Fig. 8. 5HT_2a agonist reverses blockade of CO₂ arousal caused by photoinhibition of DR^Sert terminals in the PBel, but not by deletion of PBel^CGRP neurons.

Panel a shows the experimental strategy that involved 3 h habituation to the plethysmographic chamber followed by with intraperitoneal injections of either 0.9% saline or TCB-2 (5HT_2a agonist; 5 mg/Kg), and CO₂ exposure either with photoinhibition of the DR^Sert terminals in the PBel or with deletions of the PBel^CGRP neurons. b is a representative polysomnographic record showing head twitches in the first hour after injection of TCB-2 (marked by arrows). To avoid confusion with arousal, this period was not used to analyze the latency of arousal to CO₂. c shows a representative trial of CO₂ exposure more than one hour after TCB-2 injection when animals attained stable sleep; data in this 3 h period were used for analyzing the latency to waking up to CO₂. Graphs d–f show for each group the mean (± SEM) latency of arousal (d), survival curves (e), and percent of trials (mean ± SEM) in which the animal remained asleep at various time points after onset of CO₂ exposure (f). **P < 0.01; ***P < 0.001; one-way (d) or two-way ANOVA (e and f). Silencing the DR^Sert terminals in PBel (Laser-ON) is compared here with the control (Laser-OFF), in both conditions (with injection of the 5HT_2a agonist- TCB-2 or saline; n = 3). Activation of 5HT_2a receptors completely reversed the blockade of hypercapnia-induced arousal caused by inhibiting DR^Sert terminals in the PBel (F_3,9 = 8.05; P = 0.006 for Laser-ON-saline, P = 0.91 for Laser-ON TCB-2, one-way ANOVA followed by multiple comparisons of treatment groups). In the PBel^CGRP deletion mice (n = 5), the arousal to CO₂ is compared between the saline and TCB-2 groups (F_5,17 = 15.4; P < 0.001 for saline and P = 0.002 for TCB-2, one-way ANOVA followed by multiple comparisons), where TCB-2 is ineffective in restoring the hyperapnia-induced arousal in the absence of the PBel^CGRP neurons. Effect of TCB-2 (5HT_2a receptor agonist) on respiration: g–i The respiratory airflow signals prior to CO₂ exposure and during CO₂ but prior to EEG arousal were analyzed off-line for tidal volume (mean ± SEM) (g), respiratory rate (mean ± SEM) (h), and minute ventilation (i). Laser-ON with either TCB-2 or saline injection, or Laser-OFF with TCB-2, showed identical tidal volume, respiratory rate, and minute ventilation prior to and during CO₂ exposure, indicating that neither inhibition of the DR^Sert terminals in the PBel nor restoring the CO₂ arousal response with TCB-2 affects the ventilatory response to CO₂. Data in d–i are presented as mean values ± SEM.