Central deficiency of corticotropin-releasing hormone receptor type 1 (CRH-R1) abolishes effects of CRH on NREM but not on REM sleep in mice

Abstract

Study objectives: Corticotropin-releasing hormone (CRH) is the major activator of the hypothalamic-pituitary-adrenocortical (HPA) system and orchestrates the neuroendocrine, autonomous as well as behavioral responses to stress. Many studies suggest an influence of CRH on sleep-wake regulation even in the absence of stressors. However, none of these studies yet clearly distinguished between central and peripheral effects of CRH. Therefore, we investigated in CNS-specific CRH receptor type 1 deficient mice whether centrally administered CRH could induce its sleep-wake modulatory effects without peripheral induction of HPA activity.

Design: Male mice (C57BL/6J, CNS-specific CRH-R1 knockout [CKO] mice and their control littermates [CL]) were intracerebroventricularily (i.c.v.) injected with vehicle or 3 different doses of CRH shortly before the beginning of the light period. Electroencephalogram (EEG) and electromyogram (EMG) were monitored to compare the effects of CRH on vigilance states with or without presence of central CRH-R1. To quantify HPA-axis reactivity to CRH injections in CKO and CL animals, blood samples were analyzed to determine plasma corticosterone concentrations.

Results: I.c.v. injections of CRH promoted wakefulness while decreasing NREMS in C57BL/6J and CRH-R1 CL animals, whereas such changes were not exerted in CKO mice. However, REMS suppression after CRH application persisted in all animals. I.c.v. injected CRH increased plasma corticosterone levels in both CL and CKO mice.

Conclusions: The results demonstrated that CRH has a major impact on wake and NREMS regulation which is predominantly mediated through central CRH-R1. Peripheral actions of CRH, i.e., elevated HPA activity, may interfere with its central effects on REMS but not on NREMS suppression.

Figure 1

Effects of i.c.v. CRH injections on vigilance states in C57BL/6J mice. Corticotropin-releasing hormone (CRH) dose-dependently increased wake levels during the light period for up to 10 h after application and decreased those levels for almost the entire course of the dark period. Contrariwise, NREMS was dose dependently decreased during the light period and showed increases during the successive dark period. REMS was similarly reduced immediately after CRH application and almost totally blocked for up to 4 and 6 h after injection of 1.0 μg and 3.0 μg CRH, respectively. Shortly after transition to the dark period a dose-dependent increase in REMS was observed. Depicted are percentages of the given vigilance states (indicated on the y-axis) of 2-h means ± SEM for baseline (■) and each CRH treatment (∘: 0.3 μg, 1.0μg, •: 3.0 μg). The hatched area indicates mean values ± SEM after vehicle treatment. The symbols +,_*, and # denote statistically significant differences by comparison of vehicle versus treatment with 0.3, 1.0, or 3.0 μg of CRH, respectively (tests with contrasts in ANOVA, P < 0.05). Solid lines under those symbols connect consecutive time points showing statistical differences. The open and filled bar on the x-axis indicates the light and dark period, respectively.

Figure 2

Effects of i.c.v. CRH injections on vigilance states in CL (A, B, C) and CRH-R1 CKO mice (D, E, F). CRH increased wake levels in CL mice in a dose-dependent fashion during the light period and decreased NREMS amounts at the same time. REMS reduction tended to occur also in a dose-dependent fashion, although only the highest dose of CRH induced significant reductions in comparison to vehicle treatment. In the conditional knockout animals, in contrast, wake promoting and NREMS reducing effects of CRH were almost totally blunted. However, dose-dependent REMS reduction could still be induced by CRH injections during the light period. For graph denotations please refer to Figure 1.

Figure 3

Effects of i.c.v. CRH injections on CORT levels in CL and CRH-R1 CKO mice. Compared to baseline and vehicle conditions a significant increase in plasma CORT was induced by 1.0 μg of CRH by 2 h after application in both CL (n = 8) and CRH-R1 CKO (n = 8) mice, although CL animals displayed significantly higher CORT levels after neuropeptide treatment compared to CKO animals. Symbols indicate significant differences in obtained CORT values with respect to different treatment conditions and their according genotypes (tests with contrasts in ANOVA, P < 0.05). For graph denotations please refer to Figure 1.

Supplementary Figure 1

CNS-restricted deletion of CRH-R1 expression in Crhr1^loxP/loxP nestin-cre animals. Autoradiographs of a CRH-R1-specific in situ hybridization of coronal brain sections from rostral (top) to caudal (bottom) demonstrating the spatial expression pattern of CRH-R1 throughout the brain of Crhr1^loxP/loxP control animals (CL, left). In the brain of Crhr1^loxP/loxP nestin-cre animals (CKO, right), no CRH-R1 expression is detectable.