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

SignificanceThe combination of two-photon calcium imaging and two-photon optogenetic stimulation, termed all-optical interrogation, provides spatial and temporal precision when recording and manipulating neural circuit activity in vivo. 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. Though 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. AimWe aimed to investigate whether two-photon imaging of the green fluorescent calcium reporter GCaMP6s at 920 nm increases the rate of desensitization in neurons expressing the red-shifted opsin C1V1. ApproachWe systematically varied either the inter-stimulus interval or the duration of two-photon calcium imaging during two-photon optogenetic stimulation of mouse layer 2/3 barrel cortex or visual cortex neurons. ResultsWe found that two-photon imaging at 920 nm increases the desensitization of photostimulation responses across trials in C1V1-expressing neurons - an effect that is exacerbated at shorter inter-stimulus intervals. Reduced photostimulation responses are not limited to targeted cells, and are found across the entire field of view. Such network effects are less pronounced at shorter imaging doses. ConclusionsOur results provide methodological optimizations that enable opsin desensitization to be mitigated in all-optical experiments. This will reduce an external source of trial-by-trial variability in future all-optical experiments.