Fig. 3: Active pixel power control mitigates cross-talk in neural circuits.
From: Active pixel power control for crosstalk-free all-optical neural interrogation
a Experimental strategy for evaluating cross-talk spreading in the downstream neurons. We first conduct holographic optogenetic stimulation on single or multiple ChR+ neurons while simultaneously performing volumetric calcium imaging to identify the putative downstream neurons. Next, the imaging power on ChR+ neurons is reduced using APPC, and we then conduct volumetric calcium imaging to exam the cross-talk by measuring the neuronal activity of putative downstream neurons. b 920 nm two-photon optogenetics stimulation effectively activates both ChR2 (Blue) and ChrimsonR (Red). The somatic GCaMP signals (mean ± s.d.) averaged across ten trails under the same optogenetic, with onset indicated by the red bar for 1 s. c Field of view of GCaMP6s fluorescent signal of zebrafish brain depth projection across 80–180 µm. Red patch: optogenetic stimulation target. Orange patch: off-target control stimulation position. Green: putative downstream neurons. Scale bar: 50 µm . d Heatmap displaying the activities of the optogenetic target neuron and 13 downstream neurons. The optogenetic target neuron was imaged at 5 mW, 10 mW and 15 mW, while the downstream neurons are imaged at 15 mW. The downstream neurons are determined based on the correlation between the regressor related to optogenetics stimulation (stimulation onset at red lines). e Comparison of mean calcium fluctuation (mean±s.d.) of ChR2+ group downstream neurons at different image power on upstream neuron (n = 42 neurons, 8 fish, Friedman test with Dunn’s multiple comparisons test, two-sided; no optogenetic stimulation: 0 mW: 0.53 ± 0.41, 5 mW: 0.59 ± 0.51, 10 mW: 0.60 ± 0.47, 15 mW: 0.98 ± 0.74) (f) same as (e) but for ChrimsonR+ group (n = 30 neurons, 7 fish, Friedman test with Dunn’s multiple comparisons test, two-sided; no optogenetic stimulation: 0 mW: 0.34 ± 0.34, 5 mW: 0.35 ± 0.29, 10 mW: 0.60 ± 0.50, 15 mW: 0.66 ± 0.48). g Spikes value (mean±s.d.) of ChR2+ group downstream neurons at different image power on upstream neuron (n = 42 neurons, 8 fish, Friedman test with Dunn’s multiple comparisons test, two-sided; no optogenetic stimulation: 0 mW: 13.40 ± 13.47, 5 mW: 13.18 ± 11.43, 10 mW: 17.21 ± 17.86, 15 mW: 24.13 ± 21.93). h Same as (g) but for ChrimsonR+ group (n = 30 neurons, 7 fish, Friedman test with Dunn’s multiple comparisons test, two-sided; no optogenetic stimulation: 0 mW: 14.31 ± 9.40, 5 mW: 14.00 ± 11.59, 10 mW: 18.89 ± 13.95, 15 mW: 28.67 ± 20.86).
