The effects of emotional stress are not identical to those of physical stress in mouse model of social defeat stress

Journal Pre-proof The effects of emotional stress are not identical to those of physical stress in mouse model of social defeat stress Yuko Nakatake, Hiroki Furuie, Misa Yamada, Hiroshi Kuniishi, Masatoshi Ukezono, Kazumi Yoshizawa, Mitsuhiko Yamada

PII:

S0168-0102(19)30261-5

DOI:

https://doi.org/10.1016/j.neures.2019.10.008

Reference:

NSR 4317

To appear in:

Neuroscience Research

Received Date:

7 May 2019

Revised Date:

9 October 2019

Accepted Date:

11 October 2019

Please cite this article as: Nakatake Y, Furuie H, Yamada M, Kuniishi H, Ukezono M, Yoshizawa K, Yamada M, The effects of emotional stress are not identical to those of physical stress in mouse model of social defeat stress, Neuroscience Research (2019), doi: https://doi.org/10.1016/j.neures.2019.10.008

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Neuroscience Research

The effects of emotional stress are not identical to those of physical stress in mouse model of social defeat stress Yuko Nakatakea, b, Hiroki Furuiea, Misa Yamadaa, Hiroshi Kuniishia, Masatoshi Ukezonoc, Kazumi Yoshizawab, Mitsuhiko Yamadaa* a

Department of Neuropsychopharmacology, National Institute of Mental Health, National

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Center of Neurology and Psychiatry, 4-1-1 Ogawahigashimachi, Kodaira, Tokyo 187-8553, Japan b

Laboratory of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641, Yamazaki, Noda, Chiba 278-8510, Japan

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Developmental Disorder Data Multi-level Integration Unit Medical Science Innovation Hub

Corresponding author:

Mitsuhiko Yamada, M.D., Ph.D.

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Department of Neuropsychopharmacology

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*

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Program, RIKEN, 4-1-1 Kizugawadai, Kizugawa, Kyoto 619-0225, Japan

National Institute of Mental Health,

National Center of Neurology and Psychiatry

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4-1-1 Ogawahigashimachi, Kodaira, Tokyo 187-8553, Japan Tel.: +81-42-341-2711

Fax: +81-42-346-1994

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E-mail: [email protected]

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Total number of pages: 30 pages Total number of figures: 6 figures Total number of tables: 4 tables

Highlights Mice were exposed to emotional stress by witnessing the defeat of a conspecific. Only ES mice showed anhedonia and only PS mice showed increased anxiety. 1

Emotional stress induced immune system changes 1 month after stress exposure. Fasudil did not suppress behavioral changes induced by emotional stress. The effects of emotional stress are not identical to those of physical stress.

Abstract: 194 words In this study, we investigated the effects of emotional stress and physical stress using the social defeat stress (SDS) model in mice. Male C57BL/6J mice were attacked by male

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non-retired ICR mice for 10 min daily for 10 days (physical stress; PS), while the other cohort of mice witnessed the defeat (emotional stress; ES). As a result, both PS and ES mice exhibited decreased social behavior in the social interaction test (SIT) and increased

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immobility in the forced swim test (FST). Interestingly, only ES mice exhibited decreased

sucrose preference, and only PS mice exhibited decreased time spent in the open arms in the

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elevated plus-maze test. ES mice did not exhibit increased levels of corticosterone and

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epinephrine after a single stress exposure, but showed a decrease in plasma CXCL16 levels 1 month after stress exposure. Finally, a RhoA/Rho kinase inhibitor, fasudil, which has been

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reported to attenuate the effects of chronic stress, suppressed the increased immobility in the FST in PS mice, but not in ES mice. These results demonstrate that, although ES and PS mice

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shared many characteristics, the effects of emotional stress are not identical to those of

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physical stress in mice.

Keywords

emotional stress, physical stress, social defeat stress, witness, depression, anxiety,

1. Introduction 2

The social defeat stress (SDS) model in mice has been used to investigate the neurobiological basis of stress-related mental disorders in a number of studies (Berton et al., 2006; Golden et al., 2011; Nestler & Hyman 2010). Defeated mice have been reported to show decreased social interaction, depression-like behavior, anxiety-like behavior, and immune system changes (Berton et al., 2006; Golden et al., 2011; Nestler & Hyman 2010; Ambrée et al., 2018; De Miguel et al., 2011). Therefore, the SDS model would be considered to have good construct validity and face validity.

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In the SDS model, because mice are simultaneously exposed to psychological and

physical stress, the possibility that some of the changes exhibited by defeated mice are caused by physical injuries cannot be excluded. However, in humans, stress-related mental disorders

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occur without physical stress in many cases, suggesting that physical distress is not always

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necessary for the onset of stress-related mental disorders.

Recently, a novel animal model was proposed, in which emotional stress can be

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isolated from standard SDS (Sial et al., 2016; Warren et al., 2013). In this model, animals experience emotional stress as a result of witnessing the defeat of a conspecific attacked by an

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aggressor. Witnessing the distress of conspecifics has been found to induce depression-like and anxiety-like behaviors, similar to the responses exhibited by defeated animals (Warren et

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al., 2013). These findings suggest that emotional stress itself may be capable of inducing behavioral changes in rodents.

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Interestingly, some changes were seen only in emotionally stressed animals, while

others were seen only in physically stressed animals. For example, decreased sucrose preference was observed only in emotionally stressed rats (Finnell et al., 2017). In addition, increased spine density in the nucleus accumbens was found only in physically defeated mice (Warren et al., 2014). Based on these findings, we hypothesized that the effects of emotional stress are not identical to those of physical stress. To test our hypothesis, we then investigated 3

the effects of emotional stress on behavior and immune system changes in mice using the witnessing SDS model (emotionally stressed mice: ES mice), and compared them with mice exposed to the standard SDS protocol (physically stressed mice: PS mice). When the physical attacks on the subject animals are too strong, the effects of both physical and emotional stress can plateau, which could mask the differences between physical and emotional stress. Therefore, in the current study we used less aggressive, non-retired ICR mice as the resident mice in the SDS model.

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Behavioral changes in standard SDS mice have been reported to be reversed by the chronic, but not the acute, administration of antidepressants, including imipramine and fluoxetine (Berton et al., 2006). This suggests that the SDS model should have good

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predictive validity. Recently, fluoxetine and ketamine have been reported to improve the

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decreases in social behavior induced by witnessing SDS (Warren et al., 2013; Finnell et al., 2017). Therefore, the witnessing SDS model should also have good predictive validity.

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Fasudil, a RhoA/Rho kinase (ROCK) inhibitor, was recently reported to improve depressivelike behavior induced by chronic restraint stress (García-Rojo et al., 2017) and chronic

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unpredictable mild stress (Qin et al., 2017). Therefore, we investigated pharmacological

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responses to fasudil in ES mice and compared them with responses in PS mice.

2. Materials and Methods

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2.1. Animals

Six-week-old male C57BL/6J mice (Japan SLC, Shizuoka, Japan) were used in this

study. Mice were housed five per cage at least 1 week prior to a stress session as a habituation period. Thirteen-week-old male non-retired ICR mice (Japan SLC, Shizuoka, Japan) were used as resident mice in the SDS, and were housed individually for 1 week or more prior to the stress session. All mice were housed at 23 ± 1 °C with a 12 h light-dark cycle (lights 4

switched on automatically at 8 am), and allowed free access to food (CE-2, CLEA JAPAN, Tokyo, Japan) and water, in standard polyolefin plastic cages (17 cm × 24 cm × 12 cm) (CL0103-2 Mouse TPX, CLEA JAPAN, Tokyo, Japan) containing wood shavings (soft chip, Japan SLC, Shizuoka, Japan). Five separate cohorts of mice were used for behavioral test batteries, cytokine and chemokine measurement, corticosterone measurement, epinephrine measurement, and pharmacological studies. We used different numbers of mice for different types of experiments. The largest number of mice were used for behavioral test batteries (n =

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21-24 per group) because of the higher variability within groups that was found during our

preliminary experiments compared with that for physiological analyses (n = 8-12 per group). The numbers of mice used for the pharmacological studies (n = 16-18 per group) was

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determined according to a previous report (Garcia-Rojo et al., 2017). The experiments were

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conducted in compliance with the Guidelines for the Care and use of Laboratory Animals, and approved by the National Center of Neurology and Psychiatry Animal Care and Use

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2.2. Witnessing SDS model

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Committee (2017014, 2018027).

2.2.1 Screening process for the resident ICR mice

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Non-retired ICR mice were screened using C57BL/6J mice that were not used for experiments. In the screening process, C57BL/6J mice were placed into the home cages of

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ICR mice for 3 min once a day (Golden et al., 2011; Sial et al., 2016). This process was repeated for 3 consecutive days. In each session, attack latencies and attack counts were recorded. ICR mice with attack latencies less than 1 min and more than 5 bouts over at least 2 days during the screening session were used as resident mice. We previously confirmed, in our preliminary experiment, that non-retired ICR mice exhibited reduced attack numbers against C57BL/6J mice during the stress period compared with those of retired ICR mice, 5

even though both retired and non-retired ICR mice met the criteria for the screening session (total attack numbers over 10 days: non-retired ICR 59.0 ± 5.66; retired ICR 98.2 ± 3.22).

2.2.2. Preparation of ES mice and PS mice The SDS paradigm was performed as previously described (Golden et al., 2011), with the addition of an emotional stress component (Warren et al., 2013). To start the defeat paradigm, previously screened, non-retired ICR mice were housed overnight in cages that

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were divided into two compartments by a transparent partition. ES mice were placed into the compartment adjacent to the resident ICR mouse, while PS mice were placed into the same

compartment containing the resident mouse for 10 min (Fig. 1A). PS mice were exposed to

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attacks from the resident ICR mouse, and ES mice could witness the defeat from the

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neighboring compartment. After the defeat session, ES and PS mice were separated from the resident ICR mice by a transparent partition to allow sensory contact for 24 h until the next

resident ICR mouse.

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defeat. This procedure was repeated daily for 10 consecutive days, but always with a new

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As described in the report by Sial et al., control mice were allowed to interact with their cage mate (C57BL/6J) for 10 min daily for 10 days (Sial et al., 2016), instead of being

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exposed to physical or emotional stress (Fig. 1B). They were housed in pairs with a transparent partition until the next control session. In this study, we used common control

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mice for both PS and ES mice. After the last session, all C57BL/6J mice, including control mice, were housed

individually. Body weight was measured prior to each stress or control session.

2.3. Behavioral assays When comparing the effects of emotional and physical stress on depression-like and 6

anxiety-like behaviors, mice were tested at two time points. STEP1 was assessed 24 h after and STEP2 was assessed 1 month after the last stress session (Fig. 2A).

2.3.1. Social interaction test (SIT) The SIT involved two testing sessions. In the first session, the C57BL/6J mouse was allowed to explore an open field arena (40 cm × 40 cm) for 2.5 min. Along one side of the arena was a circular (8 cm diameter) wire cage that remained empty during the first trial (no

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target). The mouse was then removed from the testing arena and a novel ICR mouse was

placed into the wire cage (target present). In the second session, C57BL/6J was placed back

into the arena and allowed to explore. The total time spent in the interaction zone (14 cm × 25

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cm wide arena surrounding the cage) was recorded and analyzed using video tracking

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software (Smart 3.0, Panlab, Barcelona, Spain) in each session. The social interaction ratio (SI ratio) was obtained by dividing the time spent in the interaction zone when the target was

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present by the time spent in the interaction zone when the target was absent (Golden et al.,

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2011).

2.3.2. Elevated plus-maze test (EPM)

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The maze consisted of two perpendicular intersecting runways. One runway had tall walls (closed arms), and the other one had no walls (open arms). The runways were 5 cm

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wide, 25 cm long and the closed walls were 15 cm tall. The maze was 50 cm from the floor. Mice were placed in the central area, facing one of the closed arms and allowed to explore for 5 min. The total time spent in the open arms was recorded and analyzed using video tracking software (Smart 3.0, Panlab, Barcelona, Spain).

2.3.3. Forced swim test (FST) 7

Mice were placed individually into 5 L beakers (27 cm × 19 cm) containing 3.5 L of water (23 ± 1 °C) for 6 min. An immobile posture was defined as stopping all active behaviors and floating in the water with minimal movement (Porsolt et al., 1979). Movements over this 6 min period were recorded using a video camera (LifeCam Studio, Microsoft, Washington, USA) and immobility was recorded blindly by the experimenters when mice spent more time in an immobile posture than performing active behaviors in each 5 sec.

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2.3.4. Sucrose preference test (SPT)

The SPT was conducted for 3 days including 2 days of training period (Fig. 2A)

according to previously established conditions, as described below. Mice were trained to drink

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from 2 separate bottles (water and 1% sucrose) for 2 days. The 2 drinking bottles were located

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on the both sides of home cage of the experimental mouse. The position of the two bottles was balanced across the experimental mice to exclude potential side preference bias. On the

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third day of SPT, mice were deprived of food, water and sucrose for 4 h. After the deprivation, mice were again provided with access to water and sucrose bottles, and their total liquid

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consumption was recorded for 1 h to obtain sucrose preference. Sucrose preference was

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obtained by dividing sucrose consumption by the total consumption (sucrose + water).

2.4. Cytokine and chemokine measurement

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Either 40 min or 1 month after the SIT, trunk blood was collected in an

ethylenediaminetetraacetic acid tube (TERUMO, Tokyo, Japan) (Fig. 2B). Blood samples were centrifuged at 3,000 × g for 10 min at 20 °C to obtain plasma, and stored at −80 °C until analysis. The plasma level of cytokines and chemokines was assayed using a mouse chemokine 33-Plex panel (Bio-Plex, Bio-Rad, CA, USA). Plasma samples were diluted five times in assay buffer and run in accordance with manufacturer’s instructions. All samples 8

were measured in duplicate.

2.5. Corticosterone measurement Blood was collected from the tail tip and collected in a heparin tube (TERUMO, Tokyo, Japan) individually 40 min after a single stress session (Fig. 2B). Blood samples were centrifuged at 3,500 × g for 90 seconds at 4 °C to obtain plasma and stored at −80 °C until use. Plasma samples were diluted 100 times in assay buffer and analyzed using a

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Corticosterone ELISA kit (Cayman Chemical, MI, USA) following the manufacturer’s instructions. All samples were measured in duplicate.

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2.6. Plasma epinephrine determination

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Trunk blood was collected in a heparin tube (TERUMO, Tokyo, Japan) immediately after a single defeat session (Fig. 2B). Blood samples were centrifuged at 3,500 × g for 90

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seconds at 4 °C to obtain plasma and stored at −80 °C until use. Plasma samples (50 μl) and an internal standard of 100 pg isoproterenol were applied onto Clean Column EG (EICOM,

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Kyoto, Japan) to extract catecholamines. The concentration of epinephrine was analyzed using high performance liquid chromatography (HPLC). The HPLC system consisted of an

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electrochemical detector (ECD-700, EICOM) including a graphite electrode (WE-3G, EICOM,) with Gasket (GS-25, EICOM), an auto-sampling injector (M-514, EICOM), and a

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reverse-phase column (EICOMPAK SC-50DS, 3.0 × 150 mm, EICOM). The mobile phase consisted of 0.1 M sodium acetate/0.1 M citric acid buffer, pH 3.5, containing 17% methanol, 200 mg/liter sodium 1-octansulfonate and 50 mg/liter EDTA-2Na. Separation was achieved at 25°C, using a flow rate of 0.5 ml/min. The electrochemical detector was set at 750 mV vs. Ag/AgCl. The concentration of epinephrine was calculated by the chromatographic peak areas using internal standard methods by a data processor (EPC500, EICOM). 9

2.7. The effects of fasudil on emotional stress and physical stress We evaluated the efficacy of fasudil hydrochloride (Tokyo Chemical Industry, Tokyo, Japan), a ROCK inhibitor, in ES and PS mice. Fasudil (10 mg/kg; i.p.) was dissolved in saline (Otsuka Pharmaceuticals, Tokushima, Japan). This dose was selected based on previous reports that fasudil reduced the increased immobility time caused by stress in the FST (Garcia-Rojo et al., 2017; Qin et al., 2017). Mice were administered saline or fasudil (10

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mg/kg) 30 min prior to a daily stress or control session for 10 days. After the last session, mice were tested in the SIT, and in the FST again the following day (Fig. 2C).

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2.8. Statistical analysis

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All data were presented as mean ± standard error of the mean (SEM). Data were analyzed using IBM SPSS statistics 21 (IBM, NY, USA). Two-way repeated-measures

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analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test was used to compare the effects of physical and emotional stress on body weight gain, SIT, EPM, FST and SPT

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performance at STEP1 and STEP2. Because of the exploratory nature of the study, one-way ANOVA followed by Dunnett’s post-hoc test was used to compare the effects of physical and

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emotional stress on the levels of plasma cytokine, chemokine, corticosterone, and epinephrine. A one-way ANOVA, followed by Dunnett’s post-hoc test, was used to compare

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the effects of fasudil treatment in ES and PS mice. Differences with p < 0.05 were considered to be statistically significant.

3. Results 3.1. Body weight gain Fig 3. shows the body weight gain during stress exposure period. Two-way ANOVA 10

revealed a significant interaction effect between stress × day (F(18,585) = 5.578, p < 0.001) and significant main effects of day (F(9,585) = 50.392, p < 0.001), but no significant main effect of stress. Post-hoc tests indicated that PS mice (n = 21) exhibited significantly higher body weight compared with control mice on the seventh day (p < 0.05), the ninth day (p < 0.01), and the tenth day (p < 0.01) of stress exposure. There was no significant change in body weight in ES mice (n = 23) compared with control mice (n = 24).

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3.2. SIT

Fig 4. shows the SI ratio at each time point in the SIT. Two-way ANOVA revealed a significant interaction effect between time point × stress (F(2,65) = 4.708, p < 0.05), and

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significant main effects of time point (F(1,65) = 25.513, p < 0.01) and stress (F(2,65) = 16.043, p

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< 0.01). Post-hoc tests showed that PS mice (n = 21) exhibited a significantly lower SI ratio compared with control mice (n = 24) and ES mice (n = 23) (CON: p < 0.01, ES: p < 0.05) at

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STEP1. ES mice exhibited a significantly lower SI ratio compared with control mice at STEP1 (p < 0.05). At STEP2, the SI ratio of PS mice was significantly lower than that of

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control mice (p < 0.05).

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3.3. EPM

Fig 5A. shows the percentage of time spent in the open arms at each time point in the

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EPM. Two-way ANOVA revealed significant main effects of time point (F(1,65) = 22.095, p < 0.01) and stress (F(2,65) = 5.895, p < 0.01), but no significant time point × stress interaction. Post-hoc testing revealed that PS mice (n = 21) exhibited a significantly lower time score in the open arms compared with control mice (n = 24) (p < 0.01). The percentage of time spent in the open arms in STEP2 was significantly lower than that in STEP1 (p < 0.01).

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3.4. FST Fig 5B. shows the immobile counts at each time point in the FST. Two-way ANOVA revealed significant main effects of time point (F(1,65) = 75.597, p < 0.01) and stress (F(2,65) = 6.413, p < 0.01), but there was no significant time point × stress interaction effect. Post-hoc testing revealed that PS (n = 21) and ES mice (n = 23) exhibited a significant increase in immobile counts compared with control mice (n = 24) (PS: p < 0.01, ES: p < 0.05). The

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immobility of STEP2 was significantly increased compared with STEP1 (p < 0.01).

3.5. SPT

Fig 5C. shows the percentage of sucrose preference at each time point in the SPT.

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Two-way ANOVA revealed a significant main effect of stress (F(2,65) = 4.095, p < 0.05), but no

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significant main effect of time point, and no significant time point × stress interaction effect. Post-hoc testing revealed that only ES mice (n = 23) displayed a significantly lower sucrose

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preference compared with control mice (n = 24) (p < 0.05).

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3.6. Corticosterone and epinephrine assay

In the corticosterone immunoassay, one-way ANOVA revealed a significant main

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effect of stress (F(2,27) = 7.321, p < 0.01). Post-hoc testing revealed that PS mice exhibited significantly increased levels of plasma corticosterone compared with control mice 40 min

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after a single stress session (n = 10 per group) (p < 0.01). In the epinephrine assay, one-way ANOVA revealed a significant main effect of stress (F(2,21) = 9.639, p < 0.01). Post-hoc testing revealed that PS mice exhibited a significant increase in the level of plasma epinephrine compared with control mice immediately after a single stress session (n = 8 per group) (p < 0.01). In contrast, the levels of plasma corticosterone and epinephrine in ES mice were unchanged compared with control mice (Table 1). 12

3.7. Cytokine and chemokine assay Table 2 shows the levels of plasma cytokine and chemokine 40 min after SIT (n = 12 per group). One-way ANOVA revealed significant main effects of stress in CXCL13 (F(2,33) = 13.747, p < 0.01), IL-4 (F(2,33) = 3.367, p < 0.05), IL-16 (F(2,33) = 21.524, p < 0.01), CCL3 (F(2,32) = 3.354, p < 0.05), CCL7 (F(2,33) = 45.985, p < 0.01), and CCL25 (F(2,32) = 3.742, p < 0.05). Plasma from PS mice exhibited significantly increased levels of CXCL13 (p < 0.01),

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IL-16 (p < 0.01), CCL7 (p < 0.01), CCL25 (p < 0.05), and a significant decrease in the level of plasma CCL3 (p < 0.05) compared with control mice. In addition, PS mice exhibited

increased levels of plasma IL-4, although this difference was not significant (p = 0.058).

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Table 3 shows the levels of plasma cytokine and chemokine 1 month after the SIT (n

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= 10 per group). One-way ANOVA revealed significant main effects of stress in CCL17 (F(2,27) = 3.481, p < 0.05) and CXCL16 (F(2,27) = 5.765, p < 0.01). PS mice exhibited

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significant decreases in the level of plasma CCL17 (p < 0.05) compared with control mice. PS and ES mice exhibited a significant decrease in the level of plasma CXCL16 (PS: p < 0.01,

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ES: p < 0.05) compared with control mice. In addition, ES mice exhibited decreased levels of

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plasma CCL17, although this difference was not significant (p = 0.066).

3.8. The effects of fasudil on stress-induced behavioral changes in PS and ES mice

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In the SIT, one-way ANOVA revealed there were no significant differences among

treatment groups in ES and PS mice (Table 4). In the FST, in ES mice a one-way ANOVA revealed significant differences among

treatment groups (n = 18 per group) (F(3,68) = 13.338, p < 0.001). Both saline and fasudiltreated ES mice displayed significant increases in the numbers of immobile counts compared with saline-treated control mice (p < 0.01) (Fig. 6A). In PS mice, a one-way ANOVA also 13

revealed significant differences among treatment groups (F(3,65) = 5.530, p < 0.01). However, the numbers of immobile counts were significantly increased in saline-treated PS mice (n = 16) compared with saline-treated control mice (n = 18) (p < 0.01), but not in fasudil-treated PS mice (n = 17) (Fig. 6B).

4. Discussion In this study, we investigated the effects of emotional stress on behavior and immune

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system changes in ES mice, and compared these changes with those observed in PS mice. To investigate this issue, we used less aggressive, non-retired ICR mice as the resident mice in the witnessing SDS paradigm to weaken the intensity of physical and emotional stress.

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During stress exposure, ES mice did not exhibit any changes in body weight

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compared with control mice, whereas PS mice exhibited significant increases in body weight. This weight gain observed in PS mice in this study was inconsistent with previous reports in

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which mice were exposed to standard SDS (Krishnan et al., 2007; Warren et al. 2013). Interestingly, mice have been reported to show body weight gain when the duration of SDS

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exposure was shortened or when the number of attacks was limited (Savignac et al., 2011; Goto et al., 2014). Based on these studies, the weakened stress intensity incurred by using less

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aggressive, non-retired ICR mice as resident mice may have contributed to the weight gain in our study. In general, mild stress causes hyperphagia, and more severe stress causes

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hypophagia (Greeno & Wing 1994). Stress has also been reported to induce the secretion of hormones involved in energy metabolism and increased body weight (Patterson et al., 2013). Further investigation is necessary to explain the mechanisms underlying the weight gain observed in PS mice in our present study. In the current experiment, not only PS mice, but also ES mice, exhibited decreases in social behavior in the SIT. This result suggests that emotional stress could induce 14

avoidance behavior, even without physical contact. The decrease in SI ratio in ES mice was smaller than that of PS mice. This finding might be expected, since PS mice had an intense fearful experience such as being chased and bitten during stress exposure, and decreased SI ratio was considered to be an avoidance behavior against an aversive stimulus. The use of the SIT to classify PS mice into susceptible (SI ratio less than 1) and resilient phenotypes (SI ratio greater than 1) may help to clarify the observed behavioral responses (Krishnan et al., 2007). The proportion of susceptible PS mice in our study (approximately 50%) was lower than that

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reported in a previous report (approximately 60 %-70%), which used a standard SDS protocol (Golden et al., 2011). Interestingly, all of the ES mice displayed the resilient feature. These

results were likely due to the use of the less aggressive, non-retired ICR mice as the resident

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mice in this study.

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One month after stress exposure, PS mice displayed a significantly lower SI ratio, as reported in a previous study (Warren et al., 2013). Interestingly, control mice also showed a

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substantial reduction in social interaction. Therefore, the results 1 month after stress exposure should be interpreted with caution. In the current study, we tested the same cohort of mice

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repeatedly in the SIT. It is possible that mice became accustomed to the ICR mice in the second SIT, and consequently did not show approach behavior toward them.

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In the EPM, PS mice spent less time in the open arms, while this anxiety-like behavior was not observed in ES mice, suggesting that anxiety-like behavior in the EPM was

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more strongly affected by physical stress than emotional stress. From the results of the SIT, we found that half of the PS mice displayed the susceptible feature, whereas all of the ES mice displayed the resilient feature. Interestingly, the anxiety-like behavior was observed in both the susceptible PS mice and in the resilient PS mice (data not shown). These results suggest that resilience and susceptibility in the SIT are not associated with anxiety-like behavior. In contrast to the current results, several previous studies reported that rodents 15

witnessing defeat displayed anxiety-like behavior (Sial et al., 2016; Warren et al., 2013; Patki et al., 2014, 2015). The weakened stress intensity incurred by using less aggressive, nonretired ICR mice as the resident mice may have contributed to the inconsistency found in our study. Further investigation is necessary to confirm the effects of physical stress and emotional stress on anxiety-like behavior using other behavioral tests, such as the open field test or the light and dark box test. In the current study, PS and ES mice exhibited increases in immobility in the FST

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that lasted up to 1 month. Compared with non-stressed controls, stressed rats typically adopt an immobile posture more quickly, extending total immobility time (Iniguez et al., 2010). These behavioral changes exhibited by stressed animals are considered to reflect greater

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sensitivity to inescapable stress conditions. Unlike the results in the SIT, the degree of

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immobility in FST in ES mice was comparable to that in PS mice, suggesting that the effects of emotional stress may be stronger than previously considered. The increased susceptibility

effects of emotional stress.

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to inescapable stress (detected by the FST) may provide a sensitive indicator of the specific

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In the SPT, decreased sucrose preference was not observed in PS mice. Considering the lower proportion of susceptible PS mice, this result was consistent with a previous report

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demonstrating that decreases in sucrose preference were observed only in susceptible mice, but not in resilient mice (Krishnan et al., 2007; Golden et al., 2011). In contrast, decreased

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sucrose preference was clearly observed in ES mice. Decreases in sucrose preference are thought to reflect dysregulation of the mesolimbic dopamine circuit known as the reward system (Nestler & Carlezon 2006). These findings suggest that emotional aspects of stress alone can strongly affect the reward system, and thereby induce anhedonia-like behavior. Surprisingly, ES mice decreased preference for sucrose, although they displayed resilient feature. These results strongly suggest that emotional stress is not simply a weaker version of 16

physical stress. To investigate the physiological effects of emotional stress, we measured the levels of plasma corticosterone and epinephrine after a single stress exposure. ES mice did not show any changes, while PS mice exhibited significant increases in the levels of both corticosterone and epinephrine. In contrast to our results, Warren et al. reported that ES mice showed increase in corticosterone (Warren et al., 2013). This discrepancy could be due to the differences in the stress intensity or the timing of blood sampling used in each experiment.

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Interestingly, despite the lack of hormonal changes, ES mice exhibited several behavioral changes. Therefore, these behavioral alterations may not always be correlated with the

changes in the levels of corticosterone and epinephrine. Because anxiety-like behavior was

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not affected by corticosterone injection (Fernandes et al., 1997), stress-induced behavioral

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changes might not be necessarily accompanied by an increase in corticosterone and epinephrine. However, it should be noted that blood was collected only after a single stress

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exposure but not at the time the behavioral changes occurred.

Although some immune system changes have also been reported in animals

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exposed to SDS, the possibility that these changes were caused by injuries during stress exposure cannot be excluded. Therefore, we investigated whether emotional stress that is not

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accompanied by injuries could induce changes in the peripheral immune system. At the time of blood sampling 40 min after the SIT, ES mice did not show any changes, while PS mice

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exhibited a significant increase in the levels of CXCL13, IL-16, CCL7 and CCL25 and a decrease in the levels of CCL3. These results may suggest that emotional stress might have little effect on the immune system at this stage. CXCL13 (B cell-attracting chemokine; BCA1) and IL-16 are known to be a B lymphocyte chemoattractant and a lymphocyte chemoattractant factor, respectively. These molecules promote the expression of proinflammatory factors and inflammatory responses (Legler et al., 1998; Gunn et al., 1998; 17

Mathy et al., 2000). CCL3 and CCL7, which have similar properties to CCL2, are classified as inflammatory, and CCL25 is classified as a homeostatic chemokine (Palomino & Marti 2015). These changes were considered to be inflammatory responses caused by wounds suffered during stress exposure, since they were observed only in PS mice. Some previous studies reported that physically defeated animals showed a range of immunological changes, including elevated levels of IL-1β, IL-6, and TNF-α (Finnell et al., 2017; Hode et al., 2014). Indeed, peripheral IL-6 has been reported to be important for SDS-

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induced avoidance behavior (Hode et al., 2014). However, these immunological changes were not observed in the current study. According to previous studies, only susceptible mice

showed increased levels of IL-1β, IL-6 and TNF-α (Dowlati et al., 2010; Hode et al., 2014;

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Finnell et al., 2017). It is possible that we did not detect above mentioned inflammatory

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responses in PS mice because the proportion of susceptible mice was relatively small, when using less aggressive non-retired ICR mice as the resident mice in our study.

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The results of a chemokine assay 1 month after the SIT is considered to reflect the steady state of the immune system after stress exposure. PS mice showed a significant

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decrease in CCL17. CCL17 has been reported to decrease in male post-traumatic stress disorder patients (Dalgard et al., 2017). Interestingly, ES mice exhibited a decrease in

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CXCL16 levels at this time point, which was also observed in PS mice. CXCL16 is a potent mediator of angiogenesis (Isozaki et al., 2013) and has been suggested to be associated with

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kidney and cardiovascular diseases (Izquierdo et al., 2014). Because the changes in CXCL16 levels displayed by PS mice were also observed in ES mice that had not been injured, these changes are likely to have been induced by emotional stress. The results suggest that CXCL16 might be a biomarker reflecting long lasting effects of stressful events. However, the association between changes in CXCL16 levels and behavioral changes is unknown. Therefore, further investigation is required to determine whether CXCL16 is associated with 18

behavioral changes long after stress exposure. To compare the responses of ES and PS mice to a drug that could attenuate the effects of chronic stress (García-Rojo et al., 2017; Qin et al., 2017), we administered fasudil to ES and PS mice and tested them in the SIT and FST. In the SIT, we did not observe any treatment effect. It is possible that the daily drug injection (i.p.) functioned as a light stressor, even in the control mice, and masked the effects of stress exposure. Unexpectedly, in the present study, fasudil suppressed increased immobility in FST in PS mice, but not in ES mice.

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This finding demonstrated that the pharmacological responses to fasudil in ES mice were

different from that observed in PS mice. Fasudil has been reported to suppress the abnormal activation of ROCK, inflammatory responses (Qin et al., 2017), and spine loss in the

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hippocampus caused by chronic stress (Garcia-Rojo et al., 2017). These results suggest that

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fasudil may have affected the hippocampus in PS mice, suppressing increased immobility in the FST. However, dysfunction of the reward system has been reported to induce increases in

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immobility in the FST (D’Aquila et al., 2004; Rincon-Cortes & Grace 2017). The SPT results in the current study suggest that emotional stress affects the mesolimbic dopamine circuit

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more strongly than physical stress. It is possible that differences in brain areas susceptible to emotional and physical stress can explain this discrepancy between ES and PS mice. Further

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investigation is needed to clarify the pathophysiological changes in the central nervous system

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in ES and PS mice after stress exposure.

5. Conclusion

In the current study, emotional stress alone induced decreases in social interaction,

changes in sensitivity to inescapable conditions and anhedonia in mice, without physical stress or contact. Interestingly, emotional stress failed to induce anxiety-like behavior, while physical stress induced anxiety-like behavior in mice. In addition, a ROCK inhibitor, fasudil 19

suppressed increased immobility in FST in PS mice, but not in ES mice. Our results demonstrate that, although ES and PS mice share many characteristics, the effects of emotional stress and physical stress are not identical.

Acknowledgments This research was financially supported by an Intramural Research Grant for Neurological and Psychiatric Disorders provided by the National Center of Neurology and

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Psychiatry, Japan (27-1, 30-1) and a grant from the Japan Foundation for Neuroscience and Mental Health. We thank Benjamin Knight, MSc. and Lisa Giles, PhD from Edanz Group

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(www.edanzediting.com/ac) for editing a draft of this manuscript.

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Table 1. Plasma levels of corticosterone and epinephrine. CON

ES

PS

corticosterone (ng/ml)

27.3 ± 6.3 (n=10)

31.9 ± 4.9 (n=10)

57.5 ± 6.7** (n=10)

epinephrine (ng/ml)

0.49 ± 0.30 (n=8)

0.37 ± 0.12 (n=8)

2.69 ± 0.65** (n=8)

Plasma samples were prepared 40 min after a single defeat session for corticosterone measurement (n = 10 per group). For epinephrine measurement, plasma samples were

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mean ± SEM. **p < 0.01 compared with control (CON) mice.

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prepared immediately after a single defeat session (n = 8 per group). All data are presented as

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Table 2. Levels of plasma cytokines and chemokines 40 min after the social interaction test. Cytokines and Chemokines (pg/mL) CON (n=12)

ES (n=12)

PS (n=12)

2607.4 ± 123.9 2655.5 ± 255.5 4073.7 ± 266.1**

CTACK/CCL27

1019.1 ± 50.1

1113.7 ± 154.5 1168.9 ± 32.6

ENA-78/CXCL5

792.3 ± 184.8

818.4 ± 217.0

1005.0 ± 316.2

Eotaxin/CCL11

432.2 ± 43.9

400.1 ± 37.8

450.6 ± 28.2

Eotaxin-2/CCL24

7987.0 ± 572.9 6918.2 ± 376.5 6979.7 ± 333.3

Fractalkine/CX3CL1

78.0 ± 3.6

79.2 ± 5.8

80.6 ± 3.0

GM-CSF

17.6 ± 0.8

17.1 ± 1.6

18.3 ± 1.1

I-309/CCL1

11.2 ± 0.4

12.0 ± 1.5

10.3 ± 0.6

IFN-γ

17.9 ± 0.9

18.1 ± 1.6

18.6 ± 0.8

IL-1β

307.3 ± 11.9

278.5 ± 23.0

340.1 ± 46.2

IL-2

8.3 ± 0.4

9.4 ± 1.4

10.2 ± 0.4

IL-4

2.5 ± 0.1

2.5 ± 0.2

3.0 ± 0.2†

IL-6

16.6 ± 0.7

19.8 ± 2.5

18.4 ± 0.8

IL-10

390.7 ± 38.5

362.0 ± 38.8

391.3 ± 36.3

IL-16

293.4 ± 9.3

335.1 ± 33.3

513.4 ± 26.6**

IP-10/CXCL10

749.6 ± 19.8

668.7 ± 38.7

702.5 ± 13.5

I-TAC/CXCL11

239.3 ± 13.7

237.9 ± 24.1

285.6 ± 13.5

KC/CXCL1

30.2 ± 0.9

35.6 ± 3.3

30.0 ± 0.6

252.3 ± 13.9

239.8 ± 20.7

225.4 ± 7.6

41.5 ± 2.7

45.1 ± 4.7

104.8 ± 7.3**

18.9 ± 0.9

20.5 ± 1.6

21.9 ± 0.9

89.4 ± 4.2

103.5 ± 7.8

100.2 ± 7.1

9.2 ± 0.4

8.7 ± 0.5

7.9 ± 0.2*

83.2 ± 2.7

79.1 ± 6.5

81.1 ± 4.1

51.9 ± 2.7

52.2 ± 5.3

53.6 ± 1.5

MIP-3α/CCL20

38.4 ± 1.1

41.7 ± 3.9

40.3 ± 2.0

MIP-3β/CCL19

283.4 ± 7.4

261.0 ± 15.6

263.2 ± 6.4

RANTES/CCL5

45.6 ± 2.1

43.8 ± 4.7

42.8 ± 2.0

SCYB16/CXCL16

292.7 ± 9.9

263.2 ± 26.2

354.0 ± 23.2

SDF-1α/CXCL12

2606.4 ± 171.5 2363.2 ± 190.4 3095.4 ± 272.0

TARC/CCL17

53.1 ± 4.0

49.7 ± 4.8

45.3 ± 6.8

TECK/CCL25

558.1 ± 62.1

679.4 ± 138.0

921.1 ± 82.3*

TNF-α

120.4 ± 5.9

114.0 ± 14.5

103.6 ± 4.4

MCP-3/CCL7

MIP-1β/CCL4

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MIP-2/CXCL2

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MCP-5/CCL12 MIP-1α/CCL3

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MCP-1/CCL2

MDC/CCL22

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BCA-1/CXCL13

All data are presented as mean ± SEM. †p < 0.1, *p < 0.05, **p < 0.01 compared with control (CON) mice (n = 12 per group). 26

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Table 3. Levels of plasma cytokines and chemokines 1 month after the social interaction test. Cytokines and Chemokines (pg/mL) CON (n=10)

ES (n=10)

BCA-1/CXCL13

4651.6 ± 333.1

4131.6 ± 215.8 4663.9 ± 478.2

CTACK/CCL27

1242.3 ± 75.8

1148.5 ± 55.9

ENA-78/CXCL5

4799.7 ± 1210.4 2695.0 ± 604.7 2794.6 ± 629.8

Eotaxin/CCL11

1371.6 ± 241.5

Eotaxin-2/CCL24

11320.6 ± 819.4 8916.0 ± 701.1 9101.3 ± 915.5

Fractalkine/CX3CL1

94.7 ± 6.8

83.6 ± 7.1

78.7 ± 12.0

GM-CSF

16.6 ± 2.2

16.3 ± 1.5

18.1 ± 3.7

I-309/CCL1

11.9 ± 1.4

10.9 ± 1.3

15.7 ± 2.4

IFN-γ

21.8 ± 1.0

19.5 ± 1.1

18.9 ± 2.6

IL-1β

476 ± 22.2

458.64 ± 28.1

478.6 ± 42.5

IL-2

9.3 ± 0.8

8.8 ± 0.5

7.9 ± 0.6

IL-4

2.8 ± 0.3

2.6 ± 0.3

2.7 ± 0.4

IL-6

22.7 ± 2.2

20.5 ± 2.1

20.9 ± 4.3

IL-10

639.5 ± 100.8

655.9 ± 119.8

615.6 ± 110.7

IL-16

442.1 ± 33.6

415.6 ± 21.2

433.7 ± 28.9

IP-10/CXCL10

950.6 ± 48.7

917.5 ± 34.8

969.9 ± 62.8

I-TAC/CXCL11

265.2 ± 38.6

245.2 ± 34.5

239.7 ± 44.9

KC/CXCL1

20.4 ± 3.3

19.1 ± 4.0

27.6 ± 10.7

356.2 ± 13.6

284.4 ± 28.9

265.0 ± 35.3

68.0 ± 5.0

60.5 ± 6.1

67.1 ± 10.0

20.9 ± 1.1

17.2 ± 1.7

20.3 ± 1.5

144.8 ± 15.7

136.0 ± 8.3

128.2 ± 16.3

8.0 ± 0.9

7.5 ± 0.8

6.1 ± 0.6

130.8 ± 7.8

124.0 ± 7.9

102.9 ± 8.4

58.2 ± 6.5

55.5 ± 7.0

45.2 ± 4.5

MIP-3α/CCL20

51.4 ± 6.0

45.5 ± 3.4

48.2 ± 6.2

MIP-3β/CCL19

187.4 ± 15.3

281.1 ± 11.0

296.0 ± 30.1

RANTES/CCL5

76.0 ± 4.6

70.7 ± 5.2

71.2 ± 11.0

SCYB16/CXCL16

347.7 ± 21.3

278.2 ± 15.5*

250.3 ± 24.9**

SDF-1α/CXCL12

5507.6 ± 866.5

4178.2 ± 618.2 4012.1 ± 520.1

TARC/CCL17

149.9 ± 23.6

98.9 ± 8.9†

95.2 ± 13.1*

TECK/CCL25

956.3 ± 160.3

848.5 ± 193.2

1009.8 ± 348.7

TNF-α

93.9 ± 10.8

209.8 ± 110.8

115.1 ± 33.2

MCP-3/CCL7

MIP-1β/CCL4

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MIP-2/CXCL2

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MCP-5/CCL12 MIP-1α/CCL3

1188.5 ± 67.6 941.6 ± 167.7

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MCP-1/CCL2

MDC/CCL22

951.8 ± 151.1

PS (n=10)

All data are presented as mean ± SEM. †p < 0.1, *p < 0.05, **p < 0.01 compared with control (CON) mice (n = 10 per group). 28

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Table 4. Effects of fasudil on SI ratio in ES and PS mice in the social interaction test. Saline

Fasudil (10 mg/kg)

CON

1.67 ± 0.11 (n=18)

1.78 ± 0.15 (n=18)

ES

1.46 ± 0.16 (n=18)

1.69 ± 0.39 (n=18)

CON

1.62 ± 0.15 (n=18)

1.53 ± 0.10 (n=17)

PS

1.43 ± 0.10 (n=16)

1.36 ± 0.08 (n=17)

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SI ratio

There were no differences among treatment groups. All data are presented as mean ± SEM.

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CON, control mice.

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Figure Legends Fig. 1. Schematic procedure of stress and control session. (A) PS mice (black) were exposed to attack by ICR mice (white), while ES mice (black with *) observed another mouse being exposed to attack for 10 min. After the stress session, PS and ES mice were housed with ICR mice across the partition. (B) Control (CON) mice (black with †) were allowed to interact with another conspecific (black without †) for 10 min. After the session, CON mice were

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housed in pairs across the partition.

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Fig. 2. Schematic schedule of analysis following stress session. (A) The schedule of stress exposure and subsequent behavioral experiments. (CON n = 24; ES n = 23; PS n = 21). (B) The schedule of blood sampling. Blood was collected immediately after a single stress session for epinephrine measurement (n = 8 per group), and 40 min after a single stress session for corticosterone measurement (n = 10 per group). For cytokine and chemokine assays, blood was collected 40 min (n=12 per group) and 1 month (n = 10 per group) after social interaction test. (C) The schedule of fasudil treatment, stress exposure, and subsequent behavioral tests

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(For ES experiments: CON-Saline n = 18; CON-Fasudil n = 18; ES-Saline n = 18; ES-Fasudil n = 18; for PS experiment: CON-Saline n = 18; CON-Fasudil n = 17; PS-Saline n = 16; PS-

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Fasudil n = 17). CON, control mice.

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Fig. 3. Effects of physical stress and emotional stress on body weight gain. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01. (CON n = 24; ES n = 23; PS n = 21). CON,

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Fig. 4. Effects of emotional stress and physical stress on the SI ratio during the social interaction test. *p < 0.05, **p < 0.01. (CON, n = 24; ES, n = 23; PS, n = 21). CON, control

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mice.

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Fig. 5. Effects of emotional stress and physical stress on the time spent in the open arms during the elevated plus-maze test (A), the number of immobile counts during the forced swim test (B), and the preference for sucrose during the sucrose preference test (C). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01. (CON, n = 24; ES, n = 23; PS, n = 21).

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CON, control mice.

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Fig. 6. Effects of fasudil on immobile counts induced by emotional stress (A) and physical stress (B) in the forced swim test. All data are presented as mean ± SEM. **p < 0.01. (For ES

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experiments: CON-Saline n = 18; CON-Fasudil n = 18; ES-Saline n = 18; ES-Fasudil n = 18; for PS experiments: CON-Saline n = 18; CON-Fasudil n = 17; PS-Saline n = 16; PS-Fasudil n

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= 17). CON, control mice.

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