Designing and deploying an expanded color palette of voltage indicators engineered for multiphoton microscopy

项目总结/摘要 神经科学家长期以来一直在寻找追踪大脑深处遗传定义的神经元电活动的能力 来解开神经回路在健康和疾病中的功能。我们寻求通过开发解决这一技术差距 基因编码的电压指示器（GEVI），这是荧光蛋白，报告电压动态的变化， 亮度松开GEVI是另一个重要的信息， 大脑中的载体。例如，GEVI承诺报告钙通常不报告的神经活动形式 典型哺乳动物皮层神经元的指标：超极化、阈下去极化和快速动作序列 潜力虽然这些基因编码的电压指示器已经显示出检测电压动态的早期前景， 需要改进它们的亮度、光稳定性和对快速电压变化的响应性，以便在 哺乳动物系统此外，当前GEVI在双光子显微镜下具有特别低的性能， 深层组织成像的首选方法。 该提案的总体目标是开发一种针对双光子优化的电压指示器的调色板 显微镜我们首先建议开发一种新的显微镜平台，可以快速筛选指示剂变体， 自动监测和分析它们对快速电压瞬变的荧光响应。第二，我们建议 开发特定于活动的GEVI，即针对特定形式的神经活动进行优化的指标。我们特别 将重点优化用于检测尖峰的GEVI和用于监测突触活动（即分级电位）的GEVI 关于静息膜电位。最后，我们建议扩展电压指示器的调色板，以实现两个- 光子多色成像和全光电生理学。具有改进性能的电压指示器将 在急性脑切片和体内的所有关键指标中进行全面表征，以促进下游部署 应用.我们预计，该研究项目将产生具有强大性能和广泛应用的电压指示器。 在神经科学中的适用性，使得能够对具有细胞类型特异性和高电压的神经元集合进行电压成像。 时空分辨率

PROJECT SUMMARY/ABSTRACT The ability to track electrical activity in genetically defined neurons deep in the brain has long been sought by neuroscientists to unravel the functions of neuronal circuits in health and disease. We seek to address this technical gap by developing genetically encoded voltage indicators (GEVIs), which are fluorescent proteins that report voltage dynamics as changes in brightness. GEVIs would be excellent completements to broadly-used indicators of calcium, another important information carrier in the brain. For example, GEVIs promise to report forms of neural activity that are not generally reported by calcium indicators in typical mammalian cortical neurons: hyperpolarizations, subthreshold depolarizations, and fast trains of action potentials. While these genetically encoded voltage indicators have shown early promise for detecting voltage dynamics, improvements in their brightness, photostability, and responsivity to fast voltage changes are needed for robust imaging in mammalian systems. Moreover, current GEVIs have particularly low performance under two-photon microscopy, the method of choice for deep-tissue imaging. The overall goal of this proposal is to develop a color palette of voltage indicators optimized for two-photon microscopy. We first propose to develop a new microscopy platform that can rapidly screen indicator variants by automatically monitoring and analyzing their fluorescence responses to fast voltage transients. Second, we propose to develop activity-specific GEVIs, that is, indicators that are optimized for specific forms of neural activity. Specifically, we will focus on optimizing a GEVI for detecting spikes and a GEVI for monitoring synaptic activity, that is, graded potentials about the resting membrane potential. Finally, we propose to expand the color palette of voltage indicators to enable two- photon multi-color imaging and all-optical electrophysiology. Voltage indicators with improved performance will be comprehensively characterized across all key metrics in acute brain slices and in vivo to facilitate deployment in downstream applications. We anticipate that this research project will produce voltage indicators with robust performance and with broad applicability in neuroscience, enabling voltage imaging of neuronal ensembles with cell type specificity and high spatiotemporal resolution.