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

681

Huijuan Jin☆, Doctor, De-

partment 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

Melatonin changes in the pineal gland of sleep- deprived 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 γ-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

INTRODUCTION

Sleep deprivation (SD) is considered a risk

factor for various disorders involving beha-

vior, emotion, attention, learning ability, and

immunological functions[1-3]

. In recent years,

the incidence rate of SD has increased with

increasing stresses of life and work, and has

attracted growing interest. It has been re-

ported that SD-induced physiological func-

tional disturbances are associated with re-

duction of melatonin[4-5]

. Therefore, clinically

administering exogenous melatonin to pa-

tients with SD or insomnia could improve the

symptoms of these diseases[1, 6]

. Animal

experiments unequivocally show that SD

can lead to melatonin reduction in the pineal

gland of rats[7]

. However, the underlying

mechanism remains unknown.

Melatonin (MT) is an indoleamine hormone,

secreted predominantly by the pineal gland.

The secretion of melatonin is mainly regu-

lated by the suprachiasmatic nucleus (SCN)

of the anterior hypothalamus, which has

been identified as the major pacemaker of

the circadian rhythm system in mammals.

The secretion of melatonin has a marked

circadian rhythm, normally peaking at

2: 00 - 3: 00 a.m. Melatonin plays a role in

the regulation of circadian rhythm by im-

pinging on the MT1 and MT2 receptors in

the SCN[8]

. As an important endogenous

synchronizer, melatonin can translate envi-

ronmental photoperiodic signals to chemical

information for the cells, and synchronize

biological rhythms[6]

. Accumulating evidence

indicates that melatonin can prolong the

total sleep time, and in particular, can im-

prove sleep quality and adjust sleep struc-

ture[9]

. Melatonin has also been proven

useful in the treatment of various sleep dis-

orders, such as irregular sleep-wake rhythm,

jet lag, shift work, and insomnia[8, 10-11]

. In

addition, melatonin has anti-stress activity,

and regulates endocrine and immune func-

tions[12]

, which are based on its actions on

the circadian rhythm.

Another structure that regulates pineal gland

activity is the thalamic habenular nucleus

(Hb)[13]

. Morphological and electrophysio-

logical data have indicated that the pineal

gland and Hb are closely linked. The Hb can

directly project vasopressin and oxytocin

fibers to the pineal gland, and can project

fibers from other structures to the dorsal part

of the pineal gland as a relay station

[14].

Electrical stimulation of the Hb can change

the firing rates of pinealocytes[15-16]

. Results

of our previous studies have shown that the

Hb is involved in the regulation of some

physiological functions[17-19]

. Hb has been

shown to be involved in regulating aspects

of the sleep-wake rhythm. For example, the

Hb shows a significant increase in glucose

usage during rapid eye movement sleep

(REMS)[20]

, and electrolytic lesion fasciculus

retroflexus can reduce the amount of time

that rats spend in REMS[21]

. In in vitro slice

preparations, the activity of Hb neurons

shows rhythmicity, which may play an important role in the regulation of circadian sys-

www.nrronline.org www.nrronline.org www.nrronline.org

Jin HJ, et al. / Neural Regeneration Research. 2011;6(9):681-685.

682

tems in mammals[22]

. Because the Hb functions to regu-

late the circadian rhythm, and because of its morpho-

logical 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 γ-aminobutyric acid

(GABA) in the Hb after SD, and observed the effect of Hb

lesions on melatonin levels to show that the Hb can re-

gulate melatonin secretion in the pineal gland during SD.

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 chromatogra-

phy-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) com-

pared 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), re-

spectively. 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 chromatogra-

phy-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).

Influence of SD on the quantification of Fos-positive

neurons in the Hb

Results from immunohistochemistry showed that com-

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).

Influence of Hb lesion on the content of melatonin in

the pineal gland

Results of HPLC-FD showed that the content of melato-

nin 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

Table 1 Content of Glu and GABA in rat Hb (x

_

±sx, n = 6, nmol/L)

Group Glu GABA

Normal 3 561.6±102.4 1 270.3±223.4

Control 3 239.0±98.7 1 132.6±78.7

SD 3 747.4±240.2 1 683.0±133.3ab

aP < 0.05, vs. normal group;

bP < 0.05, vs. control group. Glu:

Glutamate; GABA: γ-aminobutyric acid; SD: sleep deprivation; Hb: habenular nucleus.

Table 2 Quantification of Fos-positive neurons in the Hb after SD (x

_

±sx, n = 6)

Group Lateral Hb Medial Hb

Normal

Control

79.67±1.86

83.22±1.43

95.78±4.52

95.00±3.56

SD 45.22±2.44ab

40.67±1.30ab

aP < 0.01, vs. normal group;

bP < 0.01, vs. control group. SD: Sleep

deprivation; Hb: habenular nucleus.

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.

SD

gro

up

N

orm

al g

rou

p C

on

trol gro

up

Lateral Hb Medial Hb

Jin HJ, et al. / Neural Regeneration Research. 2011;6(9):681-685.

683

three rats was in the rear of the Hb; unilateral Hb was

destroyed in one rat, and the hippocampus was dam-

aged in two rats. Ultimately, 12 rats were included in the

final analysis. In the Hb lesion group, the lesioned pro-

portion 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).

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 me-

latonin 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 in-

somnia) 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 mela-

tonin in the pineal gland of SD rats decreased by 47%

compared with the control group, which is consistent with

previously reported results[7]

. 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 melato-

nin, resulting in refractory insomnia. Although experi-

mental 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 mela-

tonin 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

SD[1, 6, 25-26]

.

Hb is located on the posterior medial surface of the

caudal thalamus. It serves as a major relay station, link-

ing the midbrain with the forebrain. In recent years, in-

creasing attention has been paid to the functional rela-

tionship between the Hb and the pineal gland, giving rise

to a new recognition about the role of Hb in sleep regu-

lation. Fos protein is encoded by the c-fos pro-

to-oncogene that is present in neurons. Fos immuno-

reactivity 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 de-

creased in the medial Hb and lateral Hb by 57% and 46%,

respectively, and the content of GABA in the Hb in-

creased by 33%, with Glu remaining unchanged com-

pared with controls. These results revealed that the

neuronal activity of the Hb decreased after SD, sug-

gesting 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,

Rønnekleiv 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 sup-

presses 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 250–270 g, were pro-

vided by the Laboratory Animal Research Center of Jilin

University (SCXK (Ji) 2007-0003). Animals were main-

tained under standard conditions (22 ± 1 °C, 40 – 50%

humidity, free access to food and water, 12 hour

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.

LHb MHb

Hip

A

Hip

LHb

MHb

B

Jin HJ, et al. / Neural Regeneration Research. 2011;6(9):681-685.

684

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 stereo-

taxic apparatus (Narishige, Tokyo, Japan). The elec-

trodes were inserted into the Hb bilaterally, using ste-

reotaxic 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 sur-

rounding water. Food and water were available through a

grid placed on the top of the water tank. The water tem-

perature 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 pro-

cedure, 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 µL 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.

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 µm) [7, 32]

. The mobile phase was 40% methanol con-

taining 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 μL.

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 µL

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 µL of the

sample supernatant to a solution containing 20 µL 0.1%

dinitrofluorobenzene. The samples were incubated at

70 °C for 20 minutes, followed by PBS (pH 7.0) to 500 μL.

The samples were centrifuged at 12 000 × g for 10 mi-

nutes 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

(250 mm × 4.60 mm, 5 µm) [33]

. 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 μL. Gra-

dient 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 µm 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 in-

cubated in 0.1% rabbit anti-Fos antibody (Maixin, Fuz-

hou, China) for 24 hours at 4 °C, 0.5% biotinylated goat

anti-rabbit IgG (Maixin) for 10 minutes at room temper-

ature, 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 he-

matoxylin 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

the atlas of Paxinos and Watson[29]

were cut into 10 µm

thick sections followed by conventional Nissl staining[35]

,

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

Jin HJ, et al. / Neural Regeneration Research. 2011;6(9):681-685.

685

considered statistically significant.

Author contributions: Hua Zhao participated in the study

design, funding support, article accreditation and revision. Hui-

juan 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 pro-

vided 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)