RT Journal Article SR Electronic T1 Bicistronic Expression of a High-Performance Calcium Indicator and Opsin for All-Optical Stimulation and Imaging at Cellular Resolution JF eneuro JO eNeuro FD Society for Neuroscience SP ENEURO.0378-22.2023 DO 10.1523/ENEURO.0378-22.2023 VO 10 IS 3 A1 Paul K. LaFosse A1 Zhishang Zhou A1 Nina G. Friedman A1 Yanting Deng A1 Anna J. Li A1 Bradley Akitake A1 Mark H. Histed YR 2023 UL http://www.eneuro.org/content/10/3/ENEURO.0378-22.2023.abstract AB 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.