Glucocorticoid receptors in the locus coeruleus mediate sleep disorders caused by repeated corticosterone treatment

Stress induced constant increase of cortisol level may lead to sleep disorder, but the mechanism remains
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OPEN SUBJECT AREAS: SLEEP DISORDERS STRESS AND RESILIENCE

Received 20 January 2015 Accepted 5 March 2015 Published 24 March 2015

Correspondence and requests for materials should be addressed to Y.-H.Z. ([email protected] pku.edu.cn)

* These authors contributed equally to this work.

Glucocorticoid receptors in the locus coeruleus mediate sleep disorders caused by repeated corticosterone treatment Zi-Jun Wang*, Xue-Qiong Zhang*, Xiang-Yu Cui, Su-Ying Cui, Bin Yu, Zhao-Fu Sheng, Sheng-Jie Li, Qing Cao, Yuan-Li Huang, Ya-Ping Xu & Yong-He Zhang Department of Pharmacology, Peking University, School of Basic Medical Science, Beijing 100191, China.

Stress induced constant increase of cortisol level may lead to sleep disorder, but the mechanism remains unclear. Here we described a novel model to investigate stress mimicked sleep disorders induced by repetitive administration of corticosterone (CORT). After 7 days treatment of CORT, rats showed significant sleep disturbance, meanwhile, the glucocorticoid receptor (GR) level was notably lowered in locus coeruleus (LC). We further discovered the activation of noradrenergic neuron in LC, the suppression of GABAergic neuron in ventrolateral preoptic area (VLPO), the remarkable elevation of norepinephrine in LC, VLPO and hypothalamus, as well as increase of tyrosine hydroxylase in LC and decrease of glutamic acid decarboxylase in VLPO after CORT treatment. Microinjection of GR antagonist RU486 into LC reversed the CORT-induced sleep changes. These results suggest that GR in LC may play a key role in stress-related sleep disorders and support the hypothesis that repeated CORT treatment may decrease GR levels and induce the activation of noradrenergic neurons in LC, consequently inhibit GABAergic neurons in VLPO and result in sleep disorders. Our findings provide novel insights into the effect of stress-inducing agent CORT on sleep and GRs’ role in sleep regulation.

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tress triggers important neuroendocrine responses that enable the organism to survive and restore homeostasis. The primary responses include the rapid activation of hypothalamic–pituitary–adrenal (HPA) axis and sympathoadrenal system, leading to the release of adrenocorticotropic hormone, glucocorticoids, and catecholamines1. Sleep is an important component of human homeostasis. Sleep disorders are closely associated with significant medical, psychological, and social disturbances, such as depression2. Activation of the HPA axis or sympathetic nervous system results in wakefulness, and these hormones, including corticotropin-releasing factor, adrenocorticotropic hormone, cortisol (or corticosterone; CORT), norepinephrine, and epinephrine, are associated with attention and arousal3. Glucocorticoids are the final mediators in HPA axis cascade and critical for the pathogenesis of sustained stress-related sleep disorders. Many researchers have reported that sustained stress4,5 increases cortisol levels and can induce sleep disorders, including poor sleep quality and shorter sleep duration. Corticosteroid receptors, glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs), are highly expressed in the brain6. Researchers have demonstrated that glucocorticoids can regulate sleep directly via MRs and GRs7,8. Because of the different affinities of these two receptors, GRs play an important role when corticosteroids reach stressful levels1. Additionally, GRs are abundantly expressed in sleep-wake-related brain stem nuclei9,10. The sleep-wake-related brain nuclei contain sleep-promoting c-aminobutyric acid (GABA) neurons in ventrolateral preoptic nucleus (VLPO), and wake/active neurons including histaminergic tuberomammillary nucleus (TMN), orexinergic perifornical area (Pef), serotonergic dorsal raphe nuclei (DRN), noradrenergic locus coeruleus (LC), and cholinergic neurons in the pontine laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei11,12. Retrograde and anterograde tract-tracing studies indicated that VLPO neurons are reciprocally connected with TMN, Pef, DRN, LC and LDT/PPT13,14. Cano et al.15 reported that acute stress-induced insomnia was related to altered activity in sleep-wake regulating nuclei such as VLPO, LC. In addition, both acute stress (such as footshock or immobilization16,17) and acute hydrocortisone administration18 have been shown to suppress sleep in animal models. In the pilot studies, we demonstrated that repeated administration of glucocorticoids induced SCIENTIFIC REPORTS | 5 : 9442 | DOI: 10.1038/srep09442

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www.nature.com/scientificreports sleep changes were different. Thus, the unknown mechanism of repeated stress-induced sleep disorders needs further investigation. To better understand the underlying mechanism of long-term treatment of glucocoritcoids induced sleep disorders, we repeatedly treated rats with CORT for 7 days to mimic stress, and these animals were then evaluated with regard to sleep parameters. To correlate such behavior with possible underlying mechanisms, we measured the activity of sleep-wake-regulating nuclei, major monoamine neurotransmitters in related brain areas, GR and MR expression in LC, glutamic acid decarboxylase (GAD) in VLPO, and tyrosine hydroxylase (TH) in LC. The levels of GRs decreased in LC after 7-day CORT treatment, prompting us to test whether administration of the GR antagonist RU486 can reverse stress-related sleep disturbances.

Results Repeated administration of CORT induced sleep disorders in rats. Table 1 showed that there were significant reduction of total sleep time (TST, F1,16 5 36.987, p , 0.001), non-rapid eye movement sleep time (NREM, F1,16 5 55.206, p , 0.001) and light sleep time (LST, F1,16 5 36.624, p , 0.001) after CORT treatment; While rapid eye movement sleep time (REM) was obviously increased compared with vehicle (F1,16 5 6.086, p 5 0.026), and slow wave sleep time (SWS, F1,16 5 0.950, p 5 0.345) was unaffected. Meanwhile, the administration of CORT significantly prolonged the sleep latency (SL, F1,16 5 16.672, p 5 0.001) and shortened the REM sleep latency (REM SL, F1,16 5 23.114, p , 0.001). The REM sleep time ratio (REM%, F1,16 5 13.980, p 5 0.002) was significantly raised up as well without changing of light sleep time ratio (LST%, F1,16 5 1.613, p 5 0.223) and SWS time ratio (SWS%, F1,16 5 0.436, p 5 0.519). Repeated administration of CORT augmented neuronal activity in LC and suppressed neuronal activity in VLPO. To identify alterations in sleep-wake-related nuclei, we performed doublestaining immunohistochemistry in the VLPO, Pef, TMN, PPT, DRN, LDT, and LC. After 7 days CORT treatment (40 mg/kg, s.c.), the ratio of Fos1 and GAD1 neurons in the VLPO (Fig. 1c) was significantly decreased (t10 5 2.694, p 5 0.023), whereas the ratio of Fos1 and TH1 neurons in the LC (Fig. 1f) was significantly increased (t9 5 22.803, p 5 0.021). We did not detect noticeable differences (Fig. 2) in the Pef (t10 5 21.369, p 5 0.201), TMN (t11 5 0.151, p 5 0.882), PPT (t10 5 20.719, p 5 0.489), DRN (t8 5 20.974, p 5 0.359), or LDT (t9 5 0.019, p 5 0.985). These results indicate that changes in sleep parameters caused by sustained elevations of CORT involved alterations in neuron activity in the LC and VLPO. GR protein level in LC was lowered after repeated administration of CORT. To determine whether the increase in noradrenergic neuron activity in the LC was directly attributable to CORT Table 1 | Treatment of CORT (40 mg/kg, s.c.) for 7 days altered sleep parameters in rats Vehicle Total sleep (min) 247.2 6 6.6 NREM sleep (min) 230.2 6 6.5 Light sleep (min) 227.7 6 6.5 Slow wave sleep (min) 8.0 6 2.9 REM sleep (min) 18.2 6 1.5 Sleep latency (min) 25.9 6 3.6 REM sleep latency (min) 32.7 6 4.9 Light sleep (%) 92.1 6 3.7 Slow wave sleep (%) 3.3 6 1.2 REM sleep (%) 7.3 6 0.5

CORT (40 mg/kg, s.c., 7 days) 198.0 6 3.8** 170.1 6 4.6** 169.4 6 7.2** 4.7 6 1.6 27.8 6 1.5* 48.1 6 4.1** 6.9 6 1.2** 85.6 6 3.5 2.3 6 0.8 14.0 6 1.8**

Data are represented as mean 6 S.E.M. (n 5 8 , 9/group); *p , 0.05 and **p , 0.01 vs vehicle (Student’s t-test).

SCIENTIFIC REPORTS | 5 : 9442 | DOI: 10.1038/srep09442

administration, we analyzed total GR protein levels and GR distribution in the cytoplasm/nucleus and MR protein levels in the LC. Acute CORT administration (1-day CORT administration [CORT 1D group]; i.e., acute CORT administration on day 7 proceeded by 6 days of vehicle administration) was used as another control. After 7 days of CORT treatment (40 mg/kg, s.c.), total GR protein levels in the LC (Fig. 3a) significantly decreased (F2,11 5 8.489, p 5 0.019). Specifically, GR protein levels in the cytoplasm (Fig. 3b; F2,11 5 6.894, p 5 0.015) and nucleus (Fig. 3c; F2,11 5 10.760, p 5 0.004) significantly decreased. The normal distribution of GRs after acute exposure to CORT transferred from the cytoplasm to the nucleus, similar to the CORT 1D group (Fig. 3d; F2,11 5 4.180, p 5 0.052). However, no difference was found between the chronic CORTtreated group (CORT 7D) and vehicle group (Fig. 3d). No significant changes in MRs (F2,8 5 0.226, p 5 0.804) were observed in the LC (Fig. 3f). These results suggest that GRs in the LC might be mainly involved in mediating the sleep disturbances caused by CORT. The method of separately extracting protein from the cytoplasm and nucleus was verified in Fig. 3e. Because the highest density of GR expression was found in the LC and not in the VLPO9, we predicted that changes in GABAergic neurons in the VLPO may be attributable to the noradrenergic projection from the LC, which inhibited GABAergic neuron activity (see the HPLC results and discussion below). Microinjection of RU486 into LC reversed the sleep disruptions induced by repeated administration of CORT. To demonstrate that GR function in the LC is directly related to sleep, we microinjected the GR antagonist RU486 into the LC every day, 30 min prior to CORT administration, to test whether it can antagonize the effect of CORT on sleep. As shown in Fig....

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