PROJECT SUMMARY/ABSTRACT Two-photon all-optical electrophysiology in behaving mice Neurons communicate through electrical signals, so the ability to record membrane potential from dozens or hundreds of points simultaneously within the brain of a behaving animal would be a transformative capability for neuroscience. This proposal is to develop advanced tools-molecular reporters and microscopes for genetically targeted all-optical electrophysiology in behaving mice. Specifically, we propose to co-develop two-photon (2P)-excitable genetically encoded voltage indicators (GEVIs) and a new type of 2P voltage-imaging microscope. An important component will be to develop protocols for using these tools in vivo and to disseminate the tools to the neuroscience community. The first aim is to develop improved molecular reporters of membrane voltage, which are compatible with 2P excitation. We propose a set of detailed spectroscopic studies to understand how microbial rhodopsin-based GEVIs interact with 2P excitation. We then propose to screen opsin scaffolds from diverse naturally occurring microbial rhodopsins for improved 2P voltage sensitivity, followed by a high-throughput screen of targeted mutations to improve 2P voltage indicating properties of selected scaffolds. The output of this effort will be new 2P-excitable GEVIs with improved brightness, photostability, and voltage sensitivity. Even with the best GEVI imaginable, the signals will only be as good as the optical system used for measurement. 2P voltage imaging in vivo presents stringent technical demands due to the short duration of action potentials (1 ms), the small signals (1 – 10%), and the confinement of useful signals to the nanometers- thick cell membrane. In our second aim, we propose a new approach to high-speed scanning which can visit up to 512 points in less than 1 ms, an order of magnitude faster than other scanning systems. The third aim is to use the tools to enable qualitatively new types of measurements. We will develop protocols for (1) functional connectivity mapping in vivo, (2) measurements of microcircuit dynamics under sensory and optogenetic inputs, and (3) mapping dendritic integration and back-propagation of action potentials within individual neurons. The development of in vivo voltage imaging has historically been a challenge because the protein engineering, instrumentation, and data analysis problems are intertwined. The present proposal describes an integrated approach to turn in vivo voltage imaging into a mainstream tool for neuroscience.