Fig. 5. Optogenetic silencing of the DR^Sert neurons acutely prevents hypercapnia-induced arousal.

Schematic of a mouse (a) implanted with EEG/EMG and glass fiber targeting the DR in the experimental protocol to test CO₂ arousal, as shown in b. The 593 nm laser is on for 20 sec before and 10 sec after each 30 sec period of CO₂ stimulation during the Laser-ON condition. Panels c, d show the EEG power spectrum, EEG, EMG, CO₂ level in the chamber, and respiration during representative trials of CO₂ exposure in Laser-OFF (c) and Laser-ON (d) conditions. The mouse awoke in 17 sec in response to the rising CO₂ during Laser-OFF in c, as demonstrated by desynchronization of the EEG (decrease of power in lower frequencies; shown in top two traces), increase in EMG (third trace) and sudden increase in the rate and volume of ventilation at the time of EEG arousal (lower trace) in c. With Laser-ON (d), the mouse did not wake up to the CO₂ stimulus as can be seen by the presence of synchronized EEG, and minimal EMG activity, during the steadily increasing respiration in response to CO₂ (there are brief artifacts in the plethysmograph air flow trace at the shift in gas mixtures). Panels e–g show graphically the effect of acute silencing of the DR^Sert neurons on the latency of CO₂ arousal (mean ± SEM) (e) in Laser-OFF and Laser-ON conditions (DR^Sert ArchT, n = 6; DR^WT ArchT, n = 3), and the mice in the DR^Sert group are shown using line graphs in both Laser conditions. The survival curves (f) show the reduction in arousal (mean ± SEM) across the CO₂ exposure. Panel g shows that ~40% of the mice with silencing of DR^Sert neurons did not awaken within 30 sec (F_3,14 = 49.41, P < 0.001) of onset of CO₂ exposure, whereas without the laser the same mice woke up in every trial, and these were statistically compared using the one-way ANOVA followed by multiple comparisons for treatment groups. Data in e–g are presented as mean values ± SEM. **P < 0.01; ***P < 0.001; one-way (e, g) or two-way ANOVA (f).