A rhodopsin-based two-photon voltage indicator for all-optical mapping of synaptic connectivity in vivo

将动物的行为与不同神经细胞群体的活动联系起来是神经科学的长期目标。此外，为了理解哺乳动物大脑是如何计算和编码行为的，需要生成这个细胞群体的连接矩阵。因此，一种同时记录和调节大量神经细胞群活动的方法的发展，将使突触连接的非破坏性和慢性映射成为可能，这将改变神经科学。目前高通量监测神经元活动的最先进方法是双光子钙成像，它在过去几十年中提供了基本的见解。然而，由于钙传感器只报告膜电位的阈值以上变化，以阈值以下电压变化编码的信息仍然难以捉摸。此外，钙指示剂对细胞膜的超极化不敏感，这限制了映射到兴奋性连接。从钙离子成像到电压成像的转变将克服这些限制，似乎是神经科学的下一个挑战。理想的电压光传感器需要双光子激发，以增加成像深度、降低光毒性和背景荧光，但目前的电压传感器双光子性能较差。为了应对这一挑战，该项目将开发一种基因编码的双光子电压传感器，并将其与双光子可激活光致动器相结合。这种基因编码的双光子激活工具将允许在全光学实验中绘制突触连接性和强度。与以前的方法不同，这将使在体内以高通量和慢性方式进行单细胞分辨率的功能连接研究成为可能。

Linking the behavior of an animal to the activity of a distinct neuronal cell population is a long-standing aim of neuroscience. Additionally, generating the connectivity matrix of this cell population is needed to understand how the mammalian brain computes and encodes behavior. The development of a method to simultaneously record and modulate the activity of a large neuronal cell population, which allows non-destructive and chronic mapping of synaptic connections, will hence transform neurosciences. The current state-of-the-art method for high-throughput monitoring of neuronal activity is two-photon calcium imaging, which provided fundamental insights over the last decades. However, as calcium sensors only report supra-threshold changes in membrane potential, information encoded in sub-threshold voltage changes remains elusive. Further, calcium indicators are insensitive to hyperpolarization of the cell membrane, which limits the mapping to excitatory connections. A transition from calcium to voltage imaging would overcome those limitations and appears as the next challenge in neuroscience. To be effective, an ideal optical sensor of voltage should be used with two-photon excitation to increase imaging depth, reduce phototoxicity, and background fluorescence, but current voltage sensors have poor two-photon performances. To tackle this challenge, this project will develop a genetically-encoded two-photon voltage sensor and combine it with a two-photon activatable optogenetic actuator. This genetically-encoded, two-photon activated tool will allow to map synaptic connectivity and strength in an all-optical experiment. In contrast to previous approaches, this will enable studies of functional connections in vivo with single cell resolution in a high throughput and chronic fashion.