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: 8281248
• 负责人: Evan Walker Miller
• 金额: $ 9.06万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8281248
• 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; 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

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.

描述（由申请人提供）：荧光成像已成为用于监测限定的神经元网络中的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通过跨越大部分跨膜电压的长分子线连接到猝灭剂的荧光团。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。去极化会抑制电子转移并增亮荧光，就像 Ca2+ 结合会使 Fluo-3 等指示剂去淬灭一样。这些新型分子线电压敏感染料 (VSD) 在去极化时可大幅且快速地增加荧光，并且可以在原代培养神经元的单次试验中光学检测和解析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（哺乳动物脑切片）表征分子线 VSD 来扩展这些初步发现。先前合成的分子线 VSD 的基因靶向版本将能够对特定的神经元亚群进行询问。作为一个测试案例，外侧缰核（与抑郁行为相关的区域）中的特定神经元群将通过分子线 VSD 进行基因定位和检查。通过选择性神经元标记提高灵敏度的另一种方法是使用基因编码的传感器。在指导阶段，将检查荧光蛋白融合的分子内光诱导电子转移（PeT）速率以及电压敏感性 对该过程进行量化以确定体外电压灵敏度的最佳配置。在独立阶段，这些知识将被用来生成基于 PeT 机制的基因编码电压敏感荧光蛋白。与小分子对应物一样，基于 PeT 的电压传感方法应能以可忽略的电容负载提供大而快速的荧光变化。将通过多种策略来研究膜定位，并测量探针在活细胞中的灵敏度。最后，在独立阶段，将进行改进的分子线VSD的合理设计和合成。供体、受体和分子线的系统变化以及对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理，使未来几代 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度。该研究策略的各个组成部分共同提供了一个多学科平台，涵盖切片生理学、荧光蛋白设计和工程以及化学合成，从该平台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而精确的测量的能力将极大地增加我们对大脑内部运作的理解。 公共健康相关性：对神经元电压变化进行成像为直接询问神经元通讯提供了一种有吸引力的方法。该研究将应用新合成的分子线电压传感器来研究脑切片中的电活动，建立构建基因编码电压敏感荧光蛋白的新范例，并提高现有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通信方式的理解。