Ultra-precise all-optical manipulation of neural circuits with multifunctional Cre-dependent transgenic mice

Abstract

Causally relating the detailed structure and function of neural circuits to behavior requires the ability to precisely and simultaneously write-in and read-out neural activity. All optical systems that combine two photon (2p) calcium imaging and targeted photostimulation provide such an approach, but require co-expression of an activity indicator, such as GCaMP, and an optogenetic actuator, ideally a potent soma-targeted opsin. In the mammalian brain, such co-expression has so far been achieved by viral transduction, which is invasive and can produce variable, focal, and sometimes toxic overexpression. To overcome this challenge, we developed and validated a Cre-reporter mouse ("Ai203") that conditionally expresses a soma-targeted opsin, ChroME, fused to GCaMP7s. 1p or 2p illumination of expressing neurons in vitro and in vivo produces powerful, precise activation comparable to viral expression of ChroME. The soma-targeted GCaMP7s provides sensitive activity measurements for tracking physiological activity, and the soma-targeted ChroME provides powerful control over neural ensemble activity with holographic optogenetics. We further demonstrate the use of the Ai203 reporter line in 1p optogenetic manipulation of performance on a cortex-dependent visual task and in 2p synaptic connectivity mapping. This new transgenic line could thus greatly facilitate the study of neural circuits by providing a flexible, convenient, and stable tool for all-optical access to large, cell-type specific neural populations throughout the nervous system.

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

“…dev.) Similarly, their mean OSI was 0.62 (Kondo and Ohki, 2016) and 0.56 (Ai203; Bounds et al, 2022), while we report a mean OSI of 0.46 ± 0.28. For gOSI, they report a mean of 0.46 (Kondo and Ohki, 2016), while we report a mean gOSI of 0.31 ± 0.18.…”

“…In order to determine whether cells expressing jGCaMP8s-P2A-stChrimsonR exhibit physiologically expected sensory-evoked responses (see Kondo and Ohki, 2016; Zhang et al, 2021; Bounds et al, 2022), we used two-photon imaging to record jGCaMP8s activity from excitatory cells in layer 2/3 of mouse V1 during and between presentations of retinotopically-aligned drifting grating (Gabor patches, 15 degree full-width half-max, FWHM) stimuli. We presented gratings across eight orientations (N = 20 repetitions per orientation) in random order and computed trial-averaged ΔF/F for different orientations.…”

“…Addressing this limitation has been a core focus of past genetic approaches: successful co-expression yields the ability to measure responses to optogenetic stimulation. Recent studies typically find many, but not all, cells are responsive to photostimulation (for example, ∼80% with Ai203 transgenic line, Bounds et al, 2022; ∼90% with ChRmine, ∼85% with ChRome2s, ∼75% ChRome2f, Sridharan et al, 2022). On par with prior reports for other opsins, we found similar and greater levels of photoactivatable cells in our experiments (∼95% of cells photoactivatable).…”

Rapid learning of neural circuitry from holographic ensemble stimulation enabled by model-based compressed sensing

“…Opsin-expressing neurons could therefore be straightforwardly segmented from an image of mRuby3 expression and subsequently stimulated. However, in some cases opsin-expressing neurons do not also express a separate nuclear fluorophore for straightforward identification of photosensitive cell nuclei [Bounds et al, 2021]. We therefore considered whether our connectivity mapping system could be used in the case where neuron locations are not known a priori (i.e., when mapping is performed "blind").…”

Modeling the Diverse Effects of Divisive Normalization on Noise Correlations

“…During imaging, mice were head-fixed in a tube, and were performing an operant visual detection task [Bounds et al, 2021]. Briefly, mice were trained to withhold licking when no stimulus was present, and lick within a response window after stimulus presentation.…”