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

项目摘要/摘要 长期以来，神经学家一直在寻求追踪大脑深处基因定义的神经元的电活动的能力 揭示神经回路在健康和疾病中的功能。为了解决这一技术差距，我们开发了 基因编码的电压指示器(GEVI)，这是一种荧光蛋白，报告电压动态变化 亮度。GEVIS将是对广泛使用的钙指标的极好补充，这是另一个重要信息 大脑中的携带者。例如，GEVI承诺报告钙离子一般不报告的神经活动的形式 典型哺乳动物皮质神经元中的指示物：超极化、阈值下去极化和快速动作序列 潜力。虽然这些遗传编码的电压指示器已经显示出检测电压动态的早期希望， 需要提高它们的亮度、光稳定性和对快速电压变化的响应度，才能在 哺乳动物的系统。此外，目前的GEVI在双光子显微镜下的性能特别低， 深部组织成像的首选方法。 这项提议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。我们首先提出开发一种新的显微镜平台，它可以通过以下方式快速筛选指示剂变体 自动监测和分析其对快速电压瞬变的荧光响应。第二，我们建议 开发针对特定活动的地理信息系统，即针对特定形式的神经活动进行优化的指标。具体来说，我们 将专注于优化用于检测棘波的GEVI和用于监控突触活动的GEVI，即分级电位 关于静息的膜电位。最后，我们建议扩大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。性能更好的电压指示器将 在急性脑切片和活体中全面表征所有关键指标，以便于在下游部署 申请。我们预计这项研究项目将产生性能稳健、具有广泛应用前景的电压指标。 在神经科学中的适用性，使神经元集合的电压成像具有细胞类型特异性和高 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.