Altered sleep homeostasis after restraint stress in 5-HTT knock-out male mice: a role for hypocretins

Abstract

Restraint stress produces changes in the sleep pattern that are mainly characterized by a delayed increase in rapid eye movement sleep (REMS) amounts. Because the serotonin (5-HT) and the hypocretin (hcrt) systems that regulate REMS are interconnected, we used mutant mice deficient in the 5-HT transporter (5-HTT(-/-)) to examine the role of 5-HT and hcrt neurotransmissions in the sleep response to stress. In contrast to wild-type mice, restraint stress did not induce a delayed increase in REMS amounts in 5-HTT(-/-) mice, indicating impaired sleep homeostasis in mutants. However, pharmacological blockade of the hcrt type 1 receptor (hcrt-R1) before restraint stress restored the REMS increase in 5-HTT(-/-) mice. In line with this finding, 5-HTT(-/-) mutants displayed after restraint stress higher long-lasting activation of hypothalamic preprohcrt neurons than wild-type mice and elevated levels of the hcrt-1 peptide and the hcrt-R1 mRNA in the anterior raphe area. Thus, hypocretinergic neurotransmission was enhanced by stress in 5-HTT(-/-) mice. Furthermore, in 5-HTT(-/-) but not wild-type mice, hypothalamic levels of the 5-HT metabolite 5-hydroxyindole acetic acid significantly increased after restraint stress, indicating a marked enhancement of serotonergic neurotransmission in mutants. Altogether, our data show that increased serotonergic -and in turn hypocretinergic- neurotransmissions exert an inhibitory influence on stress-induced delayed REMS. We propose that the direct interactions between hcrt neurons in the hypothalamus and 5-HT neurons in the anterior raphe nuclei account, at least in part, for the adaptive sleep-wakefulness regulations triggered by acute stress.

Figure 1.

Effects of restraint stress on sleep–wakefulness patterns in 5-HTT^+/+ and 5-HTT^−/− mice. Sleep–wakefulness states across the light/dark cycle under control conditions (open symbols) or after RS (filled symbols) in 5-HTT^+/+ (squares, top) and 5-HTT^−/− (triangles, bottom) mice. Mice were, or were not, restrained for 90 min (5:30 P.M. to 7:00 P.M.) and then monitored for sleep and wakefulness (from 7:00 P.M. on day 1 to 5:00 P.M. on day 2; abscissa). Amounts of vigilance states, expressed as minutes per 2 h, are the mean ± SEM of eight animals in each group. The gray area corresponds to the difference between control and RS conditions. *p < 0.05, significantly different from the control conditions; paired Student's t test after ANOVA.

Figure 2.

Restraint stress alters REMS pattern in 5-HTT^+/+ and 5-HTT^−/− mice. Mice were, or were not, restrained for 90 min (5:30 P.M. to 7:00 P.M.) and then monitored for sleep and wakefulness starting at 7:00 P.M. A, REMS amounts, absolute number, and mean duration of REMS episodes for the first 2 h (7:00 P.M. to 9:00 P.M. period) after the 90 min control (C) or RS session in 5-HTT^+/+ and 5-HTT^−/− mice. B, REMS characteristics for the following 8 h period (11:00 P.M. to 7:00 A.M.) in the same control (C) and RS 5-HTT^+/+ and 5-HTT^−/− mice. Data are the mean ± SEM of independent determinations in eight animals in each group. *p < 0.05, **p < 0.01, significantly different from the control condition; paired Student's t test after ANOVA.

Figure 3.

Effects of acute treatment with the hcrt-R1 antagonist SB-334867 before restraint stress on sleep–wakefulness patterns in 5-HTT^+/+ and 5-HTT^−/− mice. A, Experimental protocol: on the first day of recording (Day 1), 5-HTT^+/+ and 5-HTT^−/− mice were injected with the vehicle at 5:00 P.M. and then subjected to the control (C) procedure for stress by disconnecting (5:30 P.M.) and connecting again (7:00 P.M.) the recording cable to the mouse. Recordings were obtained until 5:00 P.M. the next day. On the following day (Day 2), at 5:00 P.M., 5-HTT^+/+ and 5-HTT^−/− mice were injected with the vehicle or the hcrt-R1 antagonist SB-334867 (30 mg/kg, i.p.), and RS was started 30 min later (at 5:30 P.M.). Recordings of vigilance states were performed as for the preceding control step (Day 1). B, REMS amounts for 8 h starting at 11:00 P.M. under control conditions (C) or after RS in 5-HTT^+/+ and 5-HTT^−/− mice previously injected with the vehicle (veh) or SB-334867 (SB). Data are the mean ± SEM of eight to nine animals in each group. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from the respective control condition; paired Student's t test after ANOVA.

Figure 4.

Activation of hypocretin neurons after restraint stress in 5-HTT^+/+ and 5-HTT^−/− mice. A, Schematic representation of the experimental protocol. The activation of hypocretin neurons has been measured in 5-HTT^+/+ and 5-HTT^−/− mice immediately (t₀) or 5 h (t₀ + 5 h) after the end of 90 min RS and in nonstressed (control; C) paired mice killed at the same time. B, Representative immunocytochemical labeling at the level of the perifornical area of the lateral hypothalamus showing few hypocretin-IR cells (brown staining) with c-Fos-IR labeling (dark staining) in control conditions in 5-HTT^+/+ (1) and 5-HTT^−/− (2) mice. RS induced a strong and immediate (t₀) increase in the number of c-Fos-IR nuclei in hypocretin-IR neurons in both 5-HTT^+/+ (3) and 5-HTT^−/− (4) mice. Black arrows, c-Fos-IR and hypocretin-IR neurons. Scale bar, 10 μm. C, D, Percentage of hypocretin-IR neurons with c-Fos-IR labeling in control conditions (C) and after RS in 5-HTT^+/+ and 5-HTT^−/− mice. Mice were killed immediately (t₀; C) or 5 h (t₀ + 5 h; D) after RS or control conditions. Data are expressed as the mean ± SEM of seven to eight mice in each group. *p < 0.05, ***p < 0.001, significantly different from the control condition; ^#p < 0.05, 5-HTT^−/− versus wild-type mice; Fisher's PLSD test after ANOVA.

Figure 5.

Effects of restraint stress on hcrt-1 levels in the anterior raphe area of 5-HTT^+/+ and 5-HTT^−/− mice. Hcrt-1 tissue levels were measured in the anterior raphe area of 5-HTT^+/+ and 5-HTT^−/− mice in control condition (C) or immediately after RS. Hcrt-1 tissue levels are expressed as percentage of values in nonstressed 5-HTT^+/+ mice. Absolute values were as follows (ng/μg protein): 1.46 ± 0.11 (control condition in 5-HTT^+/+), 1.54 ± 0.13 (RS in 5-HTT^+/+), 2.09 ± 0.11 (control condition in 5-HTT^−/−), and 2.56 ± 0.16 (RS in 5-HTT^−/−). Data are the mean ± SEM of seven to nine animals in each group. *p < 0.05, significantly different from the control condition; ^##p < 0.01, ^###p < 0.001, 5-HTT^−/− versus wild-type mice; Fisher's PLSD test after ANOVA.

Figure 6.

Model of hcrt–5-HT interactions in 5-HTT^−/− mutants and 5-HTT^+/+ wild-type mice: influence of stress. A, In wild-type mice, restraint stress activates both hcrt and 5-HT neurons. Whereas hcrt excites 5-HT neurons through hcrt-R1 and hcrt-R2 receptors, 5-HT inhibits the activity of hcrt neurons through 5-HT_1A receptors. In this model, the inhibitory influence of the indolamine on hcrt and 5-HT neurons (via 5-HT_1A autoreceptors) will result in a new steady-state level and would allow the stress-induced sleep response (i.e., the delayed increase in REMS). B, In 5-HTT^−/− mice, hypocretinergic neurotransmission is enhanced at baseline. Such activation can result from adaptive changes in serotonergic neurotransmission and, notably, 5-HT_1A receptor downregulation and/or desensitization in the hypothalamus that is triggered by the excess of extracellular 5-HT in mutant mice. In addition, these mice exhibit strong downregulation and desensitization of 5-HT_1A autoreceptors in the dorsal raphe nucleus. We propose that this reduced 5-HT_1A-mediated inhibitory influence of 5-HT on both 5-HT and hcrt neurons contributes to higher RS-induced enhancement of hypocretinergic and serotonergic neurotransmissions in 5-HTT^−/− versus wild-type mice. Such alterations of hcrt–5-HT interactions may account for the maladaptive REMS response to stress in 5-HTT^−/− mice (i.e., the absence of the delayed increase in REMS), because pharmacological blockade of hcrt-R1 restored the delayed increase in REMS normally observed after restraint stress.