A role for cryptochromes in sleep regulation

Abstract

Background: The cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the generation of circadian rhythms, as mice lacking both of these genes (cry1,2-/-) lack circadian rhythms. We studied sleep in cry1,2-/- mice under baseline conditions as well as under conditions of constant darkness and enforced wakefulness to determine whether cryptochromes influence sleep regulatory processes.

Results: Under all three conditions, cry1,2-/- mice exhibit the hallmarks of high non-REM sleep (NREMS) drive (i.e., increases in NREMS time, NREMS consolidation, and EEG delta power during NREMS). This unexpected phenotype was associated with elevated brain mRNA levels of period 1 and 2 (per1,2), and albumin d-binding protein (dbp), which are known to be transcriptionally inhibited by CRY1,2. To further examine the relationship between circadian genes and sleep homeostasis, we examined wild type mice and rats following sleep deprivation and found increased levels of per1,2 mRNA and decreased levels of dbp mRNA specifically in the cerebral cortex; these changes subsided with recovery sleep. The expression of per3, cry1,2, clock, npas2, bmal1, and casein-kinase-1epsilon did not change with sleep deprivation.

Conclusions: These results indicate that mice lacking cryptochromes are not simply a genetic model of circadian arrhythmicity in rodents and functionally implicate cryptochromes in the homeostatic regulation of sleep.

Figure 1

Time course of sleep (upper panels) and NREMS EEG delta power (lower panel). Data from baseline (BSL), sleep deprivation (SD), and recovery (REC) are shown. Open (Cry1,2^-/-) and closed (Cry1,2^+/+) symbols designate mean hourly values ± 1 SEM. In the lower panel, thicker lines connect mean predicted delta power values based on the sleep-wake distribution in individual mice; (see Methods). Triangles mark intervals in which recovery values differed from corresponding baseline values within each genotype (triangle orientation designates direction of deviation; P < 0.05, post-hoc paired t-tests). Gray bars at the bottom of each panel mark intervals with significant genotype differences (P < 0.05, post-hoc t-tests). The baseline dark period was depicted twice to illustrate the changes at the dark-to-light transition.

Figure 2

EEG power in the 1–20 Hz range for NREMS, REM sleep (REMS) and wake during baseline. (A) EEG spectral power in cry1,2^-/- (thick lines) and wild type mice (thin lines). Differences between the genotypes are limited to NREMS delta power. (B) Delta power (1–4 Hz) during wake-to-NREMS transitions in the baseline light (left) and dark (right panel) period. Gray horizontal bars underneath the curves indicate significant genotype differences (P < 0.05; post-hoc t-tests). Error bars span ± 1 SEM.

Figure 3

Time course of sleep and NREMS EEG delta power during constant dark conditions (DD). Layout and symbols are same as in Figure 1. Gray bars at the bottom of each panel mark intervals with significant genotype differences (P < 0.05, post-hoc t-tests). The subjective day is marked with a gray horizontal bar at the top of the upper panel. The first 12-h represents the last dark-period under baseline (BSL) light-dark conditions.

Figure 4

Whole-brain mRNA levels for dbp, per1, and per2 in cry1,2^-/- and wild type mice. Levels of all three genes are elevated in cry1,2^-/- mice (KO) relative to wild type (WT) controls at ZT6 when dbp, per1, and per2 mRNAs are lowest in the forebrain of wild type mice [37]. In the lower three panels mean (± 1 SEM) expression levels are depicted. β-actin expression was used as an internal standard.

Figure 5

Sleep deprivation alters mRNA levels of per1, per2, and dbp. (A) RT-PCR analysis of the expression of five 'clock'-genes in the mouse cortex across four experimental conditions [C = control; R = recovery sleep; SD = sleep deprived; ZT = Zeitgeber time (i.e., 6 or 10 h after light-onset)]. g3pdh expression was used as an internal standard. Bars depict mean ± 1 SEM. Asterisks denote significant differences (P < 0.05) between the experimental and corresponding control group (Student-Newman-Keuls post-hoc tests; 1-way ANOVA factor 'condition': P < 0.05, for per1,2, and dbp only; n = 7/condition) (B) per1 mRNA falls significantly in rat cortex during a 2-h recovery period (R) subsequent to 6 h SD ending at ZT6 (P < 0.05, t-test). Northern analysis was performed on cortex of five sleep-deprived rats and five rats that were allowed 2 h of recovery sleep (ZT8).