Contribution of 5-HT2 receptor subtypes to sleep-wakefulness and respiratory control, and functional adaptations in knock-out mice lacking 5-HT2A receptors

Abstract

Serotonin (5-hydroxytryptamine; 5-HT) plays key roles in sleep-wakefulness regulation. Evidence indicates that 5-HT2 receptors are involved mainly in non-rapid eye movement sleep (NREMS) regulation and respiratory control. Here, we investigated the relative contribution of 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptor subtypes to NREMS and breathing during sleep, using 5-HT2 subtype-selective ligands in wild-type (5-HT(2A)+/+) and knock-out (5-HT(2A)-/-) mice that do not express 5-HT(2A) receptors. Acute blockade of 5-HT(2A) receptors induced an increase in NREMS in 5-HT(2A)+/+ mice, but not 5-HT(2A)-/- mutants, which spontaneously expressed less NREMS than wild-type animals. In 5-HT(2A)+/+ mice, 5-HT(2B) receptor blockade produced a reduction of NREMS, whereas receptor activation induced an increase in this sleep stage. These effects were less pronounced in 5-HT(2A)-/- mice, indicating a lower sensitivity of 5-HT(2B) receptors in mutants, with no change in 5-HT(2B) mRNA. Blockade of 5-HT(2C) receptors had no effect on NREMS in both strains. In addition, an increase in EEG power density after sleep deprivation was observed in 5-HT(2A)+/+ mice but not in 5-HT(2A)-/- mice. Whole-body plethysmographic recordings indicated that 5-HT(2A) receptor blockade in 5-HT(2A)+/+ mice reduced NREMS apneas and bradypneas that occurred after sighs. In contrast, in 5-HT(2A)-/- mutants, NREMS apneas were not modified, and bradypnea after sighs were more pronounced. Our results demonstrate that 5-HT exerts a 5-HT(2B)-mediated facilitation of NREMS, and an influence respectively inhibitory on NREMS and facilitatory on sleep apnea generation, via 5-HT(2A) receptors. Moreover, 5-HT(2A) gene knock-out leads to functional compensations yielding adaptive changes opposite to those caused by pharmacological blockade of 5-HT(2A) receptors in 5-HT(2A)+/+ mice.

Figure 1.

Effects of acute treatment with the 5-HT_2A receptor antagonist MDL 100907 at various doses (abscissa) on amounts of vigilance states in 5-HT_2A+/+ (dotted line) and 5-HT_2A–/– (solid line) mice. Data (mean ± SEM of 4–8 animals) are expressed as minutes during the first 3 postinjection hours. At the time of injection, there were no significant differences in amounts of vigilance states between 5-HT_2A+/+ and 5-HT_2A–/– mice. *p < 0.05, significantly different from vehicle (0 on abscissa); post hoc Bonferroni's–Dunn's test. Error bars represent SEM.

Figure 2.

Amounts of W, NREMS, and REMS across the light/dark cycle in 5-HT_2A+/+ (dotted line) and 5-HT_2A–/– (solid line) mice. Data (mean ± SEM of 10 animals per group) are expressed as minutes per 3 h during 24 h. The black bar on the abscissa represents the dark period of the light/dark cycle. ^#p < 0.05, significantly different from 5-HT_2A+/+ mice; post hoc Bonferroni's–Dunn's test. Error bars represent SEM.

Figure 3.

Effects of 6 h of sleep deprivation starting at light onset on delta power, during four consecutive 2 h intervals of the recovery period in 5-HT_2A+/+ (dotted line) and 5-HT_2A–/– (solid line) mice. Statistical analysis was performed on EEG power values, and data (mean ± SEM of 9 animals in each group) are plotted as a percentage of the paired values obtained under control conditions. *p < 0.05, significantly different from respective control value; post hoc Bonferroni's–Dunn's test. ^#p < 0.05, significantly different from 5-HT_2A+/+ value; post hoc Bonferroni's–Dunn's test. Error bars represent SEM.

Figure 4.

Effects of acute treatment with the 5-HT_2B/2C receptor antagonist SB 206553 (2.5 mg/kg, i.p.; hatched bars) on amounts of vigilance states in 5-HT_2A+/+ (white bars) and 5-HT_2A–/– (black bars) mice. Data (mean ± SEM of 7 animals per group) are expressed as minutes per hour during each of the first 3 postinjection hours. *p < 0.05, significantly different from paired-saline-treated animals; post hoc Bonferroni's–Dunn's test. Error bars represent SEM.

Figure 5.

Effects of acute treatment with the 5-HT_2C receptor antagonist SB 242084 at various doses (abscissa) on amounts of vigilance states in 5-HT_2A+/+ (dotted line) and 5-HT_2A–/– (solid line) mice. Data (mean ± SEM of 4–6 animals) are expressed as minutes during the first 3 postinjection hours. *p < 0.05, significantly different from vehicle (0 on abscissa); post hoc Bonferroni's–Dunn's test. Error bars represent SEM.

Figure 6.

A, B, Effects of acute treatment with the 5-HT_2B receptor antagonist SB 215505 (A) or the 5-HT_2B receptor agonist BW 723C86 (B) at various doses on amounts of vigilance states in 5-HT_2A+/+ (dotted line) and 5-HT_2A–/– (solid line) mice. Data (mean ± SEM of 5–11 animals per group) are expressed as minutes during the first postinjection hour. *p < 0.05, significantly different from vehicle (0 on abscissa); ^#p < 0.05, significantly different from respective 5-HT_2A+/+ value; post hoc Bonferroni's–Dunn's test. Error bars represent SEM.

Figure 7.

5-HT_2A, 5-HT_2B, and 5-HT_2C mRNA expression in the hypothalamus of 5-HT_2A+/+ (white bars) and 5-HT_2A–/– (black bars) mice. Results [relative quantity (RQ), mean ± SEM of 4 animals in each group] are expressed in arbitrary units (A.U.) after normalization to an endogenous reference gene (HPRT). Normalization with GADPH endogeneous gene (see Materials and Methods) yielded similar results. ^#p < 0.05, significantly different from respective 5-HT_2A+/+ value; unpaired Student's t test. Error bars represent SEM.

Figure 8.

Implication of 5-HT_2A receptors in the dynamics of breathing after sighs. Sighs are followed by both an immediate and a delayed decrease in respiration frequency, and apnea occurs in these two phases of bradypnea. A, Example of plethysmograph recording of a sigh (0 on abscissa). a.u., Arbitrary units. B, Average rate of respiration after a sigh. Dotted lines correspond to ± SD. C, Distribution of apneas after a sigh; the counts of apneas have been normalized to the number of sighs (bin size, 0.5 s). A–C are from the same 5-HT_2A+/+ mouse recorded from 10:00 A.M. to 4:00 P.M. D–F, The delayed decrease in breathing frequency after a sigh is almost completely suppressed after 5-HT_2A receptor blockade by MDL 100907 in 5-HT_2A+/+ mice but is more pronounced in 5-HT_2A–/– mutants. D, Average changes in respiration frequency after a sigh in 5-HT_2A+/+ during the first 3 h (between 10:00 A.M. and 1:00 P.M.) and during the next 3 h (between 1:00 P.M. and 4:00 P.M.) after MDL 100907 (2 mg/kg, i.p.) administration (n = 7). The rate of breathing is normalized to the rate before the sigh, E, Average changes in spontaneous respiration frequency after a sigh in 5-HT_2A+/+ (n=14) and 5-HT_2A–/– (n=12) mice. Recordings were made between 10:00 A.M. and 4:00 P.M. ^#,*p < 0.05, significantly different from vehicle or 5-HT_2A+/+ mice, respectively; Student's t test.