Bicistronic Expression of a High-Performance Calcium Indicator and Opsin for All-Optical Stimulation and Imaging at Cellular Resolution

Abstract

State-of-the-art all-optical systems promise unprecedented access to neural activityin 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 neuronsin vivofor two-photon holography and imaging.Significance statementNew multiphoton photostimulation methods, combined with standard two-photon calcium imaging, can yield unprecedented levels of control for dissecting brain circuit functionin 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.

Single-cell optogenetics reveals attenuation-by-suppression in visual cortical neurons

“…We use cell-specific two-photon optogenetic stimulation (Marshel et al, 2019; Dalgleish et al, 2020; LaFosse et al, 2023) to measure cells’ IO functions. By providing the same fixed input at different points along a cells’ IO function, the corresponding evoked changes in output can be used to infer the IO function shape (Fig.…”

“…Stimulated cells during our experiments were defined as those whose centroid fell within a 15 micron radius of any stimulation target’s center (5-12 targets per pattern, average 9 ± 0.5) and whose “Opto” response value exceeded a 5% ΔF/F 0 threshold (N = 15 ± 1.3 stimulated cells per pattern). Stimulation of small groups of cells typically induces smaller network effects, such as weak, broad suppression (Jouhanneau et al, 2018; Chettih and Harvey, 2019; Oldenburg et al, 2022; LaFosse et al, 2023), though these small influences are unlikely to significantly impact the much larger evoked responses we measure in targeted cells.…”

Recurrent cortical networks encode natural sensory statistics via sequence filtering

“…A titanium headplate was affixed using C&B Metabond (Parkell) and a 3 mm craniotomy was made over the primary visual cortex (-3.1 mm ML, +1.5 mm AP from lambda) and a glass optical window chronically inserted. Mice were injected with either a mixture of two AAV viruses to induce GCaMP and stChrimsonR into the cells (mixed virus injections), or a bicistronic virus expressing both GCaMP and stChrimsonR in each cell (10). For the mixed injection, the fluorescent calcium indicator, (AAV9-syn-jGCaMP8s; titers: 5.0-10x10 12 genome copies (GC)/ml) and the soma-targeted opsin (AAV9-hsyn-DIO-stChrimson-mRuby2; titer: 3.0x10 12 GC/ml) were mixed in phosphate-buffered saline.…”

“…We used a viral preparation we developed ( 10 ) to express an opsin and a calcium indicator in excitatory neurons in layer 2/3 of mouse V1, and used a stimulation laser and holographic device to deliver input to groups of chosen single neurons (Fig. 1F).…”

“…2E), we found striking off-target responses — some nonstimulated cells are activated, and some are suppressed. These off-target neural responses, in neurons that are not receiving stimulation input, are likely to be primarily due to local recurrent interactions, not due to axonal or dendrite activation, as here we used a somatically-targeted opsin (stChRimsonR, ( 10 )).…”