Molecular and optogenetic tools for studying voltage in the brain

项目概要/摘要 荧光成像已成为发展最快的技术，用于监测神经元活动的定义， 神经元网络我们最近开发了一种基于分子线的荧光传感器， 测量哺乳动物神经元的电压变化。这种新方法利用荧光团连接 通过跨越跨膜电压的大部分的长分子导线连接到猝灭器。静息 电子从猝灭剂通过导线转移到荧光团的激发态 使后者熄灭。去极化抑制电子转移并使荧光变亮，正如Ca 2+结合 会使荧光素-3这样的指示剂失活这些新的分子线电压敏感染料（VSD）提供了大的且 在去极化时荧光快速增加，并且可以光学检测和分辨诱发的， 在原代培养的神经元中的单次试验中的自发性动作电位。在辅导阶段， 拟议的研究试图通过表征分子线VSD来扩展这些初步发现， 更复杂的背景：哺乳动物的大脑切片。以前合成的基因靶向版本的 分子线VSD将使得能够询问限定的神经元亚群。作为测试案例， 外侧缰（一个与抑郁行为相关的区域）中的特定神经元群将被 基因定位用分子线VSD检测另一种提高灵敏度的方法是， 选择性神经元标记是通过使用遗传编码的传感器。在辅导阶段， 将检查荧光蛋白融合的分子内光诱导电子转移（PeT）速率， 该过程的电压灵敏度被量化以确定电压灵敏度的最佳配置， 体外在独立阶段期间，将利用该知识来生成遗传编码电压 敏感的荧光蛋白的基础上的PeT机制。与小分子对应物一样，PET- 基于电压感测的方法应该提供大的、快速的荧光变化， 即可.膜定位将通过一些策略和探针的灵敏度进行研究， 测量活细胞。最后，在自主阶段，合理设计合成了改进的分子 将进行线VSD。供体、受体和分子线的系统变异及对 探针的量子产率、电压灵敏度和溶解度将揭示设计原理 使未来几代的VSD能够在检测微小 异质大脑样本中的电压变化。总之，研究战略的组成部分提供了 一个多学科平台，涵盖切片生理学，荧光蛋白设计和工程，以及 化学合成，从这开始询问大脑切片内定义的神经元的电路。的 能够在异质神经元内的神经元亚群内进行灵敏和精确的测量， 系统将极大地增加我们对大脑内部运作的理解。

Project Summary/Abstract Fluorescence imaging has become the fastest growing technique for monitoring neuronal activity in defined networks of neurons. We have recently developed a molecular wire-based fluorescent sensor for optically measuring voltage changes in mammalian neurons. This novel method makes use of a fluorophore connected to a quencher via a long molecular wire that spans a large fraction of the transmembrane voltage. At resting potentials, electron transfer from the quencher through the wire to the excited state of the fluorophore quenches the latter. Depolarization inhibits electron transfer and brightens fluorescence, just as Ca2+ binding dequenches indicators like fluo-3. These new molecular wire voltage sensitive dyes (VSDs) provide large and fast increases in fluorescence upon depolarization and can optically detect and resolve evoked and spontaneuous action potentials in single trials in primary culture neurons. During the mentored phase, the proposed research seeks to expand upon these initial findings by characterizing molecular wire VSDs in a more complex context: mammalian brain slices. Previously synthesized genetically targeted versions of the molecular wire VSDs will enable the interrogation of defined sub-populations of neurons. As a test-case, specific neuronal populations in the lateral habenula, a region associated with depressive behavior, will be genetically targeted and examined with molecular wire VSDs . Another method for improving sensitivity via selective neuronal labeling is through the use of genetically encoded sensors. In the mentored phase, the intramolecular photoinduced electron transfer (PeT) rates of fluorescent protein fusions will be examined and the voltage sensitivity of this process quantified to determine the optimal configuration for voltage sensitivity in vitro. During the independent phase, this knowledge will be exploited to generate genetically encoded voltage sensitive fluorescent proteins based on a PeT mechanism. As with the small molecule counterparts, a PeT- based approach to voltage sensing should provide large, fast fluorescent changes with negligible capacitative load. Membrane localization will be investigated via a number of strategies and the sensitivity of the probes in live cells measured. Finally, in the independent phase, a rational design and synthesis of improved molecular wire VSDs will be carried out. Systematic variation of the donor, acceptor, and molecular wire and analysis of the resulting quantum yields, voltage sensitivities and solubilities of the probes will reveal design principles enabling future generations of VSDs to provide greater sensitivity and precision in the detection of minute voltage changes in heterogeneous brain samples. Together, the components of the research strategy provide a multidisciplinary platform, spanning slice physiology, fluorescent protein design and engineering, and chemical synthesis, from which to begin to interrogate the circuitry of defined neurons within brain slices. The ability to make sensitive and precise measurements within sub-populations of neurons within heterogeneous systems will dramatically increase our understanding of the inner workings of the brain.