Efficient Two-Photon Voltage Imaging of Neuronal Populations at Behavioral Timescales

项目总结 了解哺乳动物的新大脑皮层是如何处理信息的由来已久 神经科学中的问题。虽然动作电位是信息的基本比特，但这些 钉子编码表示法，驾驶行为尚不清楚。为了充分解决这一问题 问题已经变得很明显，在这个实验中，需要大量来自 可以在多个时间尺度上以详细和全面的方式测量神经元。直接 动作电位的测量主要是通过电生理学实现的。然而，这样的 测量不能轻易地与其他方法相结合来评估连接性和分子 神经元的特性。整合功能、解剖和遗传信息对于 了解神经元回路是如何组织和计算的。长期以来，我们一直在努力 在开发用于测量神经元活动的光学方法中，由于其与同时 使用荧光标记技术测量连接性和分子同一性。我们已经开发出一种 双光子可激发基因编码压敏指示器和超快双光子显微镜 这使得对大脑深处的动作电位进行光学测量成为可能。然而，在高分辨率下成像 由于照片漂白和照片风险，超过几分钟的信噪比仍然具有挑战性 损坏。为了让这些新技术在神经科学应用中变得更强大，它是 需要提高双光子电压成像的稳定性、可靠性和效率。至 要做到这一点，需要光学工程师、蛋白质工程师和 计算科学家为BREAD优化仪器、传感器和图像分析 传播。这项多研究人员的工作建议推进双光子电压成像，以使 在动物行为和学习的时间尺度上持续跟踪种群活动。

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. We have developed a two-photon-excitable genetically-encoded voltage-sensitive indicator and ultra-fast two-photon microscope that enables optical measurements of action potentials deep into the brain. However, imaging at high signal-to-noise beyond several minutes remains challenging due to photo-bleaching and risks of photo- damage. In order for these new technologies to become more robust for neuroscience applications, it is necessary to improve upon the stability, reliability, and efficiency of two-photon voltage imaging. To achieve this, it requires a concerted effort between optical engineers, protein engineers, and computational scientists to optimize instrumentation, sensors, and image analysis for broad dissemination. This multi-investigator effort proposes to advance two-photon voltage imaging to enable sustained tracking of population activity at timescales of animal behavior and learning.