Population Imaging of Action Potentials by Novel Two-Photon Microscopes and Genetically Encoded Voltage Indicators

项目总结 了解信息是如何在哺乳动物的新皮质中处理的一直是一个长期存在的问题 神经科学。虽然动作电位是基本的信息位，但这些尖峰是如何编码的 陈述和驾驶行为仍不清楚。为了充分解决这一问题，它已经 很明显，需要进行大量神经元活动的实验 在多个时间尺度上以详细和全面的方式衡量。直接测量 动作电位主要是通过电生理学实现的。然而，这样的测量不能 很容易与其他方法相结合来评估神经元的连通性和分子特性。 整合功能、解剖和遗传信息对于理解神经元回路如何 都是经过组织和计算的。长期以来，人们一直在努力开发光学方法，以 由于其兼容性而测量神经元活动，以同时测量连接性和分子 使用荧光标记技术进行身份识别。新设计的基因编码电压敏感型 指示器现在已经为动作电位的光学成像打开了大门。双光子显微镜有 是一种被证明是深入大脑的非侵入性成像方法。然而，快速的毫秒瞬变 动作电位和基因编码的电压敏感指标的膜定位 有助于限制光子流量的条件。这在应用两个方面带来了根本性的挑战- 用于电压成像的光子显微镜，需要以千赫帧速率和高信噪比进行扫描。 要做到这一点，需要光学工程师和蛋白质工程师共同努力，开发出新的 仪器和传感器，以达到最佳解决方案。这项由多名调查员共同参与的工作建议推进 双光子显微镜和基因编码的电压敏感指示器，实现非侵入性 单细胞空间分辨率和单峰时相动作电位的群体水平测量 精确到哺乳动物大脑中清醒的行为动物的大脑。

PROJECT SUMMARY Understanding how information is processed in the mammalian neocortex has been a longstanding question in neuroscience. While the action potential is the fundamental bit of information, how these spikes encode representations and drive behavior remains unclear. In order to adequately address this problem, it has become apparent that experiments are needed in which activity from large numbers of neurons can be measured in a detailed and comprehensive manner across multiple timescales. Direct measurements of action potentials have primarily been achieved by electrophysiology. However, such measurements cannot easily be combined with other methods to assess the connectivity and molecular properties of neurons. Integrating functional, anatomical, and genetic information is critical for understanding how neuronal circuits are organized and computed. There have been long-standing efforts in developing optical methods for measuring neuronal activity due to its compatibility to simultaneously measure connectivity and molecular identity using fluorescent labeling techniques. Newly engineered genetically-encoded voltage-sensitive indicators have now opened the door for optical imaging of action potentials. Two-photon microscopy has been a proven method for deep non-invasive imaging into the brain. However, the fast millisecond transience of action potentials and the membrane localization of genetically-encoded voltage-sensitive indicators both contribute to conditions of limited photon flux. This creates fundamental challenges in the application of two- photon microscopy for voltage imaging that requires scanning at kilohertz frame rates with high signal to noise. To achieve this requires a concerted effort between optical engineers and protein engineers to develop new instrumentation and sensors to arrive at an optimal solution. This multi-investigator effort proposes to advance two-photon microscopy and genetically-encoded voltage-sensitive indicators to enable non-invasive population-level measurements of action potentials with single-cell spatial resolution and single-spike temporal precision deep into the mammalian brain of awake behaving animals.