Bicistronic expression of a high-performance calcium indicator and opsin for all-optical stimulation and imaging at cellular resolution.

State-of-the-art all-optical systems promise unprecedented access to neural activity in vivo , using multiphoton optogenetics to allow simultaneous imaging and control of activity in selected neurons at cellular resolution. However, to achieve wide use of all-optical stimulation and imaging, simple strategies are needed to robustly and stably express opsins and indicators in the same cells. Here we describe a bicistronic adeno-associated virus (AAV) that expresses both the fast and bright calcium indicator jGCaMP8s, and a soma-targeted (st) and two-photon-activatable opsin, ChrimsonR. With this method, stChrimsonR stimulation with two-photon holography in the visual cortex of mice drives robust spiking in targeted cells, and neural responses to visual sensory stimuli and spontaneous activity are strong and stable. Cells expressing this bicistronic construct show responses to both photostimulation and visual stimulation that are similar to responses measured from cells expressing the same opsin and indicator via separate viruses. This approach is a simple and robust way to prepare neurons in vivo for two-photon holography and imaging. Significance statement New multiphoton photostimulation methods, combined with standard two-photon calcium imaging, can yield unprecedented levels of control for dissecting brain circuit function in vivo These all-optical methods rely on an interplay between optogenetics and calcium indicators, to both measure and control neural activity. However, genetic strategies to achieve reliable and stable co-expression of opsin and indicator are often challenging to execute. Here, we present a genetic tool to achieve robust co-expression of jGCaMP8s indicator and stChrimsonR opsin via a single injected virus. This approach facilitates all-optical experiments to investigate the circuit principles underlying brain activity.