All-optical electrophysiology with improved genetically encoded voltage indicators reveals interneuron network dynamics in vivo

All-optical electrophysiology can be a powerful tool for studying neural dynamics in vivo, as it offers the ability to image and perturb membrane voltage in multiple cells simultaneously. The "Optopatch" constructs combine a red-shifted archaerhodopsin (Arch)-derived genetically encoded voltage indicator (GEVI) with a blue-shifted channelrhodopsin actuator (ChR). We used a video-based pooled screen to evolve Arch-derived GEVIs with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). By combining optogenetic stimulation of individual cells with high-precision voltage imaging in neighboring cells, we mapped inhibitory and gap junction-mediated connections, in vivo. Optogenetic activation of a single NDNF-expressing neuron in visual cortex Layer 1 significantly suppressed the spike rate in some neighboring NDNF interneurons. Hippocampal PV cells showed near-synchronous spikes across multiple cells at a frequency significantly above what one would expect from independent spiking, suggesting that collective inhibitory spikes may play an important signaling role in vivo. By stimulating individual cells and recording from neighbors, we quantified gap junction coupling strengths. Together, these results demonstrate powerful new tools for all-optical microcircuit dissection in live mice.