Desensitization of opsin responses during all-optical interrogation depends on imaging parameters

Significance: . All-optical experiments often use red-shifted opsins in combination with green fluorescent reporters of neuronal activity. However, their excitation spectra still partially overlap, meaning that the imaging laser can excite the opsin. Although some care has been taken in the past to understand the effects of this spectral overlap; further work is required to understand its impact on the findings of all-optical studies. Aim: We aimed to investigate whether two-photon imaging of the green fluorescent calcium reporter GCaMP6s at 920 nm increase the rate of response desensitization in neurons targeted for two-photon stimulation at 1035 nm expressing the red-shifted opsin C1V1. Approach: We systematically varied either the inter-stimulus interval or the duration of two-photon calcium imaging during targeted two-photon optogenetic stimulation of mouse layer 2/3 barrel cortex or visual cortex neurons. Results: We found that two-photon imaging at 920 nm decreases trial-by-trial photostimulation responses in targeted C1V1-expressing neurons-an effect that is exacerbated at shorter inter-stimulus intervals. This is consistent with the imaging laser increasing the rate of opsin desensitization. Reduced photostimulation responses are not limited to targeted cells and are found across the field of view. Such network effects are less pronounced at shorter imaging doses. Conclusions: Our results provide methodological optimizations that enable trial-by-trial decreases in photostimulation response to be mitigated in all-optical experiments. This will reduce an external source of trial-by-trial variability in future all-optical experiments.