NEURAL REGENERATION RESEARCH Volume 6, Issue 9, March 2011 Cite this article as: Neural Regen Res. 2011;6(9):681-685.

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Melatonin changes in the pineal gland of sleepdeprived rats following habenular nucleus lesion**
Huijuan Jin, Meiying Song, Min Huang, Manli Wang, Hua Zhao
Department of Physiology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, Jilin Province, China

Abstract
The habenular nucleus (Hb) is an important structure that regulates the function of the pineal gland, which may affect melatonin content in the pineal gland after sleep deprivation (SD). In the present study, high performance liquid chromatography showed that the melatonin content in the pineal gland was significantly reduced, and -aminobutyric acid content in the Hb was significantly increased after SD. Furthermore, the melatonin content in the pineal gland was markedly reduced after Hb lesion under normal sleep and SD conditions. Immunohistochemistry showed that the number of Fos-positive neurons was significantly decreased in the lateral and medial Hb after SD. The findings demonstrate that the reduction of melatonin in the pineal gland after SD is related to decreased activity of Hb neurons, and that the Hb can regulate sleep-wake rhythm by influencing melatonin secretion in the pineal gland. Key Words: Habenular nucleus; melatonin; pineal gland; sleep deprivation; neural regeneration

Huijuan Jin, Doctor, Department of Physiology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, Jilin Province, China Huijuan Jin and Meiying Song contributed equally to this work. Corresponding author: Hua Zhao, Doctor, Professor, Department of Physiology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, Jilin Province, China zhua@jlu.edu.cn Supported by: the National Natural Science Foundation of China, No. 30970956*, 30570579* Received: 2010-12-12 Accepted: 2011-02-17 (N20101217001/YJ) Jin HJ, Song MY, Huang M, Wang ML, Zhao H. Melatonin changes in the pineal gland of sleep-deprived rats following habenular nucleus lesion. Neural Regen Res. 2011;6(9):681-685. www.crter.cn www.nrronline.org doi:10.3969/j.issn.1673-5374. 2011.09.008

information for the cells, and synchronize [6] biological rhythms . Accumulating evidence indicates that melatonin can prolong the Sleep deprivation (SD) is considered a risk total sleep time, and in particular, can imfactor for various disorders involving behaprove sleep quality and adjust sleep struc[9] vior, emotion, attention, learning ability, and ture . Melatonin has also been proven [1-3] immunological functions . In recent years, useful in the treatment of various sleep disthe incidence rate of SD has increased with orders, such as irregular sleep-wake rhythm, increasing stresses of life and work, and has jet lag, shift work, and insomnia[8, 10-11]. In attracted growing interest. It has been readdition, melatonin has anti-stress activity, ported that SD-induced physiological funcand regulates endocrine and immune functional disturbances are associated with retions[12], which are based on its actions on [4-5] duction of melatonin . Therefore, clinically the circadian rhythm. administering exogenous melatonin to paAnother structure that regulates pineal gland tients with SD or insomnia could improve the activity is the thalamic habenular nucleus [1, 6] [13] symptoms of these diseases . Animal (Hb) . Morphological and electrophysioexperiments unequivocally show that SD logical data have indicated that the pineal can lead to melatonin reduction in the pineal gland and Hb are closely linked. The Hb can [7] gland of rats . However, the underlying directly project vasopressin and oxytocin mechanism remains unknown. fibers to the pineal gland, and can project Melatonin (MT) is an indoleamine hormone, fibers from other structures to the dorsal part [14] secreted predominantly by the pineal gland. of the pineal gland as a relay station . The secretion of melatonin is mainly reguElectrical stimulation of the Hb can change [15-16] lated by the suprachiasmatic nucleus (SCN) the firing rates of pinealocytes . Results of the anterior hypothalamus, which has of our previous studies have shown that the been identified as the major pacemaker of Hb is involved in the regulation of some the circadian rhythm system in mammals. physiological functions[17-19]. Hb has been The secretion of melatonin has a marked shown to be involved in regulating aspects circadian rhythm, normally peaking at of the sleep-wake rhythm. For example, the 2: 00 - 3: 00 a.m. Melatonin plays a role in Hb shows a significant increase in glucose the regulation of circadian rhythm by imusage during rapid eye movement sleep pinging on the MT1 and MT2 receptors in (REMS)[20], and electrolytic lesion fasciculus [8] the SCN . As an important endogenous retroflexus can reduce the amount of time [21] synchronizer, melatonin can translate envithat rats spend in REMS . In in vitro slice ronmental photoperiodic signals to chemical preparations, the activity of Hb neurons shows rhythmicity, which may play an important role in the regulation of circadian sys-

INTRODUCTION

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[22]

tems in mammals . Because the Hb functions to regulate the circadian rhythm, and because of its morphological and functional connections with the pineal gland, it is assumed that the Hb might influence the secretion of melatonin in the pineal gland after SD. The present study measured the content of melatonin in the pineal gland, the number of Fos-positive neurons, the content of glutamate (Glu) and -aminobutyric acid (GABA) in the Hb after SD, and observed the effect of Hb lesions on melatonin levels to show that the Hb can regulate melatonin secretion in the pineal gland during SD.

pared with the normal and control groups, the number of Fos-positive neurons in the lateral Hb of the SD group was reduced by 43% and 46% ( P < 0.01), respectively, and the number of Fos-positive neurons in the medial Hb was reduced by 58% and 57% ( P < 0.01), respectively (Table 2, Figure 1).
Table 2 Quantification of Fos-positive neurons in the Hb _ after SD (x sx, n = 6)
Group Normal Control SD
a

Lateral Hb 79.67 1.86 83.22 1.43 45.22 2.44ab

Medial Hb 95.78 4.52 95.00 3.56 40.67 1.30ab

RESULTS
Quantitative analysis of experimental animals A total of 78 Wistar rats were randomly assigned to six groups: normal (n = 20), control (n = 20), SD ( n = 20), sham-surgery (n = 6), Hb lesion (n = 6) and Hb lesion + SD (n = 6) groups. Normal rats were maintained in free-sleeping conditions in home cages. Control rats were placed on a large platform. SD rats were placed on a small platform. Sham-surgery rats were placed on a large platform free of Hb lesion. Hb lesion rats were placed on a large platform and subjected to an Hb lesion. Hb lesion + SD rats were placed on a small platform and subjected to an Hb lesion. Content of melatonin in the pineal gland Results from high-performance liquid chromatography-fluorescence detector (HPLC-FD) showed that the content of melatonin was significantly decreased in the SD group (160.32 14.78 pg/pineal gland, n = 8) compared with the normal group (298.03 34.70 pg/pineal gland, n = 8) and the control group (300.50 35.50 pg/pineal gland, n = 8) by 46% and 47% ( P < 0.01), respectively. The content of melatonin was similar in the control and normal groups. Effect of SD on Glu and GABA contents in the Hb Results from high-performance liquid chromatography-UV detector (HPLC-UV) showed that the content of Glu was similar among normal, control, and SD groups, and the content of GABA was significantly increased in the SD group, compared with the normal and control groups, by 25% and 23% ( P < 0.05), respectively (Table 1).

P < 0.01, vs. normal group; bP < 0.01, vs. control group. SD: Sleep deprivation; Hb: habenular nucleus.

Lateral Hb Control group

Medial Hb

Figure 1 Distribution of Fos-positive neurons in the rat Hb (immunohistochemistry, 400). Fos-positive neurons are widely distributed in the lateral Hb and the medial Hb of the control group. Fos-positive neurons are observed in the normal group as in the control group. Fos-positive neurons are significantly decreased in the SD group. Fos-positive neurons are indicated by arrows. SD: Sleep deprivation; Hb: habenular nucleus.

Table 1
Group Normal Control SD
a

Content of Glu and GABA in _rat Hb (x sx, n = 6, nmol/L)

Glu 3 561.6102.4 3 239.098.7 3 747.4240.2 GABA 1 270.3223.4 1 132.678.7 1 683.0133.3ab

P < 0.05, vs. normal group; bP < 0.05, vs. control group. Glu: Glutamate; GABA: -aminobutyric acid; SD: sleep deprivation; Hb: habenular nucleus.

Influence of SD on the quantification of Fos-positive neurons in the Hb Results from immunohistochemistry showed that com682

Influence of Hb lesion on the content of melatonin in the pineal gland Results of HPLC-FD showed that the content of melatonin was significantly reduced in the Hb lesion group (111.22 9.79 pg/pineal gland, n = 6) compared with the control (300.50 35.50 pg/pineal gland, n = 8) and the sham-surgery groups (312.84 22.73 pg/pineal gland, n = 6) by 63% and 64% ( P < 0.01), respectively, and the content of melatonin was significantly reduced in the Hb lesion + SD group (50.16 6.51 pg/pineal gland, n = 6) compared with the Hb lesion group by 55% ( P < 0.01), and 83% and 84% (P < 0.01) compared with the control and sham-surgery groups, respectively. Hb lesion assessment A total of 18 rats were used for Hb lesion, but six were excluded from the study, because the damage location of

SD group

Normal group

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three rats was in the rear of the Hb; unilateral Hb was destroyed in one rat, and the hippocampus was damaged in two rats. Ultimately, 12 rats were included in the final analysis. In the Hb lesion group, the lesioned proportion was > 80% in two rats, > 70% in three rats, and > 60% in one rat. In the Hb lesion + SD group, the lesioned proportion was > 80% in two rats, > 70% in one rat, and > 60% in three rats (Figure 2).

A Hip

Hip

MHb

LHb

MHb LHb

Figure 2 Typical sections through the rat habenular nucleus region (Nissl staining, 40). (A) Sham-surgery Hb; (B) lesioned Hb. LHb: Lateral habenular nucleus; MHb: medial habenular nucleus; Hip: hippocampus.

DISCUSSION
SD has been extensively used in sleep studies [23-24]. The present study utilized the single small platform method to establish the SD model. A large platform was used as stress control, and normal sleep was used as normal control. The results showed no significant differences between the control and normal groups in terms of m elatonin content in the pineal gland, and the number of Fos-positive neurons and Glu and GABA contents in the Hb, and thus negating the adverse impact caused by the water environment. These results also proved that this SD method is feasible. Administration of melatonin to patients with SD (or insomnia) can improve sleep quality, and can especially prevent and improve various dysfunctions and disorders caused by insufficient sleep [1, 6, 25-26]. This proves that melatonin is related to a series of functional changes during SD[4-5]. In the present study, the content of melatonin in the pineal gland of SD rats decreased by 47% compared with the control group, which is consistent with [7] previously reported results . This result also explains why clinical SD patients can gain substantial benefits after melatonin administration. Melatonin is important in vivo for sleep regulation, and a decrease in melatonin can reduce sleeping time and cause sleep disorders. In addition, SD can further reduce the synthesis of melatonin, resulting in refractory insomnia. Although experimental SD is a model of insomnia in animals, which is induced by artificial methods, the results do clearly demonstrate that SD can impact the synthesis of melatonin in the pineal gland, and that the reduction of mela-

tonin content causes a series of functional changes evoked by SD. This is consistent with previous reports that melatonin can improve the dysfunction caused by [1, 6, 25-26] SD . Hb is located on the posterior medial surface of the caudal thalamus. It serves as a major relay station, linking the midbrain with the forebrain. In recent years, increasing attention has been paid to the functional relationship between the Hb and the pineal gland, giving rise to a new recognition about the role of Hb in sleep reg ulation. Fos protein is encoded by the c-fos proto-oncogene that is present in neurons. Fos immunoreactivity can reflect to a large extent the activities of the stressed neurons, so it is used as a marker of neuronal activity. Because they are important neurotransmitters in the brain, Glu and GABA are also related with neuronal activities. The Hb contains these neurotransmitters and related receptors. Therefore, the present study observed the number of Fos-positive neurons and the content of Glu and GABA in the Hb after SD. The results showed that after SD, the number of Fos-positive neurons decreased in the medial Hb and lateral Hb by 57% and 46%, respectively, and the content of GABA in the Hb increased by 33%, with Glu remaining unchanged compared with controls. These results revealed that the neuronal activity of the Hb decreased after SD, suggesting that the activity change of Hb is related with dysfunction caused by SD. The present study further observed that the content of melatonin in the pineal gland decreased by 63% and 84% in the Hb lesion and Hb lesion + SD groups, respectively, compared with the control group. Reciprocal connections have been found between the Hb and pineal gland using the horseradish peroxidase tracing technique[27], and electrophysiological studies also observed that the excited Hb can increase the firing rate of the pineal gland [15, 28]. In addition, Rnnekleiv et al [13] proved that the lesioned Hb can cause pineal gland degeneration. These findings prove that the Hb can excite the pineal gland. It was concluded that decreased activity of Hb neurons after SD suppresses melatonin secretion, resulting in reduced content of melatonin in the pineal gland.

MATERIALS AND METHODS

Design A randomized, controlled, animal study. Time and setting This experiment was performed at the Department of Physiology, Norman Bethune College of Medicine, Jilin University, China, from May 2007 to December 2008. Materials A total of 78 healthy, male, pathogen-free grade Wistar rats, aged 8 weeks and weighing 250270 g, were provided by the Laboratory Animal Research Center of Jilin University (SCXK (Ji) 2007-0003). Animals were maintained under standard conditions (22 1 C, 40 50% humidity, free access to food and water, 12 hour
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light-dark cycle, lights on at 7: 00 a.m.). Methods Hb lesions Rats were anesthetized with 10% chloral hydrate (400 mg/kg), as indicated by the pupil size and paw pinch reflexes. The head of the animal was fixed in a stereotaxic apparatus (Narishige, Tokyo, Japan). The electrodes were inserted into the Hb bilaterally, using stereotaxic coordinates (3.3- 3.5 mm posterior to bregma; 3.8- 4.2 mm ventral to the dura, 0.45 mm lateral to the midline, according to the atlas of Paxinos and Watson [29]. The Hb was then lesioned by an electronic stimulator (Nihon Kohden, Tokyo, Japan) with a 1.5 mA dc current for 60 seconds[17]. Sham-surgery rats were treated as above, except no current was passed. After the animals awoke, they were returned to their home cage where they were allowed to recover for 1 week. SD After 1 week of Hb lesion, the rats subjected to SD were placed on a small platform (6.5 cm diameter) located in the middle of a water tank (30 cm 25 cm 40 cm) for 96 hours. The platform was placed 2 cm above the surrounding water. Food and water were available through a grid placed on the top of the water tank. The water temperature was maintained at 21 C. When the rats reached the paradoxical phase of sleep, muscle atonia caused them to fall into the water, which woke them up. Stress-control animals were submitted to the same procedure, with platforms of 20 cm in diameter, and normal animals were housed in their home cages in the same room[30-31]. Content of melatonin in the pineal gland determined by HPLC-FD A total of 8 rats each from normal, control, and SD groups, and 6 rats each from sham-surgery, Hb lesion, and Hb lesion + SD groups were used for detection. Immediately after SD, the rats were anesthetized with diethyl ether and the pineal gland was removed on ice at 2:00-3:00 a.m. after SD. The pineal gland samples were homogenized with 200 L precooled HClO 4 (0.1 mol/L) and centrifuged at 12 000 g for 20 minutes at 4 C. The supernatants were stored at -80 C. The melatonin content was assayed by HPLC equipped with a fluorometric detector (Shimadzu, Kyoto, Japan), and a LUAN C18 HPLC column (250 mm 4.60 mm, 5 m) [7, 32]. The mobile phase was 40% methanol containing 0.1 mmol/L EDTA (pH 4.25, using glacial acetic acid). The mobile phase flow rate was set to 0.9 mL/min, and the column temperature was 30 C. The excitation and emission wavelengths were set at 285 nm and 345 nm, respectively. The injection volume was 80 L. Content of Glu and GABA in Hb tested by HPLC-UV A total of 6 rats each from normal, control, and SD groups were used for detection. Immediately after SD, the rats were anesthetized with diethyl ether and the bilateral Hb was removed on ice at 2:00 -3:00 a.m. after SD. The Hb samples were homogenized with 300 L
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precooled HClO4 (0.1 mol/L) and centrifuged at 12 000 g for 20 minutes at 4 C. The supernatants were stored at -80 C. Derivatization was performed by adding 200 L of the sample supernatant to a solution containing 20 L 0.1% dinitrofluorobenzene. The samples were incubated at 70 C for 20 minutes, followed by PBS (pH 7.0) to 500 L. The samples were centrifuged at 12 000 g for 10 minutes at 4 C, and the supernatant was used for HPLC analysis. HPLC analysis of Glu and GABA was performed using an ultraviolet detector and a LUAN C18 HPLC column [33] (250 mm 4.60 mm, 5 m) . The mobile phase (buffer A: 50 mmol/L sodium acetate, pH 6.0; buffer B: 50% acetonitrile/50% water) flow rate was set to 1 mL/min and the column temperature was at 35 C. The wavelengths were set at 360 nm. Injection volume was 20 L. Gr adient elution was performed. Quantification of Fos-positive neurons in the Hb by immunohistochemistry A total of 6 rats each from the normal, control, and SD groups were used. At 2: 00-3: 00 a.m. after SD, the rats were deeply anesthetized with diethyl ether and perfused with 150 mL precooled saline solution, followed by 250 mL paraformaldehyde (4%). Brains were rapidly removed and embedded in optimal cutting temperature compound. Coronal sections through the Hb were cut, 20 m thick, in a cryostat (Leica, Nussloch, Germany), one section was used from every six sections. The sections were incubated at room temperature for 10 minutes in 3% H2O2 and incubated in a blocking solution containing 5% normal goat serum and 0.3% Triton X-100 in PBS for 40 minutes. Sections were incubated in 0.1% rabbit anti-Fos antibody (Maixin, Fuzhou, China) for 24 hours at 4 C, 0.5% biotinylated goat anti-rabbit IgG (Maixin) for 10 minutes at room temperature, and horseradish-labeled avidin-biotin peroxidase complex (Maixin) for 10 minutes. A PBS wash was performed between each step. Sections were visualized with 0.03% diaminobenzidine, counterstained with hematoxylin for 20 seconds, washed in PBS, dehydrated in a series of alcohols, cleared with xylene, mounted with neutral gum, and observed by microscopy[34] . A total of five fields of view from the lateral Hb and medial Hb (400 magnification) were randomly selected to quantify Fos-positive neurons. Assessment of Hb lesions by Nissl staining The brains of sham-surgery rats and Hb lesion rats were fixed in 4% paraformaldehyde overnight, and embedded in paraffin. Coronal sections through the Hb according to [29] the atlas of Paxinos and Watson were cut into 10 m [35] thick sections followed by conventional Nissl staining , and observed by microscopy. Statistical analysis All data were statistically analyzed using SPSS 11.0 (SPSS, Chicago, IL, USA), and were expressed as Mean SEM. Intergroup comparison was performed by one-way analysis of variance. A value of P < 0.05 was

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considered statistically significant.

Author contributions: Hua Zhao participated in the study design, funding support, article accreditation and revision. Huijuan Jin provided, integrated, and analyzed the data, and drafted the manuscript. Meiying Song participated in the date analysis, study design, and technical support. Min Huang provided technical support. Manli Wang provided experimental data. Conflicts of interest: None declared. Funding: This study was supported by the National Natural Science Foundation of China, No. 30970956, 30570579. Ethical approval: This study obtained full approval from the Animal Ethics Committee of Jilin University, China. Acknowledgments: We thank Professor Li Zhou and Yongmao Liu from Jilin University for technical assistance.

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(Edited by Yu DW, Yue W/Su LL/Song LP)

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