Sleep Deprivation Influences Circadian Gene Expression in the Lateral Habenula

Abstract

Sleep is governed by homeostasis and the circadian clock. Clock genes play an important role in the generation and maintenance of circadian rhythms but are also involved in regulating sleep homeostasis. The lateral habenular nucleus (LHb) has been implicated in sleep-wake regulation, since LHb gene expression demonstrates circadian oscillation characteristics. This study focuses on the participation of LHb clock genes in regulating sleep homeostasis, as the nature of their involvement is unclear. In this study, we observed changes in sleep pattern following sleep deprivation in LHb-lesioned rats using EEG recording techniques. And then the changes of clock gene expression (Per1, Per2, and Bmal1) in the LHb after 6 hours of sleep deprivation were detected by using real-time quantitative PCR (qPCR). We found that sleep deprivation increased the length of Non-Rapid Eye Movement Sleep (NREMS) and decreased wakefulness. LHb-lesioning decreased the amplitude of reduced wake time and increased NREMS following sleep deprivation in rats. qPCR results demonstrated that Per2 expression was elevated after sleep deprivation, while the other two genes were unaffected. Following sleep recovery, Per2 expression was comparable to the control group. This study provides the basis for further research on the role of LHb Per2 gene in the regulation of sleep homeostasis.

Figure 1

A crystal violet staining photomicrograph of LHb lesion. (a) showed the normal Hb and (b) showed the lesion of LHb. The single arrow identifies the MHb, whereas the double arrows identify the LHb. Scale bars represent 100 μm.

Figure 2

The effect of LHb lesion on sleep-wake times following sleep deprivation in rats. (a) indicates the typical EEG and EMG recording, respectively, for wake, NREMS, and REMS times. The wake state was identified by the presence of desynchronized EEG and high EMG activity. The NREMS consisted of high-amplitude slow waves together with a low EMG relative to awake. The REMS was identified by the presence of regular θ activity coupled with low EMG relative to NREMS. (B1), (C1), and (D1), respectively, show the wake, NREMS, and REMS times following sleep deprivation before and after LHb lesion. Data from 18 h EEG recording were analyzed and presented as means ± SEM (n = 6). ^∗ P < 0.05, ^∗∗ P < 0.01, compared, respectively, difference between the wake, NREMS, and REMS times before and after sleep deprivation. ^# P < 0.05, ^## P < 0.01, compared, respectively, difference between the wake and NREMS times following deprivation before and after LHb lesion. (B2), (C2), and (D2) show, respectively, the graphs of wake, NREMS, and REMS plotted per 6 h for a 24 h period before and after lesion. ^∗ P < 0.05 compared with sleep deprivation at the same time point.

Figure 3

Sleep deprivation alters mRNA levels of clock genes. qPCR analysis of the expression of three clock genes (Per1, Per2, and Bmal1) in the LHb (a) and SCN (b) of the rat. Data was presented as mean ± SEM (n = 8/each group). ^∗ P < 0.05, compared with control group (by unpaired t-test).

Figure 4

qPCR analysis of Per1, Per2, and Bmal1 levels in the LHb following 2 hours of recovery after sleep deprivation. Data was presented as mean ± SEM (n = 8/each group).