Friedman, N. G. | bioRxiv Science

bioRxiv2022

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

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.

neuroscience↗

bioRxiv2019

The BioLuminescent-OptoGenetic in vivo Response to Coelenterazine is Proportional, Sensitive and Specific in Neocortex

BioLuminescent (BL) light production can modulate neural activity and behavior through coexpressed OptoGenetic (OG) elements, an approach termed BL-OG. Yet, the relationship between BL-OG effects and bioluminescent photon emission has not been characterized in vivo. Further, the degree to which BL-OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and non-toxic effects. We systematically tested the effects of four key components of the BL-OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin-3 (LMO3) BL-OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin-1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically non-sensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortex in vivo, there is a large CTZ range wherein BL-OG effects are specific to its active chemogenetic mechanism.

neuroscience↗