Various Types of Stress Deteriorate Spatial Memory through Inhibition of Cell Proliferation in the Hippocampus of Rats

Corresponding author: Chang-Ju Kim, Department of Physiology, College of Medicine, Kyung Hee University, 1, Hoeigi-dong, Dongdaemoon-gu, Seoul 130-701, Korea

Tel: +82-2-961-0407, E-mail: [email protected] Received August 15, 2011, Revised September 1, 2011 Accepted September 20, 2011

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2009-352-G00056).

Various Types of Stress Deteriorate Spatial Memory through Inhibition of Cell Proliferation in the Hippocampus of Rats

Departments of *Biochemistry and Molecular Biology,

Physiology, College of Medicine, Kyung Hee University,

‡

Department of Physical Education, Korea Military Academy, Seoul, Korea

Jee-Sang Yoon*, Hyung-Hwan Baik*, Su-Shin Lee

, Yu-Mi Heo

, Bo-Kyun Kim

, Mal-Soon Shin

, Sang-Won Lee

, Chang-Ju Kim

Stress is defined as physical, physiological, and psychological modifications that disrupt the homeostasis and internal balance of the body. Stress is closely associated with neuronal changes such as impaired neurogenesis, neuronal atrophy, and neuronal apoptosis in the hippocampus. In the present study, we investigated the effects of various types of stress (restraint stress, noise stress, cold stress) on spatial memory, cell proliferation, and expression of brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (TrkB) in the hippocampus of rats. The rats in the restraint stress group were restrained in a transparent cylinder.

The rats in the noise stress group were exposed to a 120 dB supersonic machine sound. The rats in the cold stress group were exposed to a cold chamber at 4

C. The rats in the control group were left undisturbed. Application of each stress was conducted for 60 min 2 times a day, and continued for 10 days. In the present results, all three types of stress induced spatial memory impairment. All three types of stress suppressed cell proliferation and inhibited expression of BDNF and TrkB in the hippocampus.

The strength of the deteriorating and suppressing effects of stresses had the following rank order: restraint stress＞noise stress＞

cold stress. Thus, impairment of spatial memory by stresses may be due to the inhibition of cell proliferation through suppressing BDNF expression in the hippocampus. (Korean J Str Res 2011;19:243∼251)

Key Words: Restraint stress, Noise stress, Cold stress, Spatial memory, Hippocampus

INTRODUCTION

Stress is defined as physical, physiological, and psychological modifications that disrupt the homeostasis and internal balance of

the body. Stress is known to be implicated in several neurodegenerative and psychiatric diseases including depression (Sung YH et al., 2010), stroke (Manolescu et al., 2011), and Alzheimer's disease (Clark et al., 2010). Stress causes structural changes and neuronal damage, especially in the hippocampus (Smith et al., 1995), which is a region that is important in learning ability and memory function. Stress is closely associated with neuronal changes such as impaired neurogenesis, neuronal atrophy, and neuronal apoptosis in the hippocampus (McEwen et al., 2001; Sung YH et al., 2010).

Neurogenesis is associated with hippocampal function,

including learning ability and memory function, and neurogenesis is modulated by a variety of factors including stress, an enriched environment, learning, serotonin, N-methyl-D-aspartate (NMDA) receptor antagonists, and stroke (Kempermann et al., 1997; Fuchs et al., 2000; Ko IG et al., 2009). Stress inhibits cell proliferation of new granular cells in the hippocampal dentate gyrus (Fuchs et al., 2001; Tanapat et al., 2001).

The neurotrophic factors include nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), and brain-de- rived neurotrophic factor (BDNF). Among these, BDNF acts as a modulator of long-term potentiation (LTP), synaptic plasticity, and neuronal plasticity in the adult central nervous system (Martin et al., 2000; Lee E et al., 2009). BDNF is found in many areas of the brain (Smith et al., 1996; Givalois et al., 2001;

Alonso et al., 2002). BDNF and its receptor, tyrosine kinase B (TrkB), are crucial for hippocampal-dependent learning ability and memory function (Patterson et al., 1996). In addition, BDNF promotes survival and growth of neurons in the hippocampus (Lowenstein et al., 1996; Bartrup et al., 1997). BDNF is known as a key molecule in stress, because BDNF expression is decreased by exposure to stress (Smith et al., 1995; Adlard et al., 2004).

Stress is known to reduce neurogenesis through the suppression of BDNF expression in the hippocampus (Lucassen et al., 2010).

However, the effects of various types of stress (restraint stress, noise stress, cold stress) on spatial memory in relation to cell proliferation have not been compared, and the exact underlying mechanisms of stress on memory function are unclear. In the present study, we investigated the effects of these kinds of stresses on spatial memory, cell proliferation, and expression of BDNF and TrkB in the hippocampus of rats. To this end, we used an 8-arm maze test for spatial memory, immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and Ki-67, and western blot analysis for BDNF and TrkB.

MATERIALS AND METHODS

1. Animals and treatments

Experimental procedures were performed in accordance with the guidelines of the National Institute of Health (NIH) and the Korean Academy of Medical Sciences. Male Sprague-Dawley rats weighing 200±10 g were used for the experiment. Food and

water were made available ad libitum. The rats were randomly divided into four groups (n=8 in each group): a control group, a restraint stress group, a noise stress group, and a cold stress group. All rats received 50 mg/kg BrdU (Sigma Chemical Co., St.

Louis, MO, USA) intraperitoneally 30 min before the start of first stress exposure, once a day for 5 consecutive days. The animals were sacrificed immediately after the completion of the behavior test.

2. Stress exposure

We applied each stress twice per day according to the previous studies (Endo et al., 1999; Adlard et al., 2004; Heine et al., 2005). The rats in the restraint stress group were restrained in a transparent Plexiglas cylinder (21 cm long, 6 cm diameter). The rats in the noise stress group were exposed to a 120 dB supersonic machine sound. The rats in the cold stress group were exposed to a cold chamber that was maintained at 4°C. The rats in the control group were left undisturbed. Application of each stress was conducted for 60 min, 2 times a day, and continued for 10 days.

3. Radial-8-arm maze test

Spatial memory was tested using a radial-8-arm maze apparatus as previously described method (Kim SE et al., 2010;

Ko IG et al., 2011). The radial-8-arm maze apparatus consisted of a central octagonal plate (30 cm in diameter) and eight radiating arms (each 50 cm in length and 10 cm in width). The apparatus was placed 1 m above the floor. A small receptacle filled with water (3 cm in diameter and 1 cm in depth) was located at the end of the arms. The rats were trained three times before the spatial learning test. The rats deprived of water for 24 h were allowed to explore for water and to drink for 5 min. On 9 days after each stress exposure, radial-8-arm maze test was performed. The time spent seeking water at the end of the arms was recorded. The test was terminated when a rat found water in all eight arms or over 5 min elapsed. Re-entering a previously visited arm was counted as an error. In addition, the number of correct choice before the first error was counted.

4. Tissue preparation

For brain tissue preparation, animals were fully anesthetized

with Zoletil 50

(10 mg/kg, i.p.; Vibac Laboratories, Carros, France), transcardially perfused with 50 mM phosphate-buffered saline (PBS), and fixed with freshly prepared solution consisting of 4% paraformaldehyde (PFA) in 100 mM phosphate buffer (PB, pH 7.4). Brains were then removed, post-fixed in the same fixative overnight, and transferred into a 30% sucrose solution for cryoprotection. Coronal sections of 40 μm thickness were made using a freezing microtome (Leica, Nussloch, Germany).

5. BrdU immunohistochemistry

For the detection of newly generated cells in the dentate gyrus, BrdU incorporation, which has been generally used as an indicator of DNA synthesis, was visualized using an immunohi- stochemical method as previously described method (Kim SE et al., 2010; Jung SY et al., 2011). Briefly, the sections were permeabilized by incubation with 0.5% Triton X-100 in PBS for 20 min, treated with 50% formamide-2× standard saline citrate (SSC) at 65

C for 2 h, denatured in 2 N HCl at 37

C for 30 min, and rinsed twice in 100 mM sodium borate (pH 8.5).

Subsequently, the sections were incubated overnight at 4

C with BrdU-specific mouse monoclonal antibody (1 : 600; Roche, Mannheim, Germany). The sections were then washed three times with PBS and incubated for 1 h with a biotinylated mouse secondary antibody (1 : 200; Vector Laboratories, Burlingame, CA, USA). Then, the sections were incubated for another 1 h with avidin–peroxidase complex (1 : 100; Vector Laboratories). For the visualization of BrdU, the sections were incubated with 50 mM Tris-HCl buffer (pH 7.6) containing 0.02% 3,3'-diamino- benzidine (DAB), nickel chloride (40 mg/ml) and 0.03% H

O

for 5 min. After BrdU-specific staining, counter-staining was performed on the same sections using a mouse anti-neuronal nuclei (NeuN) antibody (1 : 300; Chemicon International, Teme- cula, CA, USA). The sections were washed three times with PBS, incubated for 1 h with a biotinylated anti-mouse secondary antibody, and processed with VECTASTAIN

ABC kits. For staining, the sections were reacted with 0.02% DAB and 0.03%

H

O

in 50 mM Tris-HCl buffer (pH 7.6) for 5 min after which the sections were mounted onto gelatin-coated slides. The slides were air dried overnight at room temperature, and coverslips were mounted using Permount

.

6. Ki-67 immunohistochemistry

For the detection of Ki-67-positive cells in the dentate gyrus, immunohistochemistry was performed as previously described method (Sung YH et al., 2010). The sections were then incubated in 50 mM PBS for 10 min, washed 3 times using in 50 mM PBS, and then incubated in 3% H

O

for 30 min. After that, the sections were incubated overnight with mouse anti-Ki-67 antibody (1 : 500; Novocastra Laboratories, Newcastle, UK). Next day, we incubated the sections for 1 h with biotinylated anti-mouse secondary antibody (Vector Laboratories), and subsequently incubated them with an avidin-biotin-peroxidase complex (Vector Laboratories) for 1 h at room temperature.

Immunoreactivity was visualized by incubating the sections in a solution consisting of 0.05% DAB and 0.01% H

O

in 50 mM Tris-HCl buffer (pH 7.6) for approximately 3 min. The sections were then mounted on gelatin-coated glass slides, and coverslips were mounted using Permount

.

7. Western blot analysis

Western blot analysis was conducted for the determination of BDNF and TrKB expression as previously described method (Kim SE et al., 2010). Hippocampal tissues were collected and then immediately frozen at −70

C. Hippocampal tissues were homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl

ㆍ6H

O, 1 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Hercules, CA, USA). Protein (30 μg) was separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. Mouse beta actin antibody (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit BDNF and TrkB antibody (1 : 1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-rabbit antibody for BDNF and TrkB (1 : 3,000; Vector Laboratories) were used as the secondary antibodies.

Experiments were performed under normal laboratory

conditions and at room temperature, except for the transfer to

membranes. The transfer was performed at 4°C with a cold pack

and pre-chilled buffer. Band detection was performed using

enhanced chemiluminescence (ECL) detection kits (Santa Cruz Biotechnology).

8. Data analysis

The area of the hippocampal dentate gyrus was measured using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). The numbers of BrdU-positive and Ki-67-positive cells in the hippocampus were counted hemilaterally. The data are expressed as the number of cells per mm

of the hippocampus.

To compare the relative expressions of BDNF and TrKB, the detected bands were calculated densitometrically using Image-Pro Plus software (Media Cybernetics).

Statistical analysis was performed using one-way ANOVA followed by Duncan's post-hoc test. The results are presented as the mean±standard error of the mean (S.E.M.). Significance was set as p＜0.05.

RESULTS

1. Effects of various types of stress on spatial memory

To verify the effects of various types of stress on spatial memory, the radial-8-arm maze test was performed. All rats completed eight successful performances within 106.00±12.35 sec in the normal group, 253.28±14.13 sec in the restraint stress group, 226.62±22.91 sec in the noise stress group, and 154.42±14.04 sec in the cold stress group (Fig. 1A).

The number of errors made before eight successful perfor- mances was 1.28±0.47 in the normal group, 15.57±1.95 in the restraint stress group, 13.25±1.72 in the noise stress group, and 6.00±0.87 in the cold stress group (Fig. 1B).

The number of correct choices before the first error in the control group was 7.42±0.20 in the normal group, 3.28±0.42 in the restraint stress group, 3.62±0.37 in the noise stress group, and 5.57±0.42 in the cold stress group (Fig. 1C).

Fig. 1. The effects of various types of stress on spatial memory. (A)

The time for eight successful performances. (B) The number of errors

made before eight successful performances. (C) The number of correct

choices made before the first error. (a) Normal group, (b) restraint

stress group, (c) noise stress group, (d) cold stress group. The data are

mean±standard error of the mean (S.E.M.). *p＜0.05 compared to the

normal group. **p＜0.05 compared to the restraint stress group.

Fig. 2. The effects of various types of stress on cell proliferation in the hippocampus. (A) 5-Bromo-2'-deoxyuridine (BrdU)-positive cells. Upper:

Photomicrographs of BrdU-positive cells in hippocampal dentate gyrus. The scale bar is 50 μm. Lower: Number of BrdU-positive cells in the hippocampal dentate gyrus. (B) Ki-67-positive cells. Upper: Photomicrographs of Ki-67-positive cells in the hippocampal dentate gyrus. The scale bar is 50 μm. Lower: Number of Ki-67-positive cells in the hippocampal dentate gyrus. (a) Normal group, (b) restraint stress group, (c) noise stress group, (d) cold stress group. The data are mean±standard error of the mean (S.E.M.). *p＜0.05 compared to the normal group. **p＜0.05 compared to the restraint stress group.

The time of performance and the error number were increased and the correct number was decreased in all stress groups relative to the normal group (p＜0.05). The deteriorative effects of these stresses on spatial memory appeared to have the following rank order: restraint stress＞noise stress＞cold stress.

2. Effects of various types of stress on cell proliferation in the hippocampal dentate gyrus

Photomicrographs of BrdU-positive cells in the hippocampal dentate gyrus in each group are presented in Fig. 2A. The number of BrdU-positive cells was 624.32±30.88/mm

in the normal group, 281.10±14.87/mm

in the restraint stress group,

338.72±20.86/mm

in the noise stress group, and 446.91±

19.68/mm

in the cold stress group.

Photomicrographs of Ki-67-positive cells in the hippocampal dentate gyrus in each group are presented in Fig. 2B. The number of Ki-67-positive cells was 44.44±4.14/mm

in the normal group, 16.94±1.98/mm

in the restraint stress group, 16.74±1.53/mm

in the noise stress group, and 34.93±2.68/mm

in the cold stress group.

Cell proliferation in the hippocampal dentate gyrus was decreased in all stress groups relative to the normal group (p

＜0.05). The suppressing effect of these stresses on cell prolife-

ration in the hippocampal dentate gyrus appeared to have the

following rank order: restraint stress＞noise stress＞cold stress.

Fig. 3. The effects of various types of stress on the BDNF and TrkB

expression in the hippocampus. (A) Brain-derived neutrophic factor (BDNF). (B) Tyrosine kinase B (TrkB). (a) Normal group, (b) restraint stress group, (c) noise stress group, (d) cold stress group. The data are mean±standard error of the mean (S.E.M.). *p＜0.05 compared to the normal group. **p＜0.05 compared to the restraint stress group.

3. Effects of various types of stress on the expression of BDNF and TrkB in the hippo- campus

We determined the relative expression of BDNF and TrkB in the hippocampus. BDNF exists as a precursor form (32∼47.5 kDa) and a mature form (14 kDa). When the level of mature BDNF (14 kDa) in the normal group was set at 1.00, the level of mature BDNF was 0.70±0.03 in the restraint stress group, 0.78±0.00 in the noise stress group, and 0.85±0.11 in the cold stress group (Fig. 3A).

When the level of TrkB (95∼145 kDa) in the normal group was set at 1.00, the level of TrkB was 0.34±0.02 in the restraint stress group, 0.48±0.04 in the noise stress group, and 0.76±0.05 in the cold stress group (Fig. 3B).

The levels of BDNF and TrkB in the hippocampus were decreased in all stress groups relative to the normal group (p

＜0.05). The suppressing effect of these stresses on BDNF and TrkB expression in the hippocampus appeared to have the following rank order: restraint stress＞noise stress＞cold stress.

DISCUSSION

Stress is associated with deleterious changes in hippocampal excitability, long-term potentiation, cerebral blood flow, and spatial learning memory (Joëls et al., 1992; Endo et al., 1999).

Stress is known to cause deteriorative effect on cognition and memory in humans and animal models (McEwen, 1999; Stawski et al., 2006). Elizalde et al.(2008) reported that chronic mild stress induced recognition memory deficits. Exposure to chronic stress leads to dendrite atrophy and neuronal cell death associated with memory impairment and behavioral abnormalities (Luine et al., 1994; Vyas et al., 2002; Lucassen et al., 2010).

In order to determine the effect of various types of stress on spatial memory, a radial-8-arm maze test was conducted. In the radial-8-arm maze test, longer time of the successful performance, higher error number, and lower correct number indicate spatial memory disturbance (Ko IG et al., 2011). All three types of stress (restraint, noise, and cold stress) increased the time of the performance and the error number and decreased the correct number in the radial-arm maze test, showing that these stresses

induced spatial memory impairment. Restraint stress had the most potent deteriorative effect and cold stress the least deteriorative on spatial memory.

The hippocampus is a vulnerable region of the brain, and

susceptible to damage during repeated stress (McEwen, 1999).

Hippocampal neurogenesis is regulated by variable environmental factors (Fuchs et al., 2000; Gage, 2000; Gould et al., 2000).

Stress is well documented to decrease neurogenesis in the hippocampal dentate gyrus (Fuchs et al., 2001; Falconer et al., 2003). Stress also induces alteration in hippocampal morphology with loss of pyramidal neurons by elevation of corticosteroid level (McEwen et al., 1999; Sousa et al., 2000). Increased glucocor- ticoids following stress exposure decreased cell proliferation in the hippocampus (Nichols et al., 2001). The expressions of BrdU and Ki-67 have been used as markers for cell proliferation (Scholzen et al., 2000; Kim SE et al., 2010; Sung YH et al., 2010). All three types of stress (restraint, noise, and cold stress) suppressed cell proliferation in the hippocampus, indicating that these stresses inhibited new cell formation in the hippocampus. Restraint stress had the most potent suppressing effect on new cell formation in the hippocampus and cold stress the least.

Such a reducing effect of stress on cell proliferation and/or neurogenesis has been postulated to be due to the decreased expression of neurotrophic factors such as BDNF (Xu et al., 2007;

Badowska-Szalewska et al., 2010). BDNF is a neurotrophin involved in neuronal survival and plasticity that binds to high-affinity receptor, TrkB (Givalois et al., 2001). BDNF-TrkB interaction promotes the survival and differentiation of neurons and is involved in the modification of neurotransmission and synaptic plasticity of the central and peripheral nervous systems (Leibrock et al., 1989). Overexpression of BDNF and its receptor TrkB is known to improve learning or cognitive behavior, whereas inhibition of BDNF or TrkB causes memory impairment (Alonso et al., 2002; Koponen et al., 2004; Pietropaolo et al., 2007). BDNF was also suggested as the main factor for the maternal exercise-induced enhancing of memory in rat pups (Kim H et al., 2007). Jung SY et al.(2011) suggested that diabetes mellitus-induced memory impairment is closely associated with suppression of BDNF expression in the hippocampus. Stress or corticosterone treatment reduced BDNF protein level in the hippocampus (Smith et al., 1995). BDNF and TrkB are implicated in the stress response of the hippocampal neurons (Smith et al., 1996; Schaaf et al., 2000). All three types of stress (restraint, noise, and cold stress) suppressed the expression of BDNF and TrkB in the hippocampus, indicating that these stresses inhibited memory-associated neurotransmitter and its

receptor expression. Restraint stress caused the most potent inhibition of BDNF and TrkB expression in the hippocampus and cold stress the least.

Spatial memory was significantly deteriorated by restraint, noise, and cold stress. Cold stress had the least deteriorating effect on spatial memory and restraint stress the most. Cell proliferation and expression of BDNF and TrkB in the hippocampus were decreased by these stresses. Cold stress had the least suppressing effect on cell proliferation, BDNF, and TrkB and restraint stress the most. These ameliorating and suppressing effects of stresses were ranked as follows: restraint stress＞noise stress group＞cold stress. We showed that the impairment of spatial memory by stress may be due to inhibition of cell proliferation through suppression of BDNF expression in the hippocampus.

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= 국문초록 =

스트레스는 항상성과 체내 균형을 교란시키는 물리적, 생리적, 그리고 정신적 변화를 의미한다. 스트레스는 우울증, 뇌졸중, 그리고 알쯔하이머 병 등 여러 뇌 질환을 유발 시키는 인자로 알려져 있다. 해마는 스트레스에 민감한 뇌의 부위이다. 본 연구에서는 구속 스트레스, 소음 스트레스, 그리고 추위 스트레스 등 여러 종류의 스트레스가 공간기억 과 해마에서의 세포 생성에 미치는 영향을 실험하였다. 본 실험에서는 8-arm maze test, 면역조직화학법, 그리고 웨스 턴 블롯을 사용하였다. 각각의 스트레스는 하루 1시간씩 2회, 10일간 적용하였다. 본 실험결과 구속 스트레스, 소음 스트레스, 그리고 추위 스트레스는 공간기억을 감소시켰다. 또한 구속 스트레스, 소음 스트레스, 그리고 추위 스트레 스는 해마에서의 신경세포 생성을 억제하였다. 이러한 스트레스의 작용은 brain-derived neurotrophic factor (BDNF)와 tyrosine kinase receptor B (TrkB)의 발현을 억제함으로써 일어났다. 본 실험을 통하여 구속 스트레스, 소음 스트레스, 그리고 추위 스트레스 모두 신경세포의 생성을 억제하여 기억력 감퇴를 유발시킴을 알 수 있었다. 이 세가지 스트레 스 중 신경세포생성을 억제하고 공간기억력을 감퇴시키는 작용은 구속 스트레스＞소음 스트레스＞추위 스트레스의 순으로 나타났다.

중심단어: 구속 스트레스, 소음 스트레스, 추위 스트레스, 공간 기억, 신경세포생성 Neurosci. 15:1768-1777.

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