OptoConnectics

A major challenge in modern neuroscience is to determine how synaptic connectivity within neural networks shape functional outcomes, a critical step in reverse-engineering both basic and complex brain functions. Current methods, however, for assessing connectivity are limited and rely either on statistical models lacking ground truth validation or low-throughput single-cell electrophysiological recordings conducted in vivo or ex vivo. A solution to this fundamental challenge lies in all-optical in vivo electrophysiology — a powerful approach combining single-cell voltage recording with precise optogenetic stimulation. This technique enables direct measurement of synaptic weights between neurons in intact animals over multiple days, creating unprecedented opportunities for longitudinal connectivity studies. Yet, despite its transformative potential, the feasibility of this approach remains unproven. In this project, four teams of experts in in vivo functional imaging will use novel, acousto-optic deflector (AOD ) based, two-photon microscopy (resulting from the research work of one of the teams and disseminated by a French startup) to establish all-optical in vivo electrophysiology as a proof of concept. We will perform connectivity measurements in functionally and genetically defined neurons in vivo, across different cell-types and spatial scales, in both mice and zebrafish utilizing two AOD-microscopy operating modes we have developed and published. Namely, ULoVE and 3D-CASH, in 2D and 3D respectively. While we already have evidence of stable voltage recordings using the ULoVE mode to record neurons expressing the genetically encoded voltage indicator JEDI-2P, we will further optimize the combination of opsins, voltage indicators, and stimulation techniques to enhance subthreshold voltage sensitivity, reduce crosstalk during optical voltage recording, and ensure high temporal precision. We will fine-tune the parameters for utilizing both the ULoVE and 3D-CASH approaches to scale up the investigation of synaptic connectivity. We will validate our methodology at two different scales. At the local circuit level, we will probe connectivity within functionally defined neural circuits in mice and study unisensory and multisensory connectivity rules in sensory networks in the visual and auditory cortex. At the entire brain level, we will leverage the zebrafish model to estimate brain-wide connectivity, mapping interactions between hundreds of brain regions, each containing hundreds of neurons, and study how mesoscale connectivity defines endogenous spontaneous brain activity. This project aims to demonstrate the significance of this groundbreaking high-throughput methodology for mapping of neuronal connectivity in awake animals.