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bioRxiv · 10.1101/2022.09.06.506779

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

LaFosse, P. K.·Zhou, Z.·Friedman, N. G.·Deng, Y.·Akitake, B.·Histed, M. H.
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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. Significance statementThe recent advent of holographic photostimulation methods in conjunction with standard two-photon calcium imaging promises unprecedented levels of control in manipulating and dissecting brain circuitry in vivo while reading out neural activity. These all-optical methods rely on a working synergy between optogenetic strategies to both measure calcium activity through genetically-encoded calcium indicators and modulate cell activity through light-activated opsins. Genetic strategies to achieve reliable and stable co-expression of opsin and indicator remain sparse and 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 to help facilitate experiments aiming to use holography to investigate the circuit principles underlying brain activity.

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LaFosse, P. K., Zhou, Z., Friedman, N. G., Deng, Y., Akitake, B., Histed, M. H.. 2022-09-08. Bicistronic expression of a high-performance calcium indicator and opsin yields stable, robust cortical expression for holographic two-photon stimulation. https://doi.org/10.1101/2022.09.06.506779

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