All-optical neuronal circuit interrogation with reduced crosstalk between imaging and photostimulation

All-optical two-photon (2P) methods are considered of great importance to causally investigate neuronal circuits underlying mammalian behavior with near cellular resolution. These approaches typically involve a combination of green indicators (e.g., GCaMPs) and red-light-sensitive opsins (e.g., C1V1, ChRmine). However, red-shifted opsins typically display a blue-shifted tail in their absorption spectra, partially overlapping with the absorption spectra of green indicators. This overlap may lead to unwanted neuronal perturbationduring imaging periods (Packer et al., 2015; Rickgauer & Tank, 2009), resulting in biased results. Here, we developed an all-optical 2P preparation combining high-efficiency two-photon manipulation using the large-conductance, blue-light-sensitive opsin stCoChR (Forli et al., 2021) and two-photon imaging using the high-dynamic range red-shifted indicator RCaMP3 (Yokoyama et al., 2024) in layer 2/3 pyramidal neurons of primary somatosensory cortex (S1) of awake head-fixed mice. With this experimental strategy, we achieved: i) recording of network activity with a larger dynamic range and sensitivity compared to previously used red-shifted indicators; ii) effective photo-activation of neurons with low average power per cell; iii) reduced crosstalk between imaging and photostimulation; iv) functional co-expression of the opsin and indicator with a bicistronic construct developed in collaboration with Prof. Sakamoto, Kyoto University; v) investigation of the influence of local network dynamics and animal arousal level on the two-photon photostimulation neuronal responses. This novel experimental approach will enable high-efficiency recording and manipulation of neuronal networks in vivo, while granting effective spectral separation between the excitation of the opsin and of the calcium indicator.