Conditional corticotropin-releasing hormone overexpression in the mouse forebrain enhances rapid eye movement sleep

Abstract

Impaired sleep and enhanced stress hormone secretion are the hallmarks of stress-related disorders, including major depression. The central neuropeptide, corticotropin-releasing hormone (CRH), is a key hormone that regulates humoral and behavioral adaptation to stress. Its prolonged hypersecretion is believed to play a key role in the development and course of depressive symptoms, and is associated with sleep impairment. To investigate the specific effects of central CRH overexpression on sleep, we used conditional mouse mutants that overexpress CRH in the entire central nervous system (CRH-COE-Nes) or only in the forebrain, including limbic structures (CRH-COE-Cam). Compared with wild-type or control mice during baseline, both homozygous CRH-COE-Nes and -Cam mice showed constantly increased rapid eye movement (REM) sleep, whereas slightly suppressed non-REM sleep was detected only in CRH-COE-Nes mice during the light period. In response to 6-h sleep deprivation, elevated levels of REM sleep also became evident in heterozygous CRH-COE-Nes and -Cam mice during recovery, which was reversed by treatment with a CRH receptor type 1 (CRHR1) antagonist in heterozygous and homozygous CRH-COE-Nes mice. The peripheral stress hormone levels were not elevated at baseline, and even after sleep deprivation they were indistinguishable across genotypes. As the stress axis was not altered, sleep changes, in particular enhanced REM sleep, occurring in these models are most likely induced by the forebrain CRH through the activation of CRHR1. CRH hypersecretion in the forebrain seems to drive REM sleep, supporting the notion that enhanced REM sleep may serve as biomarker for clinical conditions associated with enhanced CRH secretion.

Figure 1

Time-course changes in sleep–wake patterns of corticotropin-releasing hormone (CRH)-COE-Nes mice. Data points ±s.e.m. indicate 2 h averages in time spent in waking, non-rapid eye movement (non-REM) sleep (nonREMS) and REM sleep (REMS) during a baseline recording day: open circles, CRH-COE^wt-Nes (wt), n=24; hatched area, CRH-COE^con-Nes (Nes con), n=20; gray circles with dotted line, CRH-COE^het-Nes (Nes het), n=20; black circles with solid line, CRH-COE^hom-Nes (Nes hom), n=21. Horizontal open bar, light period; horizontal filled bar, dark period. Two-way analysis of variance (ANOVA) among four genotypes; for waking during the light period F(3, 246)=3.13, P<0.05, during the dark period F(3, 246)=3.01, P<0.05; for non-REM sleep during the light period F(3, 246)=5.14, P<0.01, during the dark period F(3, 246)=1.71, n.s., for REM sleep during the light period F(3, 246)=4.32, P<0.01, during the dark period F(3, 246)=9.27, P<0.001. No statistical significances were found in vigilance between wild-type and control mice (left panels). Heterozygous and homozygous mice showed significant differences in between or compared with con mice, indicated by its respective line and ^* (right panels).

Figure 2

Changes in sleep patterns after sleep deprivation in corticotropin-releasing hormone (CRH)-COE-Nes mice. Data points ±s.e.m. indicate 2 h averages in time spent in non-rapid eye movement (non-REM) sleep (nonREMS) and REM sleep (REMS). Sleep deprivation (SD) was performed for 6 h from the beginning of the onset of the light period (Zeitgeber time 0, ZT0), indicated by a gray bar. Hatched area, CRH-COE^con-Nes (Nes con), n=14; gray circles with dotted line, CRH-COE^het-Nes (Nes het), n=12; black circles with solid line, CRH-COE^hom-Nes (Nes hom), n=14. Horizontal open bar, light period; horizontal filled bar, dark period. Two-way analysis of variance among three genotypes; for non-REM sleep during post-SD hours 6–12 (ZT6–12) in the light period F(2, 111)=4.13, P<0.05, for REM sleep during ZT6–12 in the light period F(2, 111)=9.55, P<0.001, during the 12-h dark period F(2, 222)=4.16, P<0.05. No statistical significances were found in recovery sleep between wild-type and control mice (data not shown). Heterozygous and/or homozygous mice showed significant differences compared with con mice, indicated by its respective line and ^*.

Figure 3

Sleep distributions in corticotropin-releasing hormone (CRH)-COE-Cam mice during baseline and after sleep deprivation. Data points±s.e.m. indicate 2 h averages in time spent in non-rapid eye movement (non-REM) sleep (nonREMS) and REM sleep (REMS). Sleep deprivation (SD) was performed for 6 h from the onset of the light period (Zeitgeber time 0, ZT0), indicated by a gray bar, following the baseline recording. Hatched area, CRH-COE^con-Cam (Cam con), n=9; gray squares with dotted line, CRH-COE^het-Cam (Cam het), n=10; black squares with solid line, CRH-COE^hom-Cam (Cam hom), n=11. Horizontal open bar, light period; horizontal filled bar, dark period. Two-way analysis of variance among three genotypes; on baseline day for REM sleep during the light period F(2, 162)=4.47, P<0.05, during the dark period F(2, 162)=3.34, P<0.05; on SD day for REM sleep during ZT6–12 in the light period F(2, 81)=7.94, P<0.001, during the 12-h dark period F(2, 162)=3.33, P<0.05. No statistical significances were found in respect to non-REM sleep on both days across all genotypes. Heterozygous and/or homozygous mice showed significant differences compared with con mice, indicated by its respective line and ^*.

Figure 4

Effects of sleep deprivation (SD) on plasma levels of corticosterone in corticotropin-releasing hormone (CRH)-COE-Nes and CRH-COE-Cam mice. Height±s.e.m. of columns indicates quantities of plasma corticosterone in the blood samples taken at Zeitgeber time (ZT)6 and 12 separately on baseline day and the day of SD from the same animals. White, CRH-COE^wt-Nes (wt); light gray, CRH-COE^con-Nes or CRH-COE^con-Cam (con); dark gray, CRH-COE^het-Nes or CRH-COE^het-Cam (het); black, CRH-COE^hom-Nes or CRH-COE^hom-Cam (hom). Varied numbers of samples in parentheses were due to technical reasons. ^* indicates statistical differences in the values during baseline between ZT6 and ZT12, compared with each respective genotype (P<0.05). ^* denotes the statistical significance in elevated levels of plasma corticosterone after SD, compared with the respective baseline (^*P<0.05, ^**P<0.01).

Figure 5

Effects of a corticotropin-releasing hormone receptor type 1 (CRHR1) antagonist, DMP696, on recovery sleep after sleep deprivation (SD) in CRH-COE-Nes mice. Data points ±s.e.m. indicate 1 h averages in time spent in non-rapid eye movement (non-REM) sleep (nonREMS) and REM sleep (REMS) during 7 h after 6-h SD initiated at Zeitgeber time (ZT)0. Animals received either vehicle or DMP696 per os at ZT5, at 1 h before the end of SD. Open and filled symbols indicate sleep changes after vehicle and antagonist treatment, respectively. Shaded area indicates the dark period. Numbers in brackets represent the sample size. Two-way analysis of variance for treatment effect between each genotype; CRH-COE^con-Nes for REM sleep F(1, 132)=4.55, P<0.05; CRH-COE^het-Nes for non-REM sleep F(1, 120)=5.68, P<0.05, for REM sleep F(1, 120)=8.19, P<0.01; CRH-COE^hom-Nes for non-REM sleep F(1, 132)=5.90, P<0.05, for REM sleep F(1, 132)=8.62, P<0.01. ^* denotes statistical significance in treatment (P<0.05).