Molecular and optogenetic tools for studying voltage in the brain

描述（由申请人提供）： 荧光成像已经成为在确定的神经元网络中监测神经元活动的发展最快的技术。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。这种新方法利用荧光团通过跨越跨膜电压的大部分的长分子线连接到猝灭剂。在静息电位下，电子从猝灭剂通过导线转移到荧光团的激发态，从而猝灭后者。去极化抑制电子转移并使荧光变亮，就像Ca 2+结合使指示剂如fluo-3去猝灭一样。这些新的分子线电压敏感染料（VSD）在去极化时提供荧光的大而快速的增加，并且可以在原代培养神经元的单次试验中光学检测和分辨诱发和自发动作电位。在指导阶段，拟议的研究旨在通过在更复杂的背景下表征分子线VSD来扩展这些初步发现：哺乳动物脑切片。先前合成的分子线VSD的遗传靶向版本将使得能够询问限定的神经元亚群。作为一个测试案例，外侧缰（一个与抑郁行为相关的区域）中的特定神经元群体将被遗传靶向，并使用分子线VSD进行检查。通过选择性神经元标记提高灵敏度的另一种方法是通过使用遗传编码的传感器。在指导阶段，荧光蛋白融合的分子内光诱导电子转移（PeT）速率将被检查， 量化该过程以确定体外电压灵敏度的最佳配置。在独立阶段期间，将利用这些知识基于PeT机制生成遗传编码的电压敏感荧光蛋白。与小分子对应物一样，基于PeT的电压感测方法应该提供大的、快速的荧光变化，而电容负载可以忽略不计。将通过多种策略研究膜定位，并测量活细胞中探针的灵敏度。最后，在独立设计阶段，对分子线VSD进行了合理的设计和合成。系统的供体，受体和分子线的变化和分析所产生的量子产率，电压灵敏度和溶解度的探针将揭示设计原理，使未来几代的VSD提供更高的灵敏度和精度在检测微小的电压变化在异质性的大脑样本。总之，研究策略的组成部分提供了一个多学科的平台，跨越切片生理学，荧光蛋白设计和工程，化学合成，从开始询问脑切片内定义的神经元的电路。在异质系统中的神经元亚群中进行灵敏和精确测量的能力将大大增加我们对大脑内部工作的理解。 公共卫生相关性： 神经元电压变化的成像提供了一个有吸引力的方法，直接询问神经元通信。本研究将应用新合成的分子线电压传感器研究脑片的电活动，建立一个新的范式，构建基因编码的电压敏感荧光蛋白，并提高现有的分子线电压传感器的灵敏度和摄取。这些传感器的成功应用将提高我们对神经细胞相互交流方式的理解。

DESCRIPTION (provided by applicant): 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 spontaneous 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. PUBLIC HEALTH RELEVANCE: Imaging voltage changes in neurons offers an attractive method for the direct interrogation of neuronal communication. This research will apply newly synthesized molecular wire voltage sensors to studying electrical activity in brain slices, establish a new paradigm for constructing genetically encoded voltage sensitive fluorescent proteins, and improve the sensitivity and uptake of existing molecular wire voltage sensors. Successful application of these sensors will improve our understanding of the way nerve cells communicate with one another.