Molecular and optogenetic tools for studying voltage in the brain

描述(由申请人提供)：荧光成像已成为在确定的神经元网络中监测神经元活动的最快发展技术。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。这种新的方法利用了一个荧光团，它通过一根跨越很大一部分跨膜电压的长分子导线连接到猝灭剂上。在静止电位下，电子从猝灭剂通过导线转移到荧光团的激发态，使后者猝灭。去极化会抑制电子传递并使荧光变亮，就像钙离子结合会抑制Fluo-3等指示剂一样。这些新的分子线电压敏感染料(VSD)在去极化时提供了大量和快速的荧光增强，并且可以在原代培养的神经元上单次光学检测和分辨诱发和自发动作电位。在指导阶段，拟议的研究试图通过在更复杂的背景下描述分子导线VSD来扩展这些初步发现：哺乳动物脑片。先前合成的分子导线VSD的遗传靶向版本将使对确定的神经元亚群的询问成为可能。作为一个测试案例，外侧缰核(与抑郁行为相关的区域)中的特定神经元群体将被基因定位并用分子导线VSD进行检查。另一种通过选择性神经元标记来提高敏感性的方法是通过使用遗传编码的传感器。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)率和电压敏感性。 这一过程被量化，以确定体外电压敏感性的最佳配置。在独立阶段，这一知识将被利用来产生基于pET机制的遗传编码的电压敏感荧光蛋白。与小分子对应的方法一样，基于PET的电压传感方法应该提供大的、快速的荧光变化，而电容负载可以忽略不计。膜的定位将通过一些策略进行研究，并测量活细胞中探针的灵敏度。最后，在独立阶段，对改进后的分子线VSD进行合理的设计和合成。对供体、受体和分子线的系统变化以及由此产生的量子产率、电压灵敏度和探针溶解度的分析将揭示设计原则，使未来一代的VSD能够在检测异质脑样本中的微小电压变化时提供更高的灵敏度和精确度。总而言之，研究策略的组成部分提供了一个多学科平台，横跨切片生理学、荧光蛋白质设计和工程以及化学合成，从这个平台开始询问脑片中定义的神经元的电路。在不同系统中的神经元亚群中进行灵敏和精确测量的能力将极大地增加我们对大脑内部工作原理的了解。 公共卫生相关性：成像神经元中的电压变化为直接询问神经元通信提供了一种有吸引力的方法。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，建立构建基因编码的电压敏感荧光蛋白的新范式，并提高现有分子线电压传感器的灵敏度和摄取率。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.