Bicistronic expression of a high-performance calcium indicator and opsin yields stable, robust cortical expression for holographic two-photon stimulation

Abstract

Patterns of activity across many neurons are fundamental units of neural computation. Two-photon holographic photostimulation allows both delivering input to, and imaging responses from, patterns or populations of neurons. However, to make this method an easily-deployable tool, simple methods are needed to robustly and stably express opsins and indicators in the same cells. Here we describe a bicistronic adeno-associated virus (AAV) that in transfected cells expresses both the fast and bright calcium indicator GCaMP8s, and a soma-targeted (st) and two-photon-activatable opsin, ChrimsonR. With this method, in the visual cortex of mice, stChrimsonR stimulation with two-photon holography drives robust spiking in targeted cells, and neural responses to visual sensory stimuli and spontaneous activity are strong and easy to measure. stChrimsonR is a good choice of opsin when a balance is needed between stimulation-laser activatability and avoidance of imaging laser activation. This approach is a simple and robust way to prepare neurons in vivo for two-photon holography and imaging.