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Fig. 7
NPVF neuronal activation induces tectal neuronal activity levels similar to normal sleep.
(A) Representative 2P-SPIM image of a 6 dpf Tg(elavl3:H2B-GCaMP6s) animal, ~70 μm from the dorsal surface. The white box indicates the region of the optic tectum analyzed in subsequent panels. The white oval indicates the location of NPVF neurons. The inset shows a GCaMP6s image superimposed on a brightfield image. Scale: 50 μm. (B) Neural activity, recorded as GCaMP6s fluorescence, of a representative animal during >36 h of imaging in DD. Imaging sessions were performed for 2 min at 1 Hz every 15 min. No light perturbation was applied during the first 31 s of each trial to record intrinsic brain activity. To assess arousal, a red light was turned on at t = 32 s, gradually increased in intensity until t = 93 s, then turned off. We observed increased neural activity at light onset and offset, with habituation in between. Signal is calculated as ΔF = F – F0 where F0 = lowest signal measured during the recording period. (C) Normalized neural activity, plotted as function of time, smoothed over a 5-trial sliding window, and averaged over 5–25, 32–37, and 93–98 s time windows, representing intrinsic, On-flash evoked response and Off-flash evoked response, respectively. Signal is normalized by FN = average intrinsic activity during subjective day. (D) Correlation of evoked activity to intrinsic activity. (E,F) During subjective day, animals were imaged for 15 trials, each consisting of 2 min of imaging at 1 Hz every 5 min. Conditions were identical to those used in (B). Representative neural activity recorded for individual ReaChR- and ReaChR+ animals are shown. Optogenetic stimulation began after trial 5. Signal for each animal is calculated with baseline F0 = averaged signal over the first five trials, then normalized by FN = peak of signal after red light is turned on, for the same animal. (G) Mean ± SEM of the normalized neural activity for four Tg(elavl3:H2B-GCaMP6s); Tg(npvf:ReaChR-mCitrine) larvae (red) and three Tg(elavl3:H2B-GCaMP6s) siblings (blue). (H) Mean ± SEM steady-state normalized neural activity after optogenetic activation, averaged over trials 10–15, shows 68% lower activity for ReaChR+ animals than their ReaChR- siblings (p<0.001, two-sample t-test). See also Figure 7—figure supplements 1 and 2.