Molecular and optogenetic tools for studying voltage in the brain

描述（由申请人提供）：荧光成像已成为发展最快的技术，用于监测神经元网络中的神经元活动。我们最近开发了一种基于分子线的荧光传感器，用于光学测量哺乳动物神经元的电压变化。这种新颖的方法利用一个荧光团连接到一个淬灭器通过一个长分子线，跨越跨膜电压的很大一部分。在静息电位下，电子从猝灭器通过导线转移到荧光团的激发态，猝灭后者。去极化抑制电子转移并使荧光变亮，就像Ca2+结合使fluo-3等指标熄灭一样。这些新型分子线电压敏感染料（VSDs）在去极化过程中提供了大量且快速的荧光增加，并且可以在原代培养的神经元中进行单次实验，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的基因靶向版本将能够对定义的神经元亚群进行讯问。作为一个测试案例，将对与抑郁行为相关的侧缰区（lateral habenula）的特定神经元群进行基因定位，并用分子线vsd进行检测。另一种通过选择性神经元标记来提高灵敏度的方法是使用基因编码的传感器。在指导阶段，将检测荧光蛋白融合物的分子内光致电子转移（PeT）速率和电压敏感性

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.