Molecular and optogenetic tools for studying voltage in the brain

项目摘要/摘要 荧光成像已成为监测神经元活动的最快技术 神经元网络。我们最近开发了一种基于分子导线的荧光传感器，用于光学 测量哺乳动物神经元的电压变化。这种新的方法利用连接的荧光团 通过跨越很大一部分跨膜电压的长分子导线到达猝灭剂。在休息时 电位，电子从猝灭剂通过导线转移到荧光团的激发态 平息了后者。去极化会抑制电子传递并增强荧光，就像钙离子结合一样 消除像Fluo-3这样的指示剂。这些新的分子线压敏染料(VSD)提供了大量和 去偏振时荧光快速增加，并可光学检测和解析诱发和 单次实验中原代培养神经元的自发动作电位。在指导阶段， 拟议的研究试图通过表征分子导线VSD来扩展这些初步发现 更复杂的背景：哺乳动物脑片。先前合成的基因靶向版本的 分子导线VSD将使对确定的神经元亚群的询问成为可能。作为测试用例， 外侧缰核(与抑郁行为相关的区域)中的特定神经元群体将是 基因定位并用分子线VSD进行检测。另一种通过以下方式提高灵敏度的方法 选择性神经元标记是通过使用遗传编码传感器来实现的。在指导阶段， 将检测荧光蛋白融合的分子内光诱导电子转移(PET)率和 这一过程的电压灵敏度被量化，以确定电压灵敏度的最佳配置 体外培养。在独立阶段，这一知识将被利用来产生遗传编码电压 基于宠物机制的敏感荧光蛋白。就像小分子的对应物一样，宠物- 基于电压传感的方法应该提供大的、快速的荧光变化，而电容可以忽略不计 装填。膜的定位将通过多种策略和探针的灵敏度进行研究 测量活细胞。最后，在独立阶段，合理设计和合成了改进的分子 将进行有线VSD。供体、受体和分子导线的系统变异及其分析 由此得到的探测器的量子产率、电压灵敏度和溶解度将揭示设计原则。 使下一代VSD能够在检测分钟数方面提供更高的敏感度和精度 异质脑样本中的电压变化。总而言之，研究战略的组成部分提供了 多学科平台，跨越切片生理学、荧光蛋白质设计和工程，以及 化学合成，从它开始询问脑片中特定神经元的电路。这个 能够在不同种类的神经元亚群中进行灵敏和精确的测量 系统将极大地提高我们对大脑内部工作原理的理解。

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.