SIMULTANEOUS HIGH-PERFORMANCE TWO-PHOTON IMAGING AND TWO-PHOTON MANIPULATION OF NEURONAL CIRCUITS WITH MINIMIZED CROSSTALK BETWEEN IMAGING AND PHOTOSTIMULATION

All-optical two-photon techniques are essential in the causal investigation of neuronal circuits underlying mammalian behavior at cellular resolution. These methods generally combine green fluorescent indicators (e.g., GCaMPs) and red-shifted opsins (e.g., C1V1, ChRmine). However, red-shifted opsins exhibit action spectra which significantly overlap with the excitation spectra of green fluorescent indicators. This spectral overlap can lead to unintended neuronal depolarization during imaging epochs (i.e., crosstalk between imaging and photostimulation), ultimately biasing results of all-optical experiments.
In this work, we developed an all-optical strategy combining high-efficiency two-photon photostimulation of the blue-shifted opsin stCoChR (Forli et al., 2021) with two-photon calcium imaging of the high-performance red light-sensitive indicator RCaMP3 (Yokoyama et al., 2024) in layer 2/3 principal cells of the primary somatosensory cortex of awake, head-fixed mice. We obtained functional co-expression of the indicator and the opsin using either two independent viruses or a bicistronic viral strategy. Using these approaches, we monitored network activities with largely improved dynamic range and sensitivity compared to previously employed red-shifted indicators. Moreover, this choice of indicator and opsin minimized crosstalk between imaging and photostimulation, while maintaining reliable neuronal photo-activation with low average laser power per cell.
Altogether, our results show that this novel experimental approach provides high-efficiency two-photon recording and perturbation of neuronal populations in vivo, while ensuring sufficient spectral separation between the action spectrum of the opsin and the excitation spectrum of the calcium indicator.