Central serotonin neurons are required for arousal to CO2

Abstract

There is a long-standing controversy about the role of serotonin in sleep/wake control, with competing theories that it either promotes sleep or causes arousal. Here, we show that there is a marked increase in wakefulness when all serotonin neurons are genetically deleted in mice hemizygous for ePet1-Cre and homozygous for floxed Lmx1b (Lmx1b(f/f/p)). However, this only occurs at cool ambient temperatures and can be explained by a thermoregulatory defect that leads to an increase in motor activity to generate heat. Because some serotonin neurons are stimulated by CO(2), and serotonin activates thalamocortical networks, we hypothesized that serotonin neurons cause arousal in response to hypercapnia. We found that Lmx1b(f/f/p) mice completely lacked any arousal response to inhalation of 10% CO(2) (with 21% O(2) in balance N(2)) but had normal arousal responses to hypoxia, sound, and air puff. We propose that serotonin neurons mediate the potentially life-saving arousal response to hypercapnia. Impairment of this response may contribute to sudden unexpected death in epilepsy, sudden infant death syndrome, and sleep apnea.

Fig. 1.

Lmx1b^f/f/p mice have decreased sleep at cool ambient temperatures. Representative 24-h sleep/wake histograms in male WT (Upper) and Lmx1b^f/f/p (Lower) mice at 23 °C (A and D), 30 °C (B and E), and 33 °C (C and F) are shown. The horizontal black bars in A–F indicate lights-off. (G) Bar graphs depict the percentage of the 24-h recording period spent in each vigilance state for each genotype at 23 °C (n = 8 WT, n = 8 Lmx1b^f/f/p), 30 °C (n = 9 WT, n = 8 Lmx1b^f/f/p), and 33 °C (n = 10 WT, n = 10 Lmx1b^f/f/p), as indicated. Four WT and four Lmx1b^f/f/p mice were housed at 30 °C for 10 d before being studied at each of the three recording temperatures. The remaining mice were housed at 23 °C. These data are pooled because there was no significant effect of housing temperature on sleep architecture at any given recording temperature (Fig. S3). *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001.

Fig. 2.

Genetic deletion of 5-HT neurons prevents arousal to CO₂. (A) Four-minute EEG (Top), EMG (Middle), and PCO₂ (Bottom) traces from WT and Lmx1b^f/f/p mice showing response to 7% CO₂. Arousal in the WT mouse is indicated by a decrease in EEG amplitude (and a corresponding increase in EEG frequency), with a concomitant increase in EMG amplitude. The O₂ level is 21% (balance N₂) throughout the traces. (Horizontal scale bar, 30 s; vertical scale bar, 5 μV.) (B) Thirteen-minute EEG (Top), EMG (Middle), and PCO₂ (Bottom) traces from an Lmx1b^f/f/p mouse that remained asleep throughout 7% CO₂ exposure. N, NREM; R, REM. (Horizontal scale bar, 60 s; vertical scale bar, 5 μV.) (C) Latency to arousal following gas change from RA to 7% CO₂ in WT (n = 9) and Lmx1b^f/f/p (n = 10) mice. **P < 0.0001. Arousal latency includes the delay from the gas change to gas arrival in the chamber. (D) Percentage of time spent in Wake, NREM, and REM during RA and 7% CO₂ exposure for WT (n = 9) and Lmx1b^f/f/p (n = 10) mice. *P < 0.01; **P < 0.0001. There was no significant difference between male and female mice (Fig. S6); thus, data have been pooled. (E) Hypercapnia induced arousal in a dose-dependent manner in WT mice, but Lmx1b^f/f/p mice were indifferent to every PCO₂ level (n = 6 of each genotype). Shown is arousal latency following RA (0% CO₂) and 3, 5, 7, and 10% CO₂. Arousal latency determined as in C. *P < 0.005; **P < 0.0001 between genotypes. ^†P < 0.005; ^††P < 0.0001 among WT mice compared with RA (0% CO₂). There was no significant difference among Lmx1b^f/f/p mice at any CO₂ concentration compared with RA.

Fig. 3.

Genetic deletion of 5-HT neurons does not affect the arousal response to hypoxia. (A) Ninety-second EEG (Top), EMG (Middle), and PO₂ (Bottom) traces from WT and Lmx1b^f/f/p mice indicating arousal response to hypoxia (∼8% O₂). Arousal is indicated as in Fig. 2. (Horizontal scale bar, 15 s; vertical scale bar, 5 μV.) (B) Latency to arousal following gas change from RA to ∼8% O₂ [P = 0.36; n = 9 WT (5 female, 4 male) mice, n = 8 Lmx1b^f/f/p (4 female, 4 male) mice]. There was no difference between male and female mice; thus, data were pooled.

Fig. 4.

Genetic deletion of 5-HT neurons does not affect the arousal response to air puff or sound. Thirty-second EEG (Upper) and EMG (Lower) traces from WT and Lmx1b^f/f/p mice indicate arousal response to air puff (Left) and sound (Right) stimuli. Arrows indicate stimulus presentation. Arousal is indicated as in Fig. 2. (Horizontal scale bar, 5 s; vertical scale bar, 5 μV.)