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Social isolation stress impairs passive avoidance learning in senescence-accelerated mouse (SAM)
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Research Report
Social isolation stress impairs passive avoidance learning in senescence-accelerated mouse (SAM) Yoichi Chida, Nobuyuki Sudo, Junko Mori, Chiharu Kubo ⁎ Department of Psychosomatic Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
Despite cumulative evidence showing the detrimental effect of psychosocial stress on the
Accepted 11 October 2005
learning/memory functions in dementia diseases, the precise neurobiological mechanisms
Available online 5 December 2005
behind such an effect remain unclear. Mice of the senescence-accelerated mice prone 10 (SAMP10) strain, a neurodegenerative dementia model, were chronically exposed to social
Theme:
isolation stress from the age of 5 weeks. At the age of 12 weeks, conditioning memory and
Neural basis of behavior
spatial memory were evaluated by one-trial passive avoidance and Y-maze tests,
Topic:
respectively. Chronic social isolation stress significantly reduced conditioning memory
Stress
but did not affect spatial memory. Although further behavioral tasks using an elevated plus maze and a pain threshold test exhibited stress-induced analgesia, an analysis of
Keywords:
covariance excluded the possibility that such analgesia might contribute to the stress-
Aging
induced impairment of conditioning memory. In addition, endocrinological and
Associative memory
immunohistochemical analysis revealed that isolation stress elevated the serum
Hypothalamus–pituitary–adrenal
corticosterone levels and inhibited the increase in c-Fos expression in the central
axis
amygdaloidal nucleus (CeA) that is required for conditioning memory during passive
Oxidative stress
avoidance learning. In conclusion, chronic social isolation stress exacerbated conditioning
Psychosocial stress
memory in SAM mice, probably through a glucocorticoid-mediated decrease in neural
Stress-induced analgesia
activation in the CeA. © 2005 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: +81 92 642 5336. E-mail address: [email protected] (C. Kubo). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.10.042
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Abbreviations: ANCOVA, analysis of covariance CeA, amygdaloidal nucleus GC, glucocorticoids HPA, hypothalamus–pituitary– adrenal L–D, light–dark MDA, malondialdehyde PBS, phosphate-buffered saline SAM, senescence-accelerated mice SIA, stress-induced analgesia
1.
Introduction
In encounters with environmental challenges, learning and memory are essential to the success of every living organism as they are necessary for prolonging survival and maintaining the species. Considerable evidence gained by extensive investigations of the influence of psychosocial factors on learning and memory has been reported (for review, see (Lupien and McEwen, 1997; McGaugh and Rooxendaal, 2002). Because the number of patients suffering from dementia diseases has been increasing worldwide, much attention has been focused on possible causal links between various psychosocial stresses and the development of dementia diseases such as Alzheimer's disease (Fratiglioni et al., 2004). For example, a higher susceptibility to psychological distress was reported to lead to double the risk of Alzheimer's disease (Wilson et al., 2003). However, the precise neurobiological mechanisms underlying such an injurious effect of psychosocial stress on dementia diseases remain to be elucidated. Senescence-accelerated mouse (SAM) includes senescenceacceleration-prone and -resistant substrains (SAMP and SAMR, respectively) (Takeda et al., 1991; Okuma and Nomura, 1998). Of the SAMP substrains, SAMP8 and SAMP10 mice exhibit deficits in learning and memory at a relatively early stage in their life span (Lowy et al., 1995; Numata et al., 2002; Flood and Morley, 1998). Therefore, these substrains have increasingly been used as a murine model for investigating the mechanisms underlying agerelated cognitive deficits (Maurice et al., 1996; Numata et al., 2002; Shimada et al., 2003; Strong et al., 2003; Tha et al., 2000; Ye et al., 2004). SAMP8 mice have been shown to exhibit traits similar to Alzheimer's disease and overproduce amyloid precursor protein naturally (Morley et al., 2000). More notably, SAMP10 mice have been reported to show age-dependent brain atrophy (Shimada, 1999) similar to that seen in patients suffering from neurodegenerative dementia diseases, such as Alzheimer's disease, Pick's disease, and other frontotemporal dementias. Social isolation, such as by individual housing, eliminates social interaction among animals and has been reported to induce pathophysiological changes in rodents: for example, an increased or decreased response of the hypothalamus–pituitary–adrenal (HPA) axis and sympathetic nervous system (Huong et al., 1997; Kim and Kirkpatrick, 1996; Mar Sánchez et al., 1998; Pashko et al., 1980), acceleration of the development and growth of either transplanted or chemically induced tumors (Kerr et al., 1997; Weinberg and Emerman, 1989; Wu et al., 1999), enhancement of small intestinal sensitivity to chemotherapy (Verburg et al., 2003), and exacerbation of the
autoimmune diseases of MRL/lpr mice (Chida et al., 2005). Thus, social isolation stress is considered a valuable stress paradigm for investigating the effect of chronic psychosocial stress on various pathophysiological alterations in animals. The present study, based on the social isolation stress paradigm, was done to examine the detrimental effect of psychosocial stress on conditioning memory and spatial memory in a neurodegenerative disease model using SAMP10 mice and to attempt to clarify the mechanism(s) involved.
2.
Results
2.1. The effect of social isolation stress on conditioning and spatial memory As shown in Fig. 1, no differences in body weight were found at the indicated time points between the control and stress groups of SMAP10 mice. The serum corticosterone, a wellknown critical stress hormone, levels progressively increased in the stress SAMP10 mice, but not in the control SAMP10 mice (Fig. 2). A one-trial passive avoidance test revealed that L–D latency in the retention trial was significantly reduced in the stress mice than in the control SAMP10 mice at the age of 12 weeks (Fig. 3). In addition, this behavioral assessment did not detect any significant difference in spontaneous activity between the stress and control groups of SAMP10 mice (the number of
Fig. 1 – Body weight kinetics after the beginning of stress. The stress group was exposed to social isolation stress from 5 weeks of age. All values of body weight in the survival mice were expressed as the mean ± SE (n = 14/group).
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Table 1 – Summary of the Y-maze test Behavioral index
Control (n = 14)
Stress (n = 14)
Number
5.5 ± 0.4 (43.9 ± 2.1)
6.3 ± 0.4 (39.2 ± 3.0)
4.0 ± 0.5 (28.8 ± 2.4) 3.7 ± 0.4 (27.3 ± 2.4) 60.5 ± 4.6 (37.6 ± 3.6)
3.8 ± 0.6 (31.6 ± 4.0) 3.6 ± 0.7 (29.2 ± 2.9) 68.1 ± 9.0 (41.5 ± 5.7)
70.5 ± 7.7 (42.9 ± 4.4) 32.0 ± 4.4 (19.5 ± 2.6)
57.5 ± 6.6 (34.7 ± 3.9) 39.4 ± 5.8 (23.7 ± 3.4)
Time (s)
Novel arm (%) Arm 1 (%) Arm 2 (%) Novel arm (%) Arm 1 (%) Arm 2 (%)
Data represent mean ± SE.
Fig. 2 – Serum corticosterone kinetics of mice under stress. The serum corticosterone levels were measured at the indicated time points. All values are expressed as the mean ± SE (n = 8/group). *P b 0.05 and ***P b 0.001 were considered to be significantly different between corresponding values in the control group. ##P b 0.01 and ###P b 0.001 were considered to be significantly different from the base values in each group.
times crossing the sliding door between the two compartments of the L–D box in 3 min during the initial habituation trial: Control, 11.1 ± 0.5; Stress, 12.8 ± 0.8). In the Y-maze test, neither the entry number nor the time in the novel arm was significantly different between the control and stress SAMP10 animals (Table 1). Moreover, these effects of stress on learning/memory behavior were reconfirmed at the age of 20 and 28 weeks (data not shown).
2.2. The effect of social isolation stress on anxiety and pain sensation Such a stress-induced reduction of the L–D latency in the retention trial might be accounted for by a decrease in anxiety
or pain sensation rather than an exacerbation of conditioning memory per se. Therefore, we next analyzed anxiety and pain sensation. As summarized in Table 2, an elevated plus maze test found no difference in either the entry number or the time spent in the open arm. In a pain-sensitivity test, the pain threshold levels in the stress group were significantly higher than those in the control group, thus indicating that stressinduced inhibition of pain sensation might have contributed to the decrease in latency seen in the retention trial. Actually, this latency was significantly, negatively correlated with the pain threshold levels (correlation coefficient, −0.34; P b 0.05). However, adjustment for the pain threshold levels in an ANCOVA did not diminish the significant difference in latency between the control and stress groups (L–D Latency in the retention trial: Before adjustment; Control, 67.1 ± 18.5 s; Stress, 16.7 ± 4.2 s, P = 0.031, After adjustment; Control, 61.4 ± 14.6 s; Stress, 17.2 ± 15.5 s, P = 0.048). These findings indicate that social isolation stress exacerbates conditioning memory, independently of pain sensation (Fig. 4).
2.3. Stress inhibited the increase in the number of c-Fos-positive nuclei in the CeA after the acquisition trial To further investigate possible neurobiological mechanisms behind the detrimental effect of stress on conditioning memory, we evaluated the blood and amygdala MDA levels and the number of CeA c-Fos-positive nuclei as markers of oxidative brain damage and neural activation, respectively. As shown in Table 3, the blood and amygdala MDA levels were not significantly different between the control and stress groups at the indicated time points. In contrast, the stress mice exhibited a significant decrease in the number of c-Fos-positive nuclei in the CeA 90 min following the
Table 2 – Summary of the elevated plus maze test
Fig. 3 – Isolation stress reduced light–dark latency in the retention trial. Light–dark latency in the acquisition and retention trials was evaluated in SAM mice at the age of 12 weeks, as described in Materials and methods. All values are expressed as the mean ± SE (n = 14/group). *P b 0.05 was considered to be significantly different from the control group.
Behavioral index
Control (n = 14)
Stress (n = 14)
Number
5.5 ± 0.4 (43.4 ± 2.8)
6.3 ± 0.4 (44.1 ± 3.0)
4.6 ± 0.3 (56.6 ± 2.8)
5.0 ± 0.4 (55.9 ± 2.8)
10.1 ± 0.4 67.2 ± 6.8 (22.4 ± 2.3)
11.3 ± 0.6 64.3 ± 6.4 (21.4 ± 2.1)
Time (s)
Open arms (%) Closed arms (%) Total Open arms (%)
Data represent mean ± SE.
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Fig. 4 – Effect of isolation stress on the pain threshold. The pain threshold (μA) of SAM mice was evaluated at the age of 12 weeks, as described in Materials and methods. All values are expressed as the mean ± SE (n = 14/group). *P b 0.05 was considered to be significantly different from the control group.
acquisition trial in the one-trial passive avoidance test as compared with the control mice (Fig. 5), whereas this difference was not seen in the cortex (data not shown). Given that some previous animal experiments have shown that c-Fos-expression in the CeA during conditioning is required for the process of conditioning memory (Lamprecht and Dudai, 1996; Yamamoto et al., 1997), social isolation stress might exacerbate this kind of memory due to inhibition of neural activity in the CeA.
3.
Discussion
In this study, long-term exposure to social isolation stress significantly exacerbated passive avoidance learning in SAMP10 mice, but not in SAMR1 mice, a control substrain of SAMP10 (SAMR1, L–D Latency in the retention trial: 12 W; Nonstress, 154.3 ± 15.8 s; Stress, 160.1 ± 17.2s, P = 0.344). Moreover, SAMP10 exhibited a stress-induced increase in blood corticosterone and a decrease in the c-fos expression of CeA after a retention trial, while SAMR1 mice did not (data not shown), which indicates that such stress-induced neuroendocrinal changes and memory impairment might be specific for SAMP10 mice. Passive avoidance learning represents conditioning memory (associative memory), in which certain environmental stimuli predict aversive events, and is the mechanism whereby we learn to fear people, places, objects, and animals. Evolution has crafted this form of learning to promote survival in the face of present and future threats, and it is an essential component of many defective mammalian behavior systems (Fanselow, 2004). Fear conditioning has attracted great interest in recent years because it is squarely seated at the interface of memory and emotion (LeDoux, 2000). Moreover, disturbances in fear conditioning may contribute to disorders of fear and anxiety in humans, such as panic disorder and specific phobias (Rosen and Schulkin, 1998; Wolpe, 1981). Therefore, the present finding based on a brain atrophy-prone mouse model (SAMP10)
indicates that chronic social isolation may accelerate the development of memory impairment in human neurodegenerative diseases and ultimately exacerbate emotional distress. Several possible factors may have affected L–D latency in the retention trial. First, the spontaneous activity level is critically involved in passive-avoidance performance. Hyperactivity may lead to or coincide with a learning deficit (McDonald et al., 1997). Therefore, we assessed the spontaneous activity levels by use of a one-trial passive avoidance test. However, no significant differences in the activity levels were found between the control and stress groups. Second, anxiety may have contributed to performance during the one-trial passive avoidance test. However, the elevated plus maze test did not detect any divergences of anxiety between the control and stress groups, confirming that the shortening of L–D latency was not caused by decreased anxiety in the stress mice. Third, pain sensation may affect the performance of passive avoidance learning. Many studies report that stress stimuli result in decreased pain sensation in animals via opioid-mediated and non-opioid-mediated mechanisms (stress-induced analgesia, SIA; for review, see Yamada and Nabeshima, 1995). In our experiment, isolation stress significantly reduced the pain sensation as assessed by the flinch-jump test. However, an ANCOVA excluded the possibility that SIA might have impaired the passive avoidance. Previous animal studies based on a variety of lesion techniques, including permanent (anatomical) and transient (metabolic) lesions inflicted at various time points before, during, and after aversive conditioning indicated that the amygdala was critically involved in conditioning memory (Roldan and Bures, 1994; Yamamoto et al., 1994). Recently, advanced functional neuroimaging techniques such as positron emission tomography and functional magnetic resonance imaging have shown that the amygdala is activated during aversive conditioning (Büchel and Dolan, 2000). At present, we have chosen to concentrate on the expression of cFos, a marker of neural activation, in the CeA after the acquisition trial because an increase in this expression has been reported to play a critical role in the process of conditioning memory (Lamprecht and Dudai, 1996; Yamamoto et al., 1997). In this study, social isolation stress significantly reduced the number of c-Fos-positive nuclei in
Table 3 – Serum and brain MDA levels of stress-exposed mice Sample
Group
Time course (weeks) 10
Serum (nmol/ml)
Amygdala (nmol/mg prot)
Control (n = 6) Stress (n = 6) Control (n = 4) Stress (n = 4)
13
15
12.2 ± 1.8
N.T.
14.4 ± 1.4
10.1 ± 0.6
N.T.
14.2 ± 2.5
N.T.
0.53 ± 0.02
N.T.
N.T.
0.60 ± 0.05
N.T.
N.T.; not tested, data represent mean ± SE.
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Fig. 5 – Isolation stress inhibited c-Fos expression of the central amygdaloid nucleus after the acquisition trial. Panels A and B show the c-Fos-positive nuclei (brown-stained; arrows) per high power field (×200) in the CeA (circle) 90 min after the acquisition trial. All values are expressed as the mean ± SE (n = 4/group). **P b 0.01 was considered to be significantly different from the control group.
the CeA following the acquisition trial, which suggests that stress impairs conditioning memory, probably by decreasing c-Fos-expression in the CeA. In addition, together with the well-established evidence that glucocorticoids (GC) inhibit cFos expression via cytoplasmic GC receptors (Tsigos and Chrousos, 2002) and the current findings that the serum corticosterone levels were significantly increased in the stress group compared with the control group, a putative mechanism by which a stress-induced elevation of GC suppresses cFos-expression in CeA may underlie the inhibitory effect of chronic social isolation on conditioning memory. The oxidative stress theory postulates that the oxidative effects of free radicals on biomolecules such as lipids, proteins, and DNA are responsible for the functional deterioration associated with aging, including learning/memory deficits (Goldstein et al., 1990). Consistent with this theory, oxidative stress has been reported to critically participate in aging in SAM mice (Hosokawa, 2002; Poon et al., 2004, 2005). In addition, psychological stress was shown to enhance brain lipid peroxidation, eventually causing central neural damage (Lui et al., 1996; Matsumoto et al., 1999). Therefore, we hypothesized that a stress-induced increase in oxidative damage to the brain, especially the amygdala, might result in an exacerbation of conditioning memory: however, this hypothesis seems unlikely since neither the blood nor the amygdala in the
stress-exposed mice exhibited any elevation of the MDA level, an end product of lipid peroxidation. In the present study, chronic social isolation stress did not affect spatial memory as assessed by a Y-maze test. Spatial memory has been shown to depend on the hippocampus, which has high concentrations of GC receptors (McEwen, 1998). Long-term stress causes the atrophy of dendrites of pyramidal neurons in the CA3 region of the hippocampus via a GC-involved mechanism, subsequently impairing the reliability and accuracy of spatial memory. Therefore, chronic social isolation stress, since it has the ability to elevate systemic GC levels, would be expected to impair spatial memory. In addition to GC, excitatory amino acid glutamate also exerts a key regulatory role in hippocampus-dependent memory under stress (Lowy et al., 1995), which may be related to the reason that no stress-induced impairment of spatial memory was detected in the present mouse model. In conclusion, chronic social isolation stress exacerbated the aversive conditioning memory in SAMP10 mice, probably through a GC-mediated decrease in neural activity of the CeA. Further investigation is needed to clarify the precise mechanism behind the detrimental effect of psychosocial stress on the learning/memory functions seen in dementia diseases.
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4.
Experimental procedures
4.1.
Mice
Male SAMR1 and SAMP10 mice were purchased at the age of 5 weeks from SLC Japan (Shizuoka, Japan). Immediately after arriving from the vendor and according to a method previously described (Chida et al., 2005), the mice were randomly assigned to be group-housed (control; n = 5 per cage, floor space; 18 cm × 30 cm = 540 cm2, height; 13 cm) or individually housed (stress; n = 1 per cage, floor space; 6 cm × 18 cm = 108 cm2, height; 12 cm) in such a way that the amount of floor space was the same for each mouse (108 cm2). A cardboard wall and a 4 cm distance were placed between any two cages housing individual mice, which resulted in a social isolation condition that allowed animals to have relatively normal auditory and olfactory experiences, but not to at any time see, touch, or be touched by other animals in the colony. The animal laboratory was maintained at a constant temperature (23–25 °C) with a 12 h light/dark cycle, and food and water were available ad libitum. Experiments were always done in the morning (between 09:00 and 12:00 h) to minimize variation due to circadian rhythmicity. The behavioral and pain sensory tests were performed at the age of 12 weeks in the following order: Days 1–3, a single trial passive avoidance test (conditioning memory); Day 4, a Y-maze test (spatial memory); Day 5, an elevated plus maze test (anxiety); Day 6, a flinch-jump test (pain sensation). This experiment was reviewed by the Ethics committee on Animal Experiments of the Graduate School of Medical Sciences, Kyushu University and was carried out under the control of the Guidelines for Animal Experiments of the Graduate School of Medical Sciences, Kyushu University and the Law (No. 105) and Notification (No. 6) of the Japanese Government. 4.2.
Measurement of serum corticosterone levels
Blood samples were collected from the retroorbital plexus at the indicated time points, and plasma was frozen at −80 °C until analysis. The serum level of corticosterone was measured using a commercially available RIA kit (ICN Biomedicals, Costa Mesa, CA). The serum corticosterone concentration was calculated from a standard curve and expressed in nanograms per milliliter. The detection limit of the assay was about 1 ng/ml. In a preliminary experiment, our blood-drawing procedure without an anesthetic was confirmed not to induce an elevation of the serum corticosterone level, probably because it was very rapidly performed (at most 1 min per mouse). 4.3.
One-trial passive avoidance test
One-trial passive avoidance tests were conducted as previously described (Ader et al., 1972) with some modifications. In brief, a Plexiglas apparatus was used that consisted of a two-compartment “light–dark” box (L–D box: both 14 × 14 × 25 cm). The light compartment was illuminated by a bare 40 W bulb. A 5 × 5 cm sliding door connected the two compartments. The floor of the two compartments was constructed of stainless steel rods (1 mm diameter, spaced 5 mm apart). A foot shock (0.2 mA at 1 Hz) could be delivered through the rods of the dark compartment using a shock generator-scrambler (PST-001, Star medical, Tokyo, Japan). During the initial habituation trial, mice were placed individually into the light compartment with the bare bulb turned off and the sliding door open. The mice were allowed to explore the L–D box for 3 min, during which time the number of times crossing the sliding door between the two compartments was counted to measure the spontaneous activity of the mice. On the day
following the habituation trial, an acquisition trial was conducted. The acquisition trial was performed for 2 min in a manner similar to that of the habituation trial, but the mice were placed into the light compartment with the bare bulb turned on. They received a foot shock immediately upon entrance to the dark compartment. The light–dark (step-through) latency (L–D latency) to enter the dark compartment was measured. A retention trial was conducted 24 h later, and the L–D latency was measured once again. Each trial was terminated at 300 s, and mice that did not enter the dark compartment by this time were assigned a ceiling score of 300 s. After each trial, the apparatus was cleaned using 70% alcohol. 4.4.
Y-maze test
The Y-maze apparatus (arms: 34 cm long, 8 cm wide, and 14.5 cm deep) was constructed of black Plexiglas and equipped with guillotine doors isolating each arm. The experiment consisted of two trials separated by a 1-h inter-trial time interval, as previously described (Ferguson et al., 2000) with some modifications. During the familiarization phase (trial 1), one arm (novel arm) of the Ymaze was closed with a guillotine door. We placed mice in one (arm 1) of the two remaining arms (arms 1 and 2) and allowed them to explore the maze for 3 min. During the retrieval phase (trial 2), we removed the door, and the animals had free access to all three arms. The number and duration of the explorations of each arm were recorded for each trial. The percentage of visits and time spent in the novel arm were compared to random exploration (33%) of the three arms of the maze. 4.5.
Elevated plus maze test
The elevated plus maze test was conducted as described in detail elsewhere (Hirayama et al., 2003). The maze was made of wood and consisted of a central platform 5 × 5 cm with four arms radiating from that platform in the shape of a plus. Two opposing arms (30 × 5 cm; length × width) were open, and the other two opposing arms (30 × 5 cm) were enclosed with transparent Plexiglas walls (15 cm; height) with the top and the end of the arm open. A raised edge (5 mm) was placed around the perimeter of the open arms to provide additional grip for the animals. The entire apparatus was situated at a height of 38.5 cm above the floor. To initiate the elevated plus maze test, an animal was placed on the central platform of the maze facing a closed arm. A 5-min test duration was employed, and the maze was thoroughly cleaned between tests with damp towels and 70% alcohol to remove residual odors from urine, feces, and sebum. The behavioral indices used in this study consisted of the frequency and ratio (%) of open and closed arm entries (arm entry defined as all four paws in an arm) and the duration of the stay in open and closed arms. 4.6.
Measurement of pain threshold
The pain-sensitivity test was performed as previously described (Cabib et al., 1996). Pain reactivity was measured using a modification of the flinch-jump test. The floor (30 × 30 cm) consisted of parallel stainless steel rods (1 mm diameter with gaps of 5 mm). Individual animals were placed on the floor, and six series of six shocks (1-s duration, 20, 40, 60, 80, 100, and 130 μA) were delivered at a 15-s interval through the grid floor. The shock series were administered in double alternating ascending and descending order, the first series being ascending. Shock threshold was defined as the lowest shock intensity (μA) at which an animal's hind foot left the floor. For each mouse, the mean shock threshold was calculated as the average of the six thresholds recorded in the series.
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4.7.
c-Fos immunohistochemistry
According to a previous method (Lamprecht and Dudai, 1996) with some modifications, immunohistochemistry was performed on samples collected 90 min following the acquisition trial of the one-trial passive avoidance test. Briefly, mice were killed by cervical dislocation and immediately perfused intracardially with 25 ml of 0.1 M phosphate-buffered saline (PBS) followed by 100 ml 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brain was removed and post-fixed for at least 5 h in the same fixative and cryoprotected overnight in 30% sucrose in PB. The samples were sectioned at 30 μm and then rinsed with 0.1 M PBS overnight at 4 °C. The sections were immediately processed for immunohistochemistry by a peroxidase–antiperoxidase technique using rabbit anti-Fos polyclonal antibody (1:200, sc-52, Santa Cruz Biotechnology, Santa Cruz, CA) for the detection of Fos protein. The central amygdaloid nucleus (CeA; coordinates in reference to Bregma AP −1.3 mm, L +2.4 mm, DV −4.6 mm; Paxinos and Franklin, 2001) from four animals/group was examined, and Fospositive brown nuclei were counted at a magnification of ×200. 4.8.
Measurement of malondialdehyde in the brain and serum
Malondialdehyde (MDA), a measure of lipid peroxidation, was measured by spectrophotometry (Okhawa et al., 1979) at the indicated time points. Briefly, brain tissue was homogenized with 10 times (w/v) PBS. The reagents acetic acid, 1.5 ml (20%) pH 3.5, 1.5 ml thiobarbituric acid (0.8%), and 0.2 ml sodium dodecyl sulfate (8.1%) were added to 0.1 ml of processed tissue and the serum samples. The mixture was cooled with tap water and 5 ml of nbutanol:pyridine (15:1% v/v), and 1 ml of distilled water was added. The mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was withdrawn, and absorbance was measured at 532 nm using a spectrophotometer. The brain MDA levels were normalized to the amount of protein in the sample measured by the Bio-Rad protein assay (Bio-Rad, Hercules, CA). 4.9.
Statistical analysis
All data are expressed as the means ± SE. The data were analyzed by one factor analysis of variance (ANOVA) followed by the Scheffé test. For data on the time course of the body weight, the serum corticosterone levels, and the serum MDA levels, repeated measure ANOVA followed by the Scheffé test was done. Correlation between the light–dark latency and pain threshold levels was analyzed by Pearson's correlation test. Analysis of covariance (ANCOVA) was used to examine the pain threshold levels as predictive variables for light–dark latency. All analyses were performed on a Macintosh G4 using the SAS/STAT version 8.02 (SAS Institute, Inc) statistical program. A P value b0.05 was considered to be significantly different from the corresponding value.
Acknowledgments This study was supported by grants in aid for General Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 16390200, No. 17390210, and No. 17659205).
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Crawley, J.N., 1998. Hyperactivity and learning deficits in transgenic mice bearing a human mutant thyroid hormone β1 receptor gene. Learn. Mem. 5, 289–301. McEwen, B.S., 1998. Protective and damaging effects of stress mediators. N. Engl. J. Med. 338, 171–179. McGaugh, J.L., Rooxendaal, B., 2002. Role of adrenal stress hormones in forming of lasting memories in the brain. Curr. Opin. Neurobiol. 12, 205–210. Morley, J.E., Kumar, V.B., Bernardo, A.E., Farr, S.A., Uezu, K., Tumosa, N., Flood, J.F., 2000. β-amyloid precursor polypeptide in SAMP8 mice affects learning and memory. Peptides 21, 1761–1767. Numata, T., Saito, T., Maekawa, K., Takahashi, Y., Saitoh, H., Hosokawa, T., Fujita, H., Kurasaki, M., 2002. Bcl-2-linked apoptosis due to increase in NO synthase in brain of SAMP10. Biochem. Biophys. Res. Commun. 297, 517–522. Okhawa, H., Ohishi, N., Yagi, K., 1979. Assay of lipid peroxides in animals tissue by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Okuma, Y., Nomura, Y., 1998. Senescence-accelerated mouse (SAM) as an animal model of senile dementia: pharmacological, neurochemical and molecular biological approach. Jpn. J. Pharmocol. 78, 399–404. Pashko, S., Deturck, K.H., Vogel, W.H., 1980. Use of catheterized rat in studies on social interaction and plasma-catecholamines. Pharmacol. Biochem. Bhehav. 13, 471–473. Paxinos, G., Franklin, K.J. (Eds.), 2001. The Mouse Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, San Diego. Poon, H.F., Joshi, G., Sultana, R., Farr, S.A., Banks, W.A., Morley, J.E., Calabrese, V., Butterfield, D.A., 2004. Antisense directed at the Aβ region of APP decreases brain oxidative markers in aged senescence accelerated mice. Brain Res. 1018, 86–96. Poon, H.F., Farr, S.A., Thongboonkerd, V., Lynn, B.C., Banks, W.A., Morley, J.E., Klein, J.B., Butterfield, D.A., 2005. Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders. Neurochem. Int. 46, 159–168. Roldan, G., Bures, J., 1994. Tetrodotoxin blockade of amygdala overlapping with poisoning impairs acquisition of conditioned taste aversion in rats. Behav. Brain Res. 65, 213–219. Rosen, J.B., Schulkin, J., 1998. From normal fear to pathological anxiety. Psychol. Rev. 105, 325–350. Shimada, A., 1999. Age-dependent cerebral atrophy and cognitive dysfunction in SAMP10 mice. Neruobiol. Aging 20, 125–136. Shimada, A., Keino, H., Satoh, M., Kishikawa, M., Hosokawa, M., 2003. Age-related loss of synapses in the frontal cortex of SAMP10 mouse: a model of cerebral degeneration. Synapse 48, 198–204.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
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Research Report
Social isolation stress impairs passive avoidance learning in senescence-accelerated mouse (SAM) Yoichi Chida, Nobuyuki Sudo, Junko Mori, Chiharu Kubo ⁎ Department of Psychosomatic Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
Despite cumulative evidence showing the detrimental effect of psychosocial stress on the
Accepted 11 October 2005
learning/memory functions in dementia diseases, the precise neurobiological mechanisms
Available online 5 December 2005
behind such an effect remain unclear. Mice of the senescence-accelerated mice prone 10 (SAMP10) strain, a neurodegenerative dementia model, were chronically exposed to social
Theme:
isolation stress from the age of 5 weeks. At the age of 12 weeks, conditioning memory and
Neural basis of behavior
spatial memory were evaluated by one-trial passive avoidance and Y-maze tests,
Topic:
respectively. Chronic social isolation stress significantly reduced conditioning memory
Stress
but did not affect spatial memory. Although further behavioral tasks using an elevated plus maze and a pain threshold test exhibited stress-induced analgesia, an analysis of
Keywords:
covariance excluded the possibility that such analgesia might contribute to the stress-
Aging
induced impairment of conditioning memory. In addition, endocrinological and
Associative memory
immunohistochemical analysis revealed that isolation stress elevated the serum
Hypothalamus–pituitary–adrenal
corticosterone levels and inhibited the increase in c-Fos expression in the central
axis
amygdaloidal nucleus (CeA) that is required for conditioning memory during passive
Oxidative stress
avoidance learning. In conclusion, chronic social isolation stress exacerbated conditioning
Psychosocial stress
memory in SAM mice, probably through a glucocorticoid-mediated decrease in neural
Stress-induced analgesia
activation in the CeA. © 2005 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: +81 92 642 5336. E-mail address: [email protected] (C. Kubo). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.10.042
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Abbreviations: ANCOVA, analysis of covariance CeA, amygdaloidal nucleus GC, glucocorticoids HPA, hypothalamus–pituitary– adrenal L–D, light–dark MDA, malondialdehyde PBS, phosphate-buffered saline SAM, senescence-accelerated mice SIA, stress-induced analgesia
1.
Introduction
In encounters with environmental challenges, learning and memory are essential to the success of every living organism as they are necessary for prolonging survival and maintaining the species. Considerable evidence gained by extensive investigations of the influence of psychosocial factors on learning and memory has been reported (for review, see (Lupien and McEwen, 1997; McGaugh and Rooxendaal, 2002). Because the number of patients suffering from dementia diseases has been increasing worldwide, much attention has been focused on possible causal links between various psychosocial stresses and the development of dementia diseases such as Alzheimer's disease (Fratiglioni et al., 2004). For example, a higher susceptibility to psychological distress was reported to lead to double the risk of Alzheimer's disease (Wilson et al., 2003). However, the precise neurobiological mechanisms underlying such an injurious effect of psychosocial stress on dementia diseases remain to be elucidated. Senescence-accelerated mouse (SAM) includes senescenceacceleration-prone and -resistant substrains (SAMP and SAMR, respectively) (Takeda et al., 1991; Okuma and Nomura, 1998). Of the SAMP substrains, SAMP8 and SAMP10 mice exhibit deficits in learning and memory at a relatively early stage in their life span (Lowy et al., 1995; Numata et al., 2002; Flood and Morley, 1998). Therefore, these substrains have increasingly been used as a murine model for investigating the mechanisms underlying agerelated cognitive deficits (Maurice et al., 1996; Numata et al., 2002; Shimada et al., 2003; Strong et al., 2003; Tha et al., 2000; Ye et al., 2004). SAMP8 mice have been shown to exhibit traits similar to Alzheimer's disease and overproduce amyloid precursor protein naturally (Morley et al., 2000). More notably, SAMP10 mice have been reported to show age-dependent brain atrophy (Shimada, 1999) similar to that seen in patients suffering from neurodegenerative dementia diseases, such as Alzheimer's disease, Pick's disease, and other frontotemporal dementias. Social isolation, such as by individual housing, eliminates social interaction among animals and has been reported to induce pathophysiological changes in rodents: for example, an increased or decreased response of the hypothalamus–pituitary–adrenal (HPA) axis and sympathetic nervous system (Huong et al., 1997; Kim and Kirkpatrick, 1996; Mar Sánchez et al., 1998; Pashko et al., 1980), acceleration of the development and growth of either transplanted or chemically induced tumors (Kerr et al., 1997; Weinberg and Emerman, 1989; Wu et al., 1999), enhancement of small intestinal sensitivity to chemotherapy (Verburg et al., 2003), and exacerbation of the
autoimmune diseases of MRL/lpr mice (Chida et al., 2005). Thus, social isolation stress is considered a valuable stress paradigm for investigating the effect of chronic psychosocial stress on various pathophysiological alterations in animals. The present study, based on the social isolation stress paradigm, was done to examine the detrimental effect of psychosocial stress on conditioning memory and spatial memory in a neurodegenerative disease model using SAMP10 mice and to attempt to clarify the mechanism(s) involved.
2.
Results
2.1. The effect of social isolation stress on conditioning and spatial memory As shown in Fig. 1, no differences in body weight were found at the indicated time points between the control and stress groups of SMAP10 mice. The serum corticosterone, a wellknown critical stress hormone, levels progressively increased in the stress SAMP10 mice, but not in the control SAMP10 mice (Fig. 2). A one-trial passive avoidance test revealed that L–D latency in the retention trial was significantly reduced in the stress mice than in the control SAMP10 mice at the age of 12 weeks (Fig. 3). In addition, this behavioral assessment did not detect any significant difference in spontaneous activity between the stress and control groups of SAMP10 mice (the number of
Fig. 1 – Body weight kinetics after the beginning of stress. The stress group was exposed to social isolation stress from 5 weeks of age. All values of body weight in the survival mice were expressed as the mean ± SE (n = 14/group).
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Table 1 – Summary of the Y-maze test Behavioral index
Control (n = 14)
Stress (n = 14)
Number
5.5 ± 0.4 (43.9 ± 2.1)
6.3 ± 0.4 (39.2 ± 3.0)
4.0 ± 0.5 (28.8 ± 2.4) 3.7 ± 0.4 (27.3 ± 2.4) 60.5 ± 4.6 (37.6 ± 3.6)
3.8 ± 0.6 (31.6 ± 4.0) 3.6 ± 0.7 (29.2 ± 2.9) 68.1 ± 9.0 (41.5 ± 5.7)
70.5 ± 7.7 (42.9 ± 4.4) 32.0 ± 4.4 (19.5 ± 2.6)
57.5 ± 6.6 (34.7 ± 3.9) 39.4 ± 5.8 (23.7 ± 3.4)
Time (s)
Novel arm (%) Arm 1 (%) Arm 2 (%) Novel arm (%) Arm 1 (%) Arm 2 (%)
Data represent mean ± SE.
Fig. 2 – Serum corticosterone kinetics of mice under stress. The serum corticosterone levels were measured at the indicated time points. All values are expressed as the mean ± SE (n = 8/group). *P b 0.05 and ***P b 0.001 were considered to be significantly different between corresponding values in the control group. ##P b 0.01 and ###P b 0.001 were considered to be significantly different from the base values in each group.
times crossing the sliding door between the two compartments of the L–D box in 3 min during the initial habituation trial: Control, 11.1 ± 0.5; Stress, 12.8 ± 0.8). In the Y-maze test, neither the entry number nor the time in the novel arm was significantly different between the control and stress SAMP10 animals (Table 1). Moreover, these effects of stress on learning/memory behavior were reconfirmed at the age of 20 and 28 weeks (data not shown).
2.2. The effect of social isolation stress on anxiety and pain sensation Such a stress-induced reduction of the L–D latency in the retention trial might be accounted for by a decrease in anxiety
or pain sensation rather than an exacerbation of conditioning memory per se. Therefore, we next analyzed anxiety and pain sensation. As summarized in Table 2, an elevated plus maze test found no difference in either the entry number or the time spent in the open arm. In a pain-sensitivity test, the pain threshold levels in the stress group were significantly higher than those in the control group, thus indicating that stressinduced inhibition of pain sensation might have contributed to the decrease in latency seen in the retention trial. Actually, this latency was significantly, negatively correlated with the pain threshold levels (correlation coefficient, −0.34; P b 0.05). However, adjustment for the pain threshold levels in an ANCOVA did not diminish the significant difference in latency between the control and stress groups (L–D Latency in the retention trial: Before adjustment; Control, 67.1 ± 18.5 s; Stress, 16.7 ± 4.2 s, P = 0.031, After adjustment; Control, 61.4 ± 14.6 s; Stress, 17.2 ± 15.5 s, P = 0.048). These findings indicate that social isolation stress exacerbates conditioning memory, independently of pain sensation (Fig. 4).
2.3. Stress inhibited the increase in the number of c-Fos-positive nuclei in the CeA after the acquisition trial To further investigate possible neurobiological mechanisms behind the detrimental effect of stress on conditioning memory, we evaluated the blood and amygdala MDA levels and the number of CeA c-Fos-positive nuclei as markers of oxidative brain damage and neural activation, respectively. As shown in Table 3, the blood and amygdala MDA levels were not significantly different between the control and stress groups at the indicated time points. In contrast, the stress mice exhibited a significant decrease in the number of c-Fos-positive nuclei in the CeA 90 min following the
Table 2 – Summary of the elevated plus maze test
Fig. 3 – Isolation stress reduced light–dark latency in the retention trial. Light–dark latency in the acquisition and retention trials was evaluated in SAM mice at the age of 12 weeks, as described in Materials and methods. All values are expressed as the mean ± SE (n = 14/group). *P b 0.05 was considered to be significantly different from the control group.
Behavioral index
Control (n = 14)
Stress (n = 14)
Number
5.5 ± 0.4 (43.4 ± 2.8)
6.3 ± 0.4 (44.1 ± 3.0)
4.6 ± 0.3 (56.6 ± 2.8)
5.0 ± 0.4 (55.9 ± 2.8)
10.1 ± 0.4 67.2 ± 6.8 (22.4 ± 2.3)
11.3 ± 0.6 64.3 ± 6.4 (21.4 ± 2.1)
Time (s)
Open arms (%) Closed arms (%) Total Open arms (%)
Data represent mean ± SE.
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Fig. 4 – Effect of isolation stress on the pain threshold. The pain threshold (μA) of SAM mice was evaluated at the age of 12 weeks, as described in Materials and methods. All values are expressed as the mean ± SE (n = 14/group). *P b 0.05 was considered to be significantly different from the control group.
acquisition trial in the one-trial passive avoidance test as compared with the control mice (Fig. 5), whereas this difference was not seen in the cortex (data not shown). Given that some previous animal experiments have shown that c-Fos-expression in the CeA during conditioning is required for the process of conditioning memory (Lamprecht and Dudai, 1996; Yamamoto et al., 1997), social isolation stress might exacerbate this kind of memory due to inhibition of neural activity in the CeA.
3.
Discussion
In this study, long-term exposure to social isolation stress significantly exacerbated passive avoidance learning in SAMP10 mice, but not in SAMR1 mice, a control substrain of SAMP10 (SAMR1, L–D Latency in the retention trial: 12 W; Nonstress, 154.3 ± 15.8 s; Stress, 160.1 ± 17.2s, P = 0.344). Moreover, SAMP10 exhibited a stress-induced increase in blood corticosterone and a decrease in the c-fos expression of CeA after a retention trial, while SAMR1 mice did not (data not shown), which indicates that such stress-induced neuroendocrinal changes and memory impairment might be specific for SAMP10 mice. Passive avoidance learning represents conditioning memory (associative memory), in which certain environmental stimuli predict aversive events, and is the mechanism whereby we learn to fear people, places, objects, and animals. Evolution has crafted this form of learning to promote survival in the face of present and future threats, and it is an essential component of many defective mammalian behavior systems (Fanselow, 2004). Fear conditioning has attracted great interest in recent years because it is squarely seated at the interface of memory and emotion (LeDoux, 2000). Moreover, disturbances in fear conditioning may contribute to disorders of fear and anxiety in humans, such as panic disorder and specific phobias (Rosen and Schulkin, 1998; Wolpe, 1981). Therefore, the present finding based on a brain atrophy-prone mouse model (SAMP10)
indicates that chronic social isolation may accelerate the development of memory impairment in human neurodegenerative diseases and ultimately exacerbate emotional distress. Several possible factors may have affected L–D latency in the retention trial. First, the spontaneous activity level is critically involved in passive-avoidance performance. Hyperactivity may lead to or coincide with a learning deficit (McDonald et al., 1997). Therefore, we assessed the spontaneous activity levels by use of a one-trial passive avoidance test. However, no significant differences in the activity levels were found between the control and stress groups. Second, anxiety may have contributed to performance during the one-trial passive avoidance test. However, the elevated plus maze test did not detect any divergences of anxiety between the control and stress groups, confirming that the shortening of L–D latency was not caused by decreased anxiety in the stress mice. Third, pain sensation may affect the performance of passive avoidance learning. Many studies report that stress stimuli result in decreased pain sensation in animals via opioid-mediated and non-opioid-mediated mechanisms (stress-induced analgesia, SIA; for review, see Yamada and Nabeshima, 1995). In our experiment, isolation stress significantly reduced the pain sensation as assessed by the flinch-jump test. However, an ANCOVA excluded the possibility that SIA might have impaired the passive avoidance. Previous animal studies based on a variety of lesion techniques, including permanent (anatomical) and transient (metabolic) lesions inflicted at various time points before, during, and after aversive conditioning indicated that the amygdala was critically involved in conditioning memory (Roldan and Bures, 1994; Yamamoto et al., 1994). Recently, advanced functional neuroimaging techniques such as positron emission tomography and functional magnetic resonance imaging have shown that the amygdala is activated during aversive conditioning (Büchel and Dolan, 2000). At present, we have chosen to concentrate on the expression of cFos, a marker of neural activation, in the CeA after the acquisition trial because an increase in this expression has been reported to play a critical role in the process of conditioning memory (Lamprecht and Dudai, 1996; Yamamoto et al., 1997). In this study, social isolation stress significantly reduced the number of c-Fos-positive nuclei in
Table 3 – Serum and brain MDA levels of stress-exposed mice Sample
Group
Time course (weeks) 10
Serum (nmol/ml)
Amygdala (nmol/mg prot)
Control (n = 6) Stress (n = 6) Control (n = 4) Stress (n = 4)
13
15
12.2 ± 1.8
N.T.
14.4 ± 1.4
10.1 ± 0.6
N.T.
14.2 ± 2.5
N.T.
0.53 ± 0.02
N.T.
N.T.
0.60 ± 0.05
N.T.
N.T.; not tested, data represent mean ± SE.
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Fig. 5 – Isolation stress inhibited c-Fos expression of the central amygdaloid nucleus after the acquisition trial. Panels A and B show the c-Fos-positive nuclei (brown-stained; arrows) per high power field (×200) in the CeA (circle) 90 min after the acquisition trial. All values are expressed as the mean ± SE (n = 4/group). **P b 0.01 was considered to be significantly different from the control group.
the CeA following the acquisition trial, which suggests that stress impairs conditioning memory, probably by decreasing c-Fos-expression in the CeA. In addition, together with the well-established evidence that glucocorticoids (GC) inhibit cFos expression via cytoplasmic GC receptors (Tsigos and Chrousos, 2002) and the current findings that the serum corticosterone levels were significantly increased in the stress group compared with the control group, a putative mechanism by which a stress-induced elevation of GC suppresses cFos-expression in CeA may underlie the inhibitory effect of chronic social isolation on conditioning memory. The oxidative stress theory postulates that the oxidative effects of free radicals on biomolecules such as lipids, proteins, and DNA are responsible for the functional deterioration associated with aging, including learning/memory deficits (Goldstein et al., 1990). Consistent with this theory, oxidative stress has been reported to critically participate in aging in SAM mice (Hosokawa, 2002; Poon et al., 2004, 2005). In addition, psychological stress was shown to enhance brain lipid peroxidation, eventually causing central neural damage (Lui et al., 1996; Matsumoto et al., 1999). Therefore, we hypothesized that a stress-induced increase in oxidative damage to the brain, especially the amygdala, might result in an exacerbation of conditioning memory: however, this hypothesis seems unlikely since neither the blood nor the amygdala in the
stress-exposed mice exhibited any elevation of the MDA level, an end product of lipid peroxidation. In the present study, chronic social isolation stress did not affect spatial memory as assessed by a Y-maze test. Spatial memory has been shown to depend on the hippocampus, which has high concentrations of GC receptors (McEwen, 1998). Long-term stress causes the atrophy of dendrites of pyramidal neurons in the CA3 region of the hippocampus via a GC-involved mechanism, subsequently impairing the reliability and accuracy of spatial memory. Therefore, chronic social isolation stress, since it has the ability to elevate systemic GC levels, would be expected to impair spatial memory. In addition to GC, excitatory amino acid glutamate also exerts a key regulatory role in hippocampus-dependent memory under stress (Lowy et al., 1995), which may be related to the reason that no stress-induced impairment of spatial memory was detected in the present mouse model. In conclusion, chronic social isolation stress exacerbated the aversive conditioning memory in SAMP10 mice, probably through a GC-mediated decrease in neural activity of the CeA. Further investigation is needed to clarify the precise mechanism behind the detrimental effect of psychosocial stress on the learning/memory functions seen in dementia diseases.
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4.
Experimental procedures
4.1.
Mice
Male SAMR1 and SAMP10 mice were purchased at the age of 5 weeks from SLC Japan (Shizuoka, Japan). Immediately after arriving from the vendor and according to a method previously described (Chida et al., 2005), the mice were randomly assigned to be group-housed (control; n = 5 per cage, floor space; 18 cm × 30 cm = 540 cm2, height; 13 cm) or individually housed (stress; n = 1 per cage, floor space; 6 cm × 18 cm = 108 cm2, height; 12 cm) in such a way that the amount of floor space was the same for each mouse (108 cm2). A cardboard wall and a 4 cm distance were placed between any two cages housing individual mice, which resulted in a social isolation condition that allowed animals to have relatively normal auditory and olfactory experiences, but not to at any time see, touch, or be touched by other animals in the colony. The animal laboratory was maintained at a constant temperature (23–25 °C) with a 12 h light/dark cycle, and food and water were available ad libitum. Experiments were always done in the morning (between 09:00 and 12:00 h) to minimize variation due to circadian rhythmicity. The behavioral and pain sensory tests were performed at the age of 12 weeks in the following order: Days 1–3, a single trial passive avoidance test (conditioning memory); Day 4, a Y-maze test (spatial memory); Day 5, an elevated plus maze test (anxiety); Day 6, a flinch-jump test (pain sensation). This experiment was reviewed by the Ethics committee on Animal Experiments of the Graduate School of Medical Sciences, Kyushu University and was carried out under the control of the Guidelines for Animal Experiments of the Graduate School of Medical Sciences, Kyushu University and the Law (No. 105) and Notification (No. 6) of the Japanese Government. 4.2.
Measurement of serum corticosterone levels
Blood samples were collected from the retroorbital plexus at the indicated time points, and plasma was frozen at −80 °C until analysis. The serum level of corticosterone was measured using a commercially available RIA kit (ICN Biomedicals, Costa Mesa, CA). The serum corticosterone concentration was calculated from a standard curve and expressed in nanograms per milliliter. The detection limit of the assay was about 1 ng/ml. In a preliminary experiment, our blood-drawing procedure without an anesthetic was confirmed not to induce an elevation of the serum corticosterone level, probably because it was very rapidly performed (at most 1 min per mouse). 4.3.
One-trial passive avoidance test
One-trial passive avoidance tests were conducted as previously described (Ader et al., 1972) with some modifications. In brief, a Plexiglas apparatus was used that consisted of a two-compartment “light–dark” box (L–D box: both 14 × 14 × 25 cm). The light compartment was illuminated by a bare 40 W bulb. A 5 × 5 cm sliding door connected the two compartments. The floor of the two compartments was constructed of stainless steel rods (1 mm diameter, spaced 5 mm apart). A foot shock (0.2 mA at 1 Hz) could be delivered through the rods of the dark compartment using a shock generator-scrambler (PST-001, Star medical, Tokyo, Japan). During the initial habituation trial, mice were placed individually into the light compartment with the bare bulb turned off and the sliding door open. The mice were allowed to explore the L–D box for 3 min, during which time the number of times crossing the sliding door between the two compartments was counted to measure the spontaneous activity of the mice. On the day
following the habituation trial, an acquisition trial was conducted. The acquisition trial was performed for 2 min in a manner similar to that of the habituation trial, but the mice were placed into the light compartment with the bare bulb turned on. They received a foot shock immediately upon entrance to the dark compartment. The light–dark (step-through) latency (L–D latency) to enter the dark compartment was measured. A retention trial was conducted 24 h later, and the L–D latency was measured once again. Each trial was terminated at 300 s, and mice that did not enter the dark compartment by this time were assigned a ceiling score of 300 s. After each trial, the apparatus was cleaned using 70% alcohol. 4.4.
Y-maze test
The Y-maze apparatus (arms: 34 cm long, 8 cm wide, and 14.5 cm deep) was constructed of black Plexiglas and equipped with guillotine doors isolating each arm. The experiment consisted of two trials separated by a 1-h inter-trial time interval, as previously described (Ferguson et al., 2000) with some modifications. During the familiarization phase (trial 1), one arm (novel arm) of the Ymaze was closed with a guillotine door. We placed mice in one (arm 1) of the two remaining arms (arms 1 and 2) and allowed them to explore the maze for 3 min. During the retrieval phase (trial 2), we removed the door, and the animals had free access to all three arms. The number and duration of the explorations of each arm were recorded for each trial. The percentage of visits and time spent in the novel arm were compared to random exploration (33%) of the three arms of the maze. 4.5.
Elevated plus maze test
The elevated plus maze test was conducted as described in detail elsewhere (Hirayama et al., 2003). The maze was made of wood and consisted of a central platform 5 × 5 cm with four arms radiating from that platform in the shape of a plus. Two opposing arms (30 × 5 cm; length × width) were open, and the other two opposing arms (30 × 5 cm) were enclosed with transparent Plexiglas walls (15 cm; height) with the top and the end of the arm open. A raised edge (5 mm) was placed around the perimeter of the open arms to provide additional grip for the animals. The entire apparatus was situated at a height of 38.5 cm above the floor. To initiate the elevated plus maze test, an animal was placed on the central platform of the maze facing a closed arm. A 5-min test duration was employed, and the maze was thoroughly cleaned between tests with damp towels and 70% alcohol to remove residual odors from urine, feces, and sebum. The behavioral indices used in this study consisted of the frequency and ratio (%) of open and closed arm entries (arm entry defined as all four paws in an arm) and the duration of the stay in open and closed arms. 4.6.
Measurement of pain threshold
The pain-sensitivity test was performed as previously described (Cabib et al., 1996). Pain reactivity was measured using a modification of the flinch-jump test. The floor (30 × 30 cm) consisted of parallel stainless steel rods (1 mm diameter with gaps of 5 mm). Individual animals were placed on the floor, and six series of six shocks (1-s duration, 20, 40, 60, 80, 100, and 130 μA) were delivered at a 15-s interval through the grid floor. The shock series were administered in double alternating ascending and descending order, the first series being ascending. Shock threshold was defined as the lowest shock intensity (μA) at which an animal's hind foot left the floor. For each mouse, the mean shock threshold was calculated as the average of the six thresholds recorded in the series.
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4.7.
c-Fos immunohistochemistry
According to a previous method (Lamprecht and Dudai, 1996) with some modifications, immunohistochemistry was performed on samples collected 90 min following the acquisition trial of the one-trial passive avoidance test. Briefly, mice were killed by cervical dislocation and immediately perfused intracardially with 25 ml of 0.1 M phosphate-buffered saline (PBS) followed by 100 ml 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brain was removed and post-fixed for at least 5 h in the same fixative and cryoprotected overnight in 30% sucrose in PB. The samples were sectioned at 30 μm and then rinsed with 0.1 M PBS overnight at 4 °C. The sections were immediately processed for immunohistochemistry by a peroxidase–antiperoxidase technique using rabbit anti-Fos polyclonal antibody (1:200, sc-52, Santa Cruz Biotechnology, Santa Cruz, CA) for the detection of Fos protein. The central amygdaloid nucleus (CeA; coordinates in reference to Bregma AP −1.3 mm, L +2.4 mm, DV −4.6 mm; Paxinos and Franklin, 2001) from four animals/group was examined, and Fospositive brown nuclei were counted at a magnification of ×200. 4.8.
Measurement of malondialdehyde in the brain and serum
Malondialdehyde (MDA), a measure of lipid peroxidation, was measured by spectrophotometry (Okhawa et al., 1979) at the indicated time points. Briefly, brain tissue was homogenized with 10 times (w/v) PBS. The reagents acetic acid, 1.5 ml (20%) pH 3.5, 1.5 ml thiobarbituric acid (0.8%), and 0.2 ml sodium dodecyl sulfate (8.1%) were added to 0.1 ml of processed tissue and the serum samples. The mixture was cooled with tap water and 5 ml of nbutanol:pyridine (15:1% v/v), and 1 ml of distilled water was added. The mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was withdrawn, and absorbance was measured at 532 nm using a spectrophotometer. The brain MDA levels were normalized to the amount of protein in the sample measured by the Bio-Rad protein assay (Bio-Rad, Hercules, CA). 4.9.
Statistical analysis
All data are expressed as the means ± SE. The data were analyzed by one factor analysis of variance (ANOVA) followed by the Scheffé test. For data on the time course of the body weight, the serum corticosterone levels, and the serum MDA levels, repeated measure ANOVA followed by the Scheffé test was done. Correlation between the light–dark latency and pain threshold levels was analyzed by Pearson's correlation test. Analysis of covariance (ANCOVA) was used to examine the pain threshold levels as predictive variables for light–dark latency. All analyses were performed on a Macintosh G4 using the SAS/STAT version 8.02 (SAS Institute, Inc) statistical program. A P value b0.05 was considered to be significantly different from the corresponding value.
Acknowledgments This study was supported by grants in aid for General Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 16390200, No. 17390210, and No. 17659205).
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