Molecular and optogenetic tools for studying voltage in the brain

用于研究大脑电压的分子和光遗传学工具

• 批准号: 8728414
• 负责人: Evan Walker Miller
• 金额: $ 24.66万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8728414
• 关键词: Action Potentials; Affinity; Antibodies; Behavior; Binding; Biological Assay; Brain; Cells; Chimeric Proteins; Communication; Complex; Detection; Development; Dissection; Dyes; Electron Transport; Electrons; Fluo-3; Fluorescence; Fluorescence Microscopy; Future Generations; Habenula; Hippocampus (Brain); Image; In Vitro; Investigation; Knowledge; Label; Lateral; Length; Life; Light; Measurement; Measures; Membrane; Membrane Potentials; Mentors; Methods; Molecular; Monitor; Neurobiology; Neurons; Pathology; Phase; Physiology; Population; Positioning Attribute; Primary Cell Cultures; Process; Proteins; Rattus; Reaction Time; Relative (related person); Reporting; Research; Sampling; Side; Slice; Solid; Solubility; Solutions; Speed; Staining method; Stains; System; Techniques; Testing; Variant; Work; abstracting; attenuation; base; chemical synthesis; chromophore; depressive symptoms; design; engineering design; fluorescence imaging; fluorophore; improved; multidisciplinary; novel; optogenetics; patch clamp; quantum; relating to nervous system; response; sensor; small molecule; tool; uptake; voltage; water solubility

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.

项目概要/摘要 荧光成像已成为监测特定领域神经元活动发展最快的技术 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，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了连接的荧光团 通过跨越大部分跨膜电压的长分子线到达猝灭剂。休息时 电势，电子从猝灭剂通过导线转移到荧光团的激发态 淬灭后者。去极化会抑制电子转移并增亮荧光，就像 Ca2+ 结合一样 终止荧光素 3 等指示剂。这些新型分子线电压敏感染料 (VSD) 提供了大且 去极化后荧光快速增加，可以光学检测和解析诱发和 原代培养神经元单次试验中的自发动作电位。在辅导阶段， 拟议的研究旨在通过表征分子线 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。之前合成的基因靶向版本 分子线 VSD 将能够对特定的神经元亚群进行询问。作为测试用例， 外侧缰核（与抑郁行为相关的区域）中的特定神经元群将被 使用分子线 VSD 进行基因靶向和检查。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基因编码的传感器来实现的。在辅导阶段， 将检查荧光蛋白融合物的分子内光诱导电子转移（PeT）速率并 量化该过程的电压灵敏度以确定电压灵敏度的最佳配置 体外。在独立阶段，这些知识将被用来生成基因编码电压 基于 PeT 机制的敏感荧光蛋白。与小分子对应物一样，PeT- 基于电压传感的方法应提供大而快速的荧光变化，而电容可忽略不计 加载。将通过多种策略和探针的灵敏度来研究膜定位 测量活细胞。最后，在独立阶段，合理设计并合成改进的分子 将进行有线 VSD。供体、受体和分子线的系统变异及其分析 由此产生的量子产率、电压灵敏度和探针的溶解度将揭示设计原理 使下一代 VSD 能够在微小的检测中提供更高的灵敏度和精度 异质脑样本中的电压变化。研究策略的各个组成部分共同提供 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，从中开始询问脑切片内定义的神经元的电路。这 能够在异质神经元亚群中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。