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

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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.