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

Area of Science:

• Neuroscience
• Optogenetics
• Calcium Imaging

Background:

• All-optical interrogation combines two-photon calcium imaging and optogenetic stimulation for precise neural circuit analysis.
• Spectral overlap between imaging lasers and opsins can affect experimental outcomes.
• Understanding this spectral overlap is crucial for accurate all-optical studies.

Purpose of the Study:

• To investigate if 920 nm two-photon calcium imaging of GCaMP6s affects response desensitization in C1V1-expressing neurons targeted for 1035 nm optogenetic stimulation.
• To quantify the impact of imaging parameters on neural responses during all-optical experiments.

Main Methods:

• Systematic variation of inter-stimulus interval and imaging duration during two-photon calcium imaging and optogenetic stimulation.
• Targeted stimulation of mouse layer 2/3 barrel cortex and visual cortex neurons.
• Measurement of photostimulation responses in targeted and non-targeted neurons.

Main Results:

• Two-photon imaging at 920 nm decreased trial-by-trial photostimulation responses in targeted C1V1 neurons, an effect worsened by shorter inter-stimulus intervals.
• This desensitization, attributed to imaging laser-induced opsin excitation, was observed across the field of view.
• Network effects were less pronounced with shorter imaging durations.

Conclusions:

• Methodological optimizations can mitigate trial-by-trial decreases in photostimulation response during all-optical experiments.
• Reducing imaging-induced opsin desensitization minimizes external variability in future all-optical studies.
• Findings provide practical guidance for designing more robust all-optical interrogation experiments.