The interaction between acute oligomer Aβ1–40 and stress severely impaired spatial learning and memory

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The interaction between acute oligomer Ab

1–40

and stress severely impaired

spatial learning and memory

Hei-Jen Huang

a,b

_{, Keng-Chen Liang}

c

_{, Yen-Yu Chang}

b

_{, Hsing-Chieh Ke}

_a,*

_{, Jia-Yu Lin}

b

_{, Hsiu Mei Hsieh-Li}

_a,*

a

Department of Life Science, National Taiwan Normal University, Taipei 116, Taiwan

b

Department of Nursing, Mackay Medicine, Nursing and Management College, Taipei, Taiwan

c

Department of Psychology, National Taiwan University, Taipei, Taiwan

a r t i c l e

i n f o

Article history: Received 1 May 2009 Revised 24 July 2009 Accepted 29 July 2009 Available online xxxx Keywords: Stress Olgomer Ab1–40 GR/MR ratio Oxidative stress

Spatial learning and memory

a b s t r a c t

In this study, we investigated whether stress can enhance the toxicity of oligomer Ab1–40in the mouse

brain. Stress was applied to the animals, consisting of a 2-day inescapable foot shock followed by 3-weekly situation reminders (SRs). We found that stress signiﬁcantly affected not only the amygdala-dependent (anxiety) but also the hippocampal-amygdala-dependent (spatial learning and memory) behaviors through the oxidative damage caused in these two regions. However, oligomer Ab1–40treatment alone

did not induce behavioral impairment. In addition, combined oligomer Ab1–40 and stress treatment

increased the glucocorticoid receptor (GR)/mineralocorticoid receptor (MR) ratio and the expression of corticotrophin releasing factor 1 (CRF-1) receptor in the hippocampus. Changes in the components of the hypothalamic–pituitary–adrenal (HPA) axis, such as the GR/MR ratio and CRF-1 level, were observed, accompanied by increasing Ab accumulation, oxidative stress, nuclear transcription factor (NF-jB) hypo-activity, and apoptotic signaling in the hippocampus, and decreasing calbindin D28K and NMDA receptor 2A/2B (NR2A/2B) in the hippocampus, along with alteration of the cholinergic neurons (ChAT) in the medium septum/diagnoid band (MS/DB), noradrenergic neurons (TH) in the locus coeruleus (LC), and serotonergic neurons (5-HT) in the Raphe nucleus. Therefore, apoptosis and synaptic dysfunction in the hippocampus severely induced the impairment of spatial learning and memory. These results suggest that stress may play an important role in the early stages of Alzheimer’s disease (AD), and an antioxidant strategy might be a potential therapeutic approach for stress-mediated disorders.

1. Introduction

Stress has been suggested to be one of the environmental fac-tors that can inﬂuence the pathogenesis of Alzheimer’s disease

(AD) (Hasegawa, 2007; Wilson et al., 2006). Several reports have

identiﬁed increased plasma corticosterone levels in patients with

dementia of an Alzheimer’s type (Dong et al., 2008; Peskind,

Wilkinson, Petrie, Schellenberg, & Raskind, 2001; Weiner, Vobach, Olsson, Svetlik, & Risser, 1997). However, increased glucocorticoids do not absolutely lead to AD, because a number of disorders result in increased cortisol, such as Cushing’s disease, and yet no evidence has been published demonstrating a link between these patients

and AD (Swaab, Bao, & Lucassen, 2005). Therefore, whether an

increased glucocorticoid level is a risk factor for AD needs further investigation. One recent study suggested that stress works as a

co-factor to induce vulnerability in hippocampal neurons (McDonald,

Craig, & Hong, 2008). Other evidences further suggest that stress increases Ab1–42deposition (Dong et al., 2008; Kang, Cirrito, Dong,

Csernansky, & Holtzman, 2007). Several reports have shown that, under general conditions, a higher proportion of Ab is produced in the form of Ab1–40than Ab1–42, and an increased Ab1–40/Ab1–42

ratio in cerebrospinal ﬂuid (CSF) has been identiﬁed during the

early stages of AD (Hu, Smith, Walsh, & Rowan, 2008; Kanai

et al., 1998). However, whether stress also regulates Ab1–40level

and toxicity has not yet been elucidated in present.

The hippocampus, which is involved in central brain functions such as cognition, learning, and memory, is very sensitive to vari-ous neurological insults, such as behavioral stressors (Morra et al., 2008) and Ab deposition in AD (Behl, 1998). There are two types of adrenal steroid receptors in the hippocampus, MR and GR. An early study showed that the balance of the GR/MR ratio regulates the homeostasis of calcium, NR2A/NR2B, NF-jB, and the considerable input mediated by biogenic amines such as acetylcholine,

nor-adrenalin, and serotonin into hippocampal neurons (De Kloet,

Vreugdenhil, Oitzl, & Joels, 1998). In addition, recent studies have also suggested that neurodegenerative and psychiatric disorders induce brain oxidative stress (Matos, Augusto, Oliveira, & Agostinho, 2008; Rammal, Bouayed, Younos, & Soulimani, 2008). Therefore, we attempted to examine the GR/MR ratio, calcium binding

* Corresponding author. Fax: +886 2 29312904. E-mail address:hmhsieh@ntnu.edu.tw(H.-C. Ke).

Contents lists available atScienceDirect

Neurobiology of Learning and Memory

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y n l m e

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mem-protein, Ab1–40accumulation, oxidative stress, nuclear

transcrip-tion factor NF-jB, NR2A/2B, and related neurotransmitters of the hippocampus in order to evaluate the effects of acute oligomer Ab1–40treatment after chronic stress.

In this study, we demonstrated that mice under an inescapable electric foot shocks followed by 3-weekly situational reminders (SRs), exhibited increased levels of plasma corticosterone, Ab1–40

deposition in the hippocampus, and oxidative stress in the hippo-campus and amygdale, as well as avoidance of the shock box, and a decreased calcium binding signal in the hippocampus and de-creased related neurotransmitter projection into the hippocampus. In addition, we also found that stress affected not only amygdale-dependent but also hippocampal-amygdale-dependent behaviors through oxidative damage in the hippocampus and amygdale. Therefore, the stress manipulation is reliable and provides a long-lasting stress animal model. Notably, the interactive effects of oligomer

Ab1–40and stress altered the components of the HPA axis, such

as GR, MR, and CRF-1 in the hippocampus. Accompanying effects

such as intracellular calcium dysregulation, Ab1–40accumulation,

oxidative stress enhancement, NF-jB hypoactivity, and apoptotic signal expression in the hippocampus were also observed in these animals. In addition to apoptosis, synaptic dysfunction may also be modiﬁed through the decreasing of NR2A/2B in the hippocampus along with the alteration of neurotransmitter systems projection into the hippocampus. Therefore, the interaction between oligomer

Ab1–40and stress-induced apoptosis and synaptic dysfunction in

the hippocampus subsequently signiﬁcantly resulted in the severe impairment of spatial learning and memory.

2. Materials and methods 2.1. Subjects

A total of 120 male C57BL/6J mice (6–8 weeks old) were pur-chased from the National Breeding Center for Laboratory Animals (Taipei, Taiwan). The mice were randomly divided into four groups, each containing 30 animals: (i) non-stress/CA1 administration of

vehicle; (ii) non-stress/CA1 administration of oligomer Ab1–40;

(iii) stress/CA1 administration of vehicle; and (iv) stress/CA1

administration of oligomer Ab1–40. The experiments were

per-formed during the light phase between 9 AM and 5 PM. All exper-imental procedures involving animals were performed according to the guidelines established by the Institutional Animal Care and Use Committee of National Taiwan Normal University, Taipei, Tai-wan. The mice were housed at 20–25 °C and 60% relative humidity under a 12-h light/dark cycle (light turned on at 7 AM), and food and water were made available ad libitum.

2.2. Experiment timeline and establishment of the stress model

The stress model was established as previously described (Li,

Murakami, Wang, Maeda, & Matsumoto, 2006). As depicted in

Fig. 1, the male mice (n = 120) were acclimatized in their home cages on days 1–5 and were handled once a day during this 5-day habituation period. Handling consisted of holding the animal in gloved hands for 2 min. After adaptation, mice received either

shocks (a total of 15 intermittent inescapable electric foot shocks of 0.8 mA intensity, 10 s interval, and 10 s duration; n = 60) or no shocks (n = 60) on days 6 and 7, respectively. Prior to shock treat-ment, the mice were allowed a 10-s adaptation period in the shock box (the dark compartment of the light–dark transition test box). The non-stress groups received the same treatment, but with the shock mechanism inactivated. The mice were re-exposed to the same chamber but without foot shock treatment on days 8, 13, and 20 (SR 1, 2 and 3, respectively). After each SR, a blood sample was collected from each mouse and analyzed in order to measure the corticosterone level. On day 21, after SR3, 60 mice in each group were randomly assigned to receive an injection of oligomer Ab1–40(n = 30) or vehicle (n = 30) in the CA1 subregion of the

hip-pocampus. After a 5-day recovery period, mouse behavior was evaluated by locomotor, light–dark transition, and water maze tasks on days 27, 28, and 29–35, respectively. Twenty-four hours after SR4 (conducted on day 36), the mice were sacriﬁced for Wes-tern blot and immunohistochemistry analyses.

2.3. Analysis of corticosterone level

Animals (n = 3 for each group) were sacriﬁced immediately after each SR and blood samples collected into heparin-treated tubes, which were then placed on wet ice. After centrifugation

(2000g), blood plasma was isolated and stored at 20 °C until

assaying. All experiments were performed in the morning (9– 11 AM) to minimize the possibility of interference from circadian alteration in the corticosterone level. A corticosterone competitive ELISA Kit (AssayPro system, GENTAUR) was used to measure the corticosterone level as per the manufacturer’s protocol.

2.4. Preparation of oligomer

Ab1–40was dissolved in 1 ml of double-distilled water to a

con-centration of 0.23 mM and incubated at 37 °C for 7 days to allow

oligomer formation, as previously described (Hoshi et al., 2003;

Huang, Liang, Chen, Chen, & Hsieh-Li, 2007). 2.5. Animal surgery

To avoid the side effects caused by interaction between brain operation and shock, amyloid application was performed after SR3 (day 21), which consisted of an acute injection of Ab into the CA1 of the mouse brain. Mice were anesthetized with avertin (0.016 ml/g, Sigma, MO, USA) and placed in a stereotaxic instru-ment (DKI-900, David Kopf Instruinstru-ments, CA, USA). An incision was made in the scalp and a hole drilled into the skull over the injection site, and a 30-gauge-needle was then lowered into the dorsal hippocampus. The coordinates for the anterior–posterior (from bregma), medial–lateral (from midline), and dorsal–ventral

(from surface of the skull) axes were 2.3, ±2.5, and 1.5 mm,

respectively. Bilateral intrahippocampal infusion was administered via a 10.0-ll Hamilton microsyringe with a 30-gauge needle ﬁtted

to the arm of the stereotaxic instrument. About 0.6

ll of oligomer

Ab1–40or vehicle was slowly infused at a rate of 0.2

ll/min. The

oli-gomer conformation of Ab1–40was conﬁrmed by Western blotting

Fig. 1. The experimental timeline of this study. Mice were exposed to inescapable electric foot shocks (0.8 mA, 10 s) followed by 3-weekly SRs. After the training sessions (days 6–20), mice were submitted to a testing session composed of three different behavioral tests, motor activity test, light–dark transition test, and the Morris water maze. Please cite this article in press as: Huang, H.-J., et al. The interaction between acute oligomer Ab and stress severely impaired spatial learning and

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mem-(data not shown). After an additional 5 min to ensure adequate dif-fusion, the needle was slowly retracted from the animal.

2.6. Locomotor

On day 27, the motor activity of the animals was recorded for 10 min. Motor tracking was performed using a video camera in-stalled on the ceiling, which fed directly into a personal computer

running a Noldus EthoVision video tracking system (Spink,

Tege-lenbosch, Buma, & Noldus, 2001) in subtraction mode. Prior to the start of recording, the animals were placed in Plexiglas cages (30 30 30 cm; one animal per testing cage) to which they were not habituated. The motor activity in the ﬁrst and second 5-min periods was then recorded, which was the same duration as the measurement period used for the light–dark transition test. 2.7. Light–dark transition test

The apparatus, modiﬁed from a previous study (Costall, Jones,

Kelly, Naylor, & Tomkins, 1989), consisted of a Plexiglas chamber

subdivided into two compartments, a dark compartment

(300 300 350 mm high, of the same scale, color, and odor as the chamber in which the foot shocks were delivered), and a light compartment (450 300 350 mm high, totally different from the dark compartment) illuminated by a white bulb (60 W), set 40 cm above the ﬂoor. The compartments were connected by a small divider (50 50 mm). On day 28, each animal was placed into the light compartment facing the wall opposite the divider. The latency of the ﬁrst entry into the dark compartment, the time spent in the dark compartment, and the numbers of transitions were assessed for a 5-min period.

2.8. Morris water maze (MWM)

Spatial memory was evaluated using a conventional Morris water maze (MWM), a device commonly used for studying cogni-tive deficits in APP transgenic mice (Janus & Westaway, 2001). The water maze apparatus consisted of a circular pool (1.2 m diam-eter and 0.47 m high) made of white plastic; the pool was filled to a depth of 40 cm with water (24–25 °C) and made opaque by the addition of nontoxic white paint. During conventional MWM train-ing, an escape platform (10 cm in diameter) made of white plastic, with a grooved surface for better grip, was submerged 0.5 cm underneath the water level. Cues of various types provided distal landmarks in the testing area of the room. The swim path of the mouse during each trial was recorded by a video camera sus-pended 2.5 m above the center of the pool and connected to a video tracking system. On the day prior to spatial training, all mice underwent pre-training in order to assess their swimming ability and to acclimatize them to the pool. Each mouse was first placed on a visible platform located at the center of the pool and allowed to stay there for 20 s. In the following three 60-s trials, the mouse was released into the water facing the wall of the pool from semi-randomly chosen cardinal compass points. If the mouse failed to swim to the platform or stay on it for 20 s, it would be placed on the platform by an experimenter. The mice were given a 4-day training session consisting of four 60-s training trials (inter-trial interval: 20–30 min) per day. The hidden platform was always placed at the same location of the pool (Northeast quadrant) throughout the training period. During each trial, from quasi-ran-domly chosen cardinal compass points, the mouse was released into the water facing the pool wall. After climbing onto the plat-form, the mouse was allowed to rest on it for 20 s. After the last training trial, all mice were given three testing trials to assess the time taken to climb onto the hidden platform. 24 h after the

last testing trial, all mice were given three probe trials to evaluate their spatial memory regarding the platform.

2.9. Immunohistochemistry

Immediately after the water maze test, nine mice per group were anesthetized (avertin, 0.016 ml/g) and transcardially per-fused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Mouse brains were removed and post-ﬁxed with 4% parafor-maldehyde overnight and then placed in 30% sucrose in PBS for 2 days, followed by continuous serial cryostat sectioning at

30

lm. Rabbit anti-Ab

1–40 polyclonal antibody (Chemicon, CA,

1:200 dilution) staining was used to detect the presence of Ab. Spe-cific antibodies were used to assess oxidative stress (MnSOD, Up-state, CA, 1:200 dilution), apoptosis (Caspase3, Chemicon, 1:40 dilution), and neurotransmitters by rabbit anti-ChAT polyclonal antibody (choline acetyltransferase, cholinergic specific, 1:500; Chemicon), rabbit anti-TH polyclonal antibody (tyrosine hydroxy-lase, noradrenergic-specific, 1:1000; Chemicon), and rat anti-sero-tonin monoclonal antibody (5-HT, serotonergic-specific, 1:100; Chemicon).

Free-ﬂoating sections were immunostained for immunohisto-chemical analysis. In brief, sections were washed in PBS three times (10 min/wash). Nonspeciﬁc epitopes were then blocked by incubation in 5% normal goat serum or normal rat serum and 0.1% Triton X-100 in PBS for 1 h. Sections were incubated in pri-mary antibodies overnight at room temperature, washed with PBS, and incubated in the secondary antibodies (1:200 dilution in blocking solution, Vector Laboratories, CA, USA) for 1 h, then in an avidin–biotin complex for 1 h at room temperature. The reac-tion was developed using a DAB-Kit (diaminobenzidine) from Vec-tor. All sections were mounted on coated slides and cover-slipped for light microscopy. The images obtained were loaded in a re-search-based digital image analysis software (Image Pro Plus, Med-ia Cybernetics, MD, USA) and the DAB pixel counts measured by setting the threshold to the same value for each section. Pixel counts were taken as the average from three adjacent sections per animal, and the data are presented inTable 1.

2.10. Western blot analysis

Western blot analysis was conducted on proteins extracted from the hippocampus with antibodies for GR, MR, CRF-1, NF-jB, NR2A/ 2B, and b-actin. The antibodies used were rabbit polyclonal GR (1:200; Santa Cruz), rabbit polyclonal MR (1:100; Santa Cruz), rabbit polyclonal CRF-1 (1:100; Santa Cruz), rabbit polyclonal NF-jB

(1:2000, Chemicon), rabbit polyclonal NR2A/2B (1:100;

Chemicon), and mouse monoclonal b-actin (1:2000; Chemicon); the secondary antibodies used were rabbit IgG HRP-linked anti-body (1:10,000; Cell Signaling, USA) for GR, MR, CRF-1, NF-jB, and NR2A/2B, and anti-mouse IgG HRP-linked antibody (1:10,000; Cell signaling) for b-actin. The speciﬁc antibody–antigen complex was detected by an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The intensity of Western blots was quantiﬁed using the LAS-3000 imaging system (Fuji, Japan), and was expressed as the ratio relative to b-actin protein.

2.11. Data analysis

Data were analyzed by two-way analysis of variance (ANOVA) by group (stress and non-stress) and CA1 (vehicle and oligomer Ab1–40) administration, followed by Student’s post hoc t-testing in

order to compare the effects of all treatments. In addition, the light–dark transition test and Western blotting results were analyzed by nonparametric multiple independent samples testing.

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mem-Results are expressed as mean ± SEM. Differences were considered statistically signiﬁcant if_{p < 0.05,}_{p < 0.01, and}_{p < 0.001.}

3. Results

3.1. Stress enhanced the plasma corticosterone level of treated animals The stress resulting from the shock treatment followed SRs sig-niﬁcantly enhanced the plasma corticosterone concentration in the

mice. As shown inFig. 2, the plasma corticosterone levels were

greatly increased in the stressed mice but not in the non-stressed mice (F(1, 35) = 182.0578, p < 0.001). The corticosterone levels were not signiﬁcantly different between the SR (F(3, 35) = 2.4206, p > 0.05) and stress SR interaction groups (F(3, 35) = 0.4116, p > 0.05). Furthermore, the plasma corticosterone concentration in the non-stressed mice remained at the basal morning level throughout the experiment.

3.2. Avoidance into the dark compartment increased with aversive experience in the stressed mice

Animals that had been previously exposed to stress exhibited an increase in the latency of escape from the light compartment

to the dark compartment (p < 0.05;Fig. 3A), a decrease in the time spent in the dark compartment (p < 0.05;Fig. 3B), and a decrease in the number of transitions during the testing period (p < 0.05;

Fig. 3C). These results showed that the stressed mice learned to avoid the aversive-like compartment (the dark compartment), indicating that they exhibited a fear response to the context asso-ciated with traumatic events.

3.3. Motor activity was not affected in stressed mice, regardless of oligomer Ab1–40or vehicle treatment

The effects of oligomer Ab1–40and vehicle on motor activity in

mice with stress/non-stress were shown inFig. 4. We used the first and second 5 min for analysis because that 5-min duration is the same as the measurement period used in the light–dark transition test. There were no significant alterations in the motor activity identified in the stressed (F(1, 33) = 3.0382, p > 0.05), oligomer-treated (F(1, 33) = 2.3299, p > 0.05), and combined stress oligo-mer Ab1–40-treated mice (F(1, 33) = 0.0829, p > 0.05) during the

ﬁrst 5 min of testing (Fig. 4A). The motor activity of each group during the second 5 min of the experiment was even more con-stant than in the ﬁrst 5 min (Fig. 4B). The statistical results for the different treatment groups are F(1, 33) = 1.1478 (p > 0.05) for the stressed group, F(1, 33) = 3.8654 (p > 0.05) for the oligomer-treated group, and F(1, 33) = 0.0070 (p > 0.05) for the combined treatment group. These results reveal that the motor activity was not affected in stressed mice.

3.4. Alteration of cholinergic, noradrenergic, and serotonergic immunoreactive neurons in stressed and oligomer Ab1–40-treated

animals

We observed the alteration of cholinergic immunoreactive neu-rons (ChAT-ir), tyrosine hydroxylase immunoreactive neuneu-rons (TH-ir), and serotonergic immunreactive neurons (5-HT-ir) in the mouse brain after different treatments (Fig. 5). The quantitative re-sults are summarized inTable 1. Stress signiﬁcantly altered ChAT-ir in the medium septum/diagnoid band (MS/DB) (F(1, 8) = 1516.467, p < 0.001), TH-ir in the locus coeruleus (LC) (F(1, 8) = 68.2954, p < 0.05), and 5-HT-ir in the Raphe nucleus (F(1, 8) = 77.8087, p < 0.05). Oligomer treatment also signiﬁcantly altered the levels of ChAT-ir in the MS/DB (F(1, 8) = 706.3953, p < 0.05), 5-HT-ir in the Raphe nucleus (F(1, 8) = 51.7464, p < 0.05), and TH-ir in the locus coeruleus (F(1, 8) = 58.5573, p < 0.05). The interactive effect of stress and oligomer Ab1–40treatment severely decreased the ChAT-ir in the

MS/DB (F(1, 8) = 61.3969, p < 0.001), TH in the LC (F(1,8) = 5.6313,

Table 1

The quantiﬁcation of the immunohistochemical analyses in the different treatment mice.

Non-stress-vehicle Non-stress-Ap Stress-vehicle Stress-Ap

APlJM(CA1) 24.0 ± 3.61 242.0 ± 4.93b,* 122.0 ± 2.65a,* 552.7 ± 6.01c,* MnSOD(CA1) 23.0 ± 4.04 115.0 ± 3.79b,* 59.3 ± 3.18a,* 190.0 ± 10.82c,*** MnSOD (Amy) 235.0 ± 3.46 259.3 ± 7.69 748.3 ± 29.87a,*** _{797.0 ± 7.93}a,* Caspase 3 (CA1) 0.0 ± 0.0 5.3 ± 0.45 0.0 ± 0.0 33.3 ± 4.03c,*** Calbindin (CA1) 175.3 ± 3.48 133.7 ± 7.22b,* _{105.0 ± 4.04}a,* _{53.3 ± 5.04}c,*** ChAT(MS) 193.4 ± 4.05 125.0 ± 5.03b,* _{80.0 ± 4.93}a,* _{64.7 ± 5.46}c,*** ChAT(DB) 164.7 ± 6.57 87.7 ± 3.28b,* _{77.2 ± 4.09}a,* _{16.3 ± 2.60}c,*** TH (LC) 1150.7 ± 48.35 871.7 ± 25.33b,* _{778.3 ± 9.56}a,* _{543.7 ± 17.65}c,*** 5-HT (Raphe nucleus) 37.4 ± 4.61 18.7 ± 1.44b,* 22.2 ± 1.49a,* 0 ± 0c,*** Each value represent the mean ± SEM (n = 3 for each group).

a

Indicated as non-stress versus stress.

b

Indicated as Ap versus vehicle.

c

Indicated as interaction between stress and Ap.

*_{p < 0.05.} **

p < 0.01.

***

p < 0.001.

Fig. 2. Plasma corticosterone levels in the stressed and non-stressed mice. Regardless of SR, plasma corticosterone levels were signiﬁcantly higher in the stressed mice than in the non-stressed mice after 24 h of SR.

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mem-p < 0.001), and 5-HT-ir in the Ramem-phe nucleus (F(1, 8) = 5.609, p < 0.001).

3.5. Increasing Ab accumulation, oxidative stress, and apoptotic signals in the stressed and oligomer Ab1–40-treated mice

Immunohistochemical analyses of Ab1–40accumulation,

oxida-tive stress, and apoptotic signals are shown inFig. 6andTable 1. We observed that stress, oligomer treatment, and combined interac-tion (stress oligomer Ab1–40) signiﬁcantly enhanced the

accumu-lation of Ab1–40in the CA1 of the hippocampus (F(1, 8) = 2076.072,

p < 0.05; F(1, 8) = 5230.547, p < 0.05; and F(1, 8) = 562.2155, p < 0.001, respectively). In addition, we also found that stress signifi-cantly enhanced the oxidative stress signal MnSOD in the CA1 of the hippocampus (F(1, 8) = 78.5606, p < 0.05) and the basolateral part of the amygdale (BLA) (F(1, 8) = 1076.376, p < 0.001); however, oligomer treatment significantly induced oxidative stress only in the CA1 (F(1, 8) = 314.2423, p < 0.05), and not in the BLA (F(1, 8) = 5.1928, p > 0.05). Significant oxidative stress was also identified in the CA1 (F(1, 8) = 9.4761, p < 0.001), but not in the BLA (F(1, 8) =

0.577, p > 0.05), induced by combined stress oligomer Ab1–40

treatment. In addition, all the treatments signiﬁcantly induced the apoptotic signal caspase 3 in the CA1: for stress, (F(1, 8) = 37.5319, p < 0.001); for oligomer treatment, F(1, 8) = 71.5745, p < 0.001;

and for stress oligomer Ab1–40 interaction, F(1, 8) = 37.5319,

p < 0.001. However, only in stress-treated oligomer Ab1–40 mice

Fig. 3. Anxiety evaluation of stressed and non-stressed mice after acute CA1 administration with oligomer Ab1–40 and vehicle. After treatment, the mice

underwent the light–dark transition test for 5 min on day 28. The latency of the ﬁrst entry into the dark compartment (A), time spent in the dark compartment (B), and number of transitions (C) of the animals were recorded. These results show that the stressed mice exhibited elevated anxiety levels and speciﬁc avoidance of the context associated with stress, regardless of oligomer Ab1–40or vehicle treatment.

Fig. 4. The effects of oligomer Ab1–40and vehicle on the motor activity in mice with

stress or non-stress treatment. (A) The distance traveled within the chamber in the first 5 min of testing. (B) The distance traveled within the chamber in the second 5 min of testing. Motor activity did not differ significantly between groups in either the first or second 5-min testing period.

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mem-was a signiﬁcant difference in apoptosis of the CA1 induced as com-pared with the other three groups, from the post hoc Student’s t-test analysis.

3.6. Reduction of calbindin immunoreactive neurons in stress and oligomer Ab1–40-treated mice

The calbindin immunoreactive neurons were signiﬁcantly re-duced in the CA1 of the hippocampus in the stress- (F(1, 8) = 294.2402, p < 0.05;Fig. 7andTable 1) and oligomer Ab1–40-treated

mice (F(1,8) = 123.9265, p < 0.05;Fig. 7andTable 1); a signiﬁcant

decrease in the stress oligomer Ab1–40 interaction group

(F(1, 8) = 5.6667, p < 0.001;Fig. 7andTable 1) was also observed. In addition, from the post hoc Student’s t-test, the stress and oligo-mer Ab1–40-treated mice exhibited the most severe loss of

calbin-din immunopositive neurons in the CA1 of the hippocampus as compared with the other three groups.

3.7. Increases in the GR/MR ratio and CRF-1 expression, and decreases in NF-jB and NR 2A/2B expression were observed in the hippocampus of the combined stress and oligomer Ab1–40-treated mice

We examined the GR/MR ratio, CRF-1, NF-jB, and NR2A/2B expression in the whole hippocampus in the animals (n = 9 for each group), and found a significant alteration in the GR/MR ratio, CRF-1, NF-jB, and NR2A/2B expression in the hippocampus of the combined-treated mice (p < 0.05;Fig. 8D). However, no significant effect on the GR/MR ratio, CRF-1, NF-jB, or NR2A/2B expression was identified in animals that received stress or oligomer Ab treat-ment alone (p > 0.05;Fig. 8).

3.8. Spatial reference learning and memory was severely impaired in the stress and oligomer Ab1–40-treated mice

During the testing phase, the stressed mice took a signiﬁcantly longer time than the non-stressed mice to reach the hidden platform (F(1, 36) = 31.3427, p < 0.001; Fig. 9A), especially the oligomer Ab1–40-treated mice (F(1, 36) = 6.5382, p < 0.05;Fig. 9A). However,

no signiﬁcant effect was identiﬁed in the combined stress oligo-mer Ab1–40-treated animals (F(1, 36) = 3.1349, p > 0.05;Fig. 9A).

During the training phase, a substantial delay in learning was ob-served in the stressed mice as compared with the mice in the non-stressed groups (Fig. 9B). On training day 1 of the spatial learning task, stress was found to signiﬁcantly damage the acquisition of spa-tial learning (F(1, 36) = 4.2773, p < 0.05), as did oligomer treatment (F(1, 36) = 4.0673, p < 0.05), but no signiﬁcant interaction was found

in the combined stress oligomer Ab1–40-treated mice

(F(1, 36) = 0.8443, p > 0.05). However, on training days 2–4, signiﬁ-cant impairment in the spatial learning task was observed in the stressed (p < 0.05), oligomer-treated (p < 0.05), and combined stress oligomer Ab1–40-treated (p < 0.05) mice. The acquisition of

spatial learning was not impaired in the non-stressed groups,

although the oligomer Ab1–40-treated mice needed more time to

search for the platform than the vehicle-treated mice (p < 0.05;

Fig. 9B). By two-factor ANOVA analysis, no treatment effect (F(3, 244) = 0.6129, p > 0.05;Fig. 9C) but a significant quadrant effect (F(3, 244) = 27.4603, p < 0.001;Fig. 9C) was identified, and signifi-cant interaction between the different treatments and quadrants (F(9, 244) = 5.6336, p < 0.001;Fig. 9C) was noted. Post hoc compari-son showed a significant decrease in the target quadrant as com-pared with the other three quadrants for the combined stress and

oligomer Ab1–40-treated mice. Furthermore, the swimming velocity

Fig. 5. Reduction of cholinergic, noradrenergic, and serotoninergic immunoreacitve neurons in treated mice. Representative immunostaining results of ChAT in the MS (a–d) and VDB/HDB (e–h), TH in the LC (i–l), and 5-TH in the Raphe nucleus (m–p). Whether combined with oligomer Ab1–40treatment or not, stress treatment caused a signiﬁcant

reduction in immunoreactive neurons in the MS/DB, LC, and Raphe nucleus; however, combined treatment induced an even more severe reduction in immunoreactive neurons in these regions of treated mice (n = 3 for each group). Scale bar = 50lm.

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mem-in the probe test was not signiﬁcantly different between the groups of mice (data not shown).

4. Discussion

The aim of this study was to evaluate the effects of stress, acute

oligomer Ab1–40and the combined interaction of these two factors

in C57BL/6J male mice by assessing plasma corticosterone concen-tration, serial behavioral, immunohistochemical data and western blot analyses results. First, a stress paradigm was established by administering 2 days of inescapable foot shocks followed by re-peated SRs. After SR administration, we found that the plasma cor-ticosterone level in the stressed mice was signiﬁcantly enhanced, a result consistent with those of previous studies (Bland et al., 2005;

Fig. 7. Calbindin expression in the CA1 subregion of the hippocampus in treated mice. Mice subjected to oligomer Ab1–40, stress or combined treatment exhibited signiﬁcantly

reduced calbindin immunopositive neurons as compared with the non-stressed and vehicle-treated mice. Arrowheads indicate signals. Scale bar = 50lm.

Fig. 6. Immunohistochemistry of Ab1–40accumulation, oxidative stress and apoptosis in treated mice. Representative immunostaining results of Ab1–40immunoreactive

neurons in the CA1 (a–d), MnSOD in the CA1 (e–h), and BLA (i–l) and caspase 3 in the CA1 (m–p). Oligomer Ab1–40treatment induced Ab1–40accumulation and oxidative stress

in the CA1; stress induced Ab1–40accumulation in the CA1, and oxidative stress in the CA1 and BLA. Furthermore, the interaction between acute administration of oligomer

Ab1–40and stress accelerated Ab1–40accumulation, oxidative stress, and apoptosis in the CA1. Arrows indicate signals. Scale bar = 50lm.

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mem-Fig. 8. GR/MR ratio, CRF-1, NF-jB, and NR2A/2B expression in the hippocampus of the treated mice. (A) Expression and quantification of the GR/MR ratio in the whole hippocampus. (B) Expression and quantification of CRF-1 in the whole hippocampus. (C) Expression and quantification of NF-jB in the whole hippocampus. (D) Expression and quantification of NR2A/2B in the whole hippocampus. After stress, mice treated with oligomer Ab1–40exhibited a significant change in the GR/MR ratio, CRF-1, NF-jB, and

NR2A/2B expression in the hippocampus as compared with the other three groups. To conﬁrm equal protein loading, the same blots were hybridized with antibodies speciﬁc for b-actin.

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mem-Jin, mem-Jin, Zhou, Li, & Chi, 1997; Louvart et al., 2006; Ottenweller, Natelson, Pitman, & Drastal, 1989). In terms of behavioral analysis, stress not only induced anxiety, as shown in previous studies (Koba et al., 2001; Li et al., 2006; Louvart et al., 2006), but also im-paired spatial learning and memory, as reported in an early study (Ladera-Fernandez, 2001). In addition, we also found that stress had no inﬂuence on motor activity, as found in previous studies (Li et al., 2006; Pynoos, Ritzmann, Steinberg, Goenjian, & Prisecaru, 1996; Yan et al., 2004), although some early evidence suggested that animals exhibit hypoactivity after exposure to electrical foot shocks (Van den Berg, Lamberts, Wolterink, Wiegant, & Van Ree, 1998; van Dijken, Mos, van der Heyden, & Tilders, 1992). The rea-son for these contradictory results may be the different experimen-tal paradigms, animal strains, or shock intensities used in each study. From the immunostaining results, it was found that stress

signiﬁcantly increased Ab1–40deposition in the CA1 subregion of

the hippocampus, and oxidative stress in the CA1 subregion and the BLA, and decreased 5-HT-ir in the Raphe nucleus, ChAT-ir in the MS/DB, TH-ir in the LC, and calbindin D28K in the CA1 subre-gion. Previous studies have reported that stress enhanced the Ab level (Dong et al., 2008; Hasegawa, 2007), oxidative stress (Ng, Berk, Dean, & Bush, 2008; Zaﬁr & Banu, 2008), and dysregulation in neurotransmitter regulation in serotonergic, cholinergic and noradrenergic neurons (Berridge & Waterhouse, 2003; Brand, Gro-enewald, Stein, Wegener, & Harvey, 2008; Forster, Schoenfeld, Marmar, & Lang, 1995; Helm, Ziegler, & Gallagher, 2004), and

de-creased calcium binding protein calbindin D28K (Keifer, Brewer,

Meehan, Brue, & Clark, 2003). Therefore, these data suggest that inescapable foot shock treatment followed by SRs is a reliable and long-lasting stress animal model,

In this study, acute oligomer Ab1–40treatment alone induced

Ab1–40 accumulation, oxidative stress, calcium binding signal

reduction in the CA1, and related neurotransmitter dysregulated projection into the hippocampus; however, it did not induce any behavioral impairment in the animals. These results are consistent

with those of recent studies (Huang et al., 2007; Schmid et al.,

2008). Recent evidence also suggests that alterations in

neuro-transmitters alone cannot induce alteration of behavior (Alves

et al., 2008; Lamour, Bassant, Potier, Billard, & Dutar, 1994). Taken

together, these results suggest that acute oligomer Ab1–40

treat-ment alone is not sufﬁcient to induce behavioral impairtreat-ment; how-ever, interaction with other factors, such as stress, as shown in our study, is sufﬁcient to impair spatial learning and memory.

The different physiological responses exhibited by mice

receiv-ing stress and acute oligomer Ab1–40treatment were evidenced by

the level of plasma corticosterone and the target sites of oxidative stress. In this study, we found that stress, and not acute oligomer

Ab1–40treatment, induced anxiety and the impairment of spatial

learning and memory through oxidative stress in the amygdala and hippocampus. Several pieces of evidence have shown that reduction in the activities of some antioxidant enzymes can be di-rectly caused by high levels of corticosterone in the brain ( McIn-tosh, Hong, & Sapolsky, 1998; Zaﬁr & Banu, 2008). In addition, an oxidative pathophysiology in stress disorders is strongly supported

by animal models and human biochemical data (Ng et al., 2008).

Recent evidence further shows that dysfunction in the connection between the hippocampus and amygdale induces modiﬁcation of synaptic plasticity in the hippocampus (Hegde et al., 2008; Reagan, Grillo, & Piroli, 2008; Tsoory et al., 2008). These data suggest that oxidative mechanisms may be involved in the common pathogenic pathways of stress, which implies new targets for the development of therapeutic interventions for stress-mediated disorders.

In addition, we found that elevated corticosteroid levels

dramatically increased the toxic effect of oligomer Ab1–40in the

Fig. 9. Interactive effects of oligomer Ab1–40and stress on mouse performance in

the Morris water maze task. After four training sessions on days 30–33, three testing trials were carried out on day 34, and the probe trial was performed 24 h after the last testing trial. (A) Task acquisition in the stressed mice was signiﬁcantly slower than in the non-stressed mice. Acquisition of the task in the oligomer Ab1–40-treated mice was also signiﬁcantly slower than in the

vehicle-treated mice. (B) Oligomer Ab1–40treatment in association with stress severely

induced impairment in the spatial learning task. (C) The stressed mice spent less time in the target quadrant than the non-stressed mice, especially under oligomer Ab1–40treatment.

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mem-hippocampus, such as accumulation of Ab, the level of oxidative stress, the GR/MR ratio, the apoptosis signals, expression of CRF-1, NR2A/2B, NF-jB, calbindin, cholinergic neurons in the MS/DB, serotonergic neurons in the Raphe nucleus, and noradrenergic neu-rons in the LC, which subsequently induced a severe impairment of spatial learning and memory. Recent studies have noted that corti-costerone exacerbates the accumulation of the Ab in Tg2576

Alz-heimer’s transgenic mice (Green, Billings, Roozendaal, McGaugh,

& LaFerla, 2006; Lee et al., 2009). Therefore, we suggest that stress

enhances the toxicity of oligomer Ab1–40. In addition, the GR/MR

ratio, CRF-1 expression, Ab accumulation, and oxidative stress

lev-els were increased after combined stress and oligomer Ab1–40

treatment. A previous study suggested that changes in the compo-nents of the HPA axis induce Ab accumulation (Dong et al., 2008); recent evidence further implies that accumulation of oxidative stress and elevation of Ab level may exacerbate a vicious cycle in

progressive Ab accumulation (Misonou, Morishima-Kawashima, &

Ihara, 2000; Shen et al., 2008; Tabner et al., 2005; Tamagno et al., 2005; Tong et al., 2005). Therefore, we suggest that positive feed-back may be established in the relationships between the

cortico-sterone concentration, GR/MR ratio, CRF-1 receptor, Ab

accumulation, and oxidative stress. In this study, we also found

that the interactive effect of stress and oligomer Ab1–40induced

NF-jB hypoactivity and apoptotic signaling in the hippocampus. This result was consistent with that of a recent study, which found that intracerebroventricular Ab injection downregulated NF-jB

and induced apoptosis in the hippocampus (Ji et al., 2008). With

the exception of apoptosis, our results further indicated that expression of NR2A/2B and calbindin in the hippocampus was

sig-niﬁcantly decreased by oligomer Ab1–40 treatment on previous

aversive experience. These results were consistent with previous evidence that has shown that stress increases the accumulation of Ab and oxidative stress and further induces deregulation of

NMDA receptors (Harvey, Oosthuizen, Brand, Wegener, & Stein,

2004; Parameshwaran, Dhanasekaran, & Suppiramaniam, 2008). Therefore, these results demonstrated that positive feedback among corticosterone concentration, GR/MR ratio, CRF-1 expres-sion, Ab accumulation and oxidative stress-induced apoptosis through the inhibition of NF-jB activity in the hippocampus and synaptic dysfunction through decreasing the NR2A/2B and calcium binding protein levels in the hippocampus, which eventually se-verely induced impairment of spatial learning and memory.

In summary, the results of this study ﬁrst indicate that 2 days of inescapable foot shock treatment followed by 3-weekly SRs is a reliable and long-lasting stress animal model, in which an in-creased plasma corticosterone level and avoidance of the shock compartment were observed. In addition, we found that oxidative damage in the CA1 and BLA was induced by stress, and propose that oxidative damage may be involved in the common pathogenic pathways of stress. Furthermore, interaction between stress and

oligomer Ab1–40induced a positive feedback relationship among

corticosterone concentration, GR/MR, CRF-1, Ab1–40accumulation,

oxidative stress, and NF-jB, which resulted in apoptosis in the hip-pocampus. Finally, the interaction severely impaired hippocampal-dependent behavior, in addition to decreasing NR2A/2B, calcium-binding signaling, and the projection of related neurotransmitters into the hippocampus. Therefore, our ﬁndings suggest that stress may play an important role in the early stages of Alzheimer’s dis-ease (AD), and antioxidant therapy might attenuate stress and the impairment of spatial learning and memory.

Acknowledgments

Our gratitude goes to the Academic Paper Editing Clinic, NTNU. We thank Zheng-Ru Fang for her assistance in animal care, and Dr. Chien Chen Lu for ELISA technological assistance. This work was

supported in part by research grants from the National Taiwan Normal University (96TOP001) and National Science Council (NSC97-2320-B-003-003-MY3).

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