为研究人员提供研究大脑中电活动的通用工具，从而促进对精神疾病和神经疾病的理解。通过研究特定类型神经元的电活动，研究人员将了解不同类型的细胞如何对大脑功能做出贡献。这将使临床科学家能够决定针对哪些类型的神经元使用药物来纠正神经功能的异常。

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.