Effects of chronic mild stress on behavioral and neurobiological parameters — Role of glucocorticoid

    Effects of chronic mild stress on behavioral and neurobiological parameters — Role of glucocorticoid Jiao Chen, Zhen-zhen Wang, Wei Zuo, Suai Zhang, Shi-feng Chu, Naihong Chen PII: DOI: Reference:

S0018-506X(15)30178-1 doi: 10.1016/j.yhbeh.2015.11.006 YHBEH 3989

To appear in:

Hormones and Behavior

Received date: Revised date: Accepted date:

20 April 2015 5 November 2015 20 November 2015

Please cite this article as: Chen, Jiao, Wang, Zhen-zhen, Zuo, Wei, Zhang, Suai, Chu, Shi-feng, Chen, Nai-hong, Effects of chronic mild stress on behavioral and neurobiological parameters — Role of glucocorticoid, Hormones and Behavior (2015), doi: 10.1016/j.yhbeh.2015.11.006

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ACCEPTED MANUSCRIPT Effects of chronic mild stress on behavioral and neurobiological parameters – role of glucocorticoid

State Key Laboratory of Bioactive Substances and Functions of Natural Medicines,

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Jiao Chen a, Zhen-zhen Wang a, Wei Zuo a, Suai Zhang a, Shi-feng Chu a, Nai-hong Chen a,b, 

Institute of Materia & Neuroscience Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

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Hunan University of Chinese Medicine, Changsha, China

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ABSTRACT

when

impairments

occur

in

mood-related

brain

regions.

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particularly

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Major depression is thought to originate from maladaptation to adverse events,

Hypothalamus-pituitary-adrenal (HPA) axis is one of the major systems involved in

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physiological stress response. HPA axis dysfunction and high glucocorticoid concentrations play an important role in the pathogenesis of depression. In addition, astrocytic disability and dysfunction of neurotrophin brain-derived neurotrophin factor (BDNF) greatly influence the development of depression and anxiety disorders. Therefore, we investigated whether depressive-like and anxiety-like behaviors manifest in the absence of glucocorticoid production and circulation in adrenalectomized (ADX) rats after chronic mild stress (CMS) exposure and its potential molecular mechanisms. The results demonstrate that glucocorticoid-controlled rats showed anxiety-like 

Correspondence: Professor N-H Chen, Tel: + 86 10 63165177, Fax: + 86 1063165177, E-mail:[email protected]

ACCEPTED MANUSCRIPT behaviors but not depression-like behaviors after CMS. Molecular and cellular changes included the decreased BDNF in the hippocampus, astrocytic dysfunction with

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connexin43 (cx43) decreasing and abnormality in gap junction in prefrontal cortex

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(PFC). Interestingly, we did not find any changes in glucocorticoid receptor (GR) or its chaperone protein FK506 binding protein 51 (FKBP5) expression in the hippocampus or PFC in ADX rats subjected to CMS. In conclusion, the production and circulation of

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glucocorticoids are one of the contributing factors in the development of depression-like

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behaviors in response to CMS. In contrast, the effects of CMS on anxiety-like behaviors are independent of the presence of circulating glucocorticoids. Meanwhile, stress

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decreased GR expression and enhanced FKBP5 expression via higher glucocorticoid

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anxiety-like behaviors.

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exposure. Gap junction dysfunction and changes in BDNF may be associated with

KEYWORDS: Depression; Chronic mild stress; GR; FKBP5;

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junction

BDNF; Gap

Introduction

Major depression is a common and disabling disease which is affecting a growing number of people in the world. Stress represents an environmental contributor to the development of depression. Exposure to prolonged stress resulted in hyper-activity of the stress system, defective glucocorticoid-negative feedback, and eventually caused structural and functional changes in mood-related brain regions including the prefrontal

ACCEPTED MANUSCRIPT cortex (PFC) and hippocampus (Maccari and Morley, 2007; Christopher and Ronald, 2008). Many literatures have documented high concentration level of cortisol in severe

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depression patients (Edward et al., 1979). In laboratory animals, repeated corticosterone

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(CORT) injection produced depression-like behaviors in the forced swim test (Gregus et al., 2005; Johnson et al., 2006). However, the question that whether CORT elevation is the result or the cause of major depression (Pariante and Lightman, 2008) remains

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unknown, and the cellular and molecular mechanisms underlying depression need to be

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further elucidated. Interestingly, the investigators have demonstrated that repeated CORT injections had no significant effect on anxiety in the open-field or social

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interaction tests (Gregus et al., 2005). This evidence reminds us of the possibility that

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stress-induced increase in glucocorticoid level may regulate the depression-like

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behaviors but not anxiety-like behaviors, and the mechanisms in the action of this hormone remain unclear.

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Recently, some literatures have reported that glucocorticoid receptor disability plays an important role in the development of major depression. Stress response may exert its influence through a series of mechanisms including reduced the affinity of GR to the ligands or down-regulating GR expression or defecting the effect of GR co-chaperone protein FK506 binding protein 51 (FKBP5) on glucocorticoid-feedback loop (Florian, 2000; Gianluigi et al., 2013). Also, brain-derived neurotrophin factor (BDNF) has been suggested to be involved in the etiopathology of depression. Indeed, recent studies have provided evidence that the BDNF disability in specific brain regions such as hippocampus may be related to the pathology of major depression(Cunha et al.,

ACCEPTED MANUSCRIPT 2006; Taliaz et al., 2010; Govindarajan et al., 2006). However, the questions that all those correlates of depression are consequences of stress or they predispose the

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individual to depression remain unclear.

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Furthermore, our previous work demonstrated the fact that rats exposed to chronic mild stress (CMS) show gap junction dysfunction in the PFC, which could contribute to the pathophysiology of major depression (Jian et al., 2011; Banasr et al., 2008; Dost et

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al., 1998). Gap junctional channels (GJC) are permeable for endogenous bioactive

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cytoplasmic molecules, and therefore the GJC-based astrocytic syncytium provides homeostatic and metabolic support likely essential for physiological neuronal function

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(Kimelberg, 2007; Giaume et al., 2010). Gap junctional channels are composed of

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connexin (Cx) proteins. One of them, Cx43, is mostly acknowledged to be synthesized

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in astrocytes. Therefore, it is interesting to ask if this dysfunction of astrocytes is the direct consequence of glucocorticoid exposure or may be induced through other

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mechanisms related to stress. Based on the above, we applied a behavioral assay to test the effect of chronic mild stress (CMS), a well-documented animal model of depression (Gianluigi et al., 2013; Calabrese et al., 2012). Then the ultrastructure of astrocytic gap junction and alteration of Cx43 in the rat PFC were detected after surgically manipulating CORT levels via adrenalectomy (ADX) or sham operation. Moreover, another interesting question was that how did the neurotrophin factor BDNF change in rats exposed to CMS, which was in the absence of glucocorticoid production and circulation. Meanwhile, the GR and FKBP5 expression in the hippocampus and PFC were also detected to investigate the

ACCEPTED MANUSCRIPT possible mechanism.

Chronic stress paradigm and experiment design

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Materials and methods

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Control animals (no stress) were housed under standard conditions, whereas

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stressed animals were exposed to the chronic mild stress procedure (CMS) for a period of 56 consecutive days, a manipulation that leads to a chronic depression-like state that

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develops gradually over time (Calabrese et al., 2012). The CMS regimen consisted of

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once or twice daily exposure to different stressors including food deprivation, crowding,

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isolation, soiled caged, 4 h immobilization, 1 h shaker stress (160 r.p.m), 1 min tail pinch, cage tilt 45 degree overnight. Figure1 presents the experimental schedule and

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timeline for the study. Two weeks after surgery, the rats were subjected to the CMS daily for 8 weeks. The behavioral performance of the animals in sucrose preference test (SPT), novelty suppressed feeding test (NSFT), forced swim test (FST), and elevated plus maze (EPM) was measured after 8 weeks‟ CMS. In order to exclude the effect of surgery on ADX rats, the anti-fatigue test by weight-loaded forced swim (WLFS) was performed.

Animals

ACCEPTED MANUSCRIPT Male Sprague-Dawley rats (Vital River Laboratories, Beijing, China) were housed under a 12-h light/12-h dark cycle at constant temperature (22 ℃) with free access to

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food and water except when animals were subjected to stress. The animals (250-300g)

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were divided into four groups with 12 animals in each group. All experiments were performed in accordance with the guidelines established by the National Institutes of Health for the care and use of laboratory animals and were approved by the Animal

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Care Committee of the Peking Union Medical College and Chinese Academy of

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Medical Sciences.

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Surgery

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The bilateral ADX or sham-operated surgery was performed under chloral hydrate anesthesia (400 mg/kg, i.p.). The completeness of the adrenalectomy was verified by

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visual inspection during surgery and later by the assessment of plasma CORT immediately after adrenalectomy. After surgery, ADX rats were supplied with CORT (sigma, USA) in their drinking solution at a concentration of

25 μg/ml. This

supplementation of CORT could get to basal glucocorticoid level (about 30% below that of sham rats) (Qiu et al., 2012; Akana et al., 1997), which have previously been reported to be sufficient to maintain the survival of mature hippocampal granule neurons (Cameron and Gould, 1994). In the sham surgery, the adrenals were touched by the surgical instrument but not removed. At the time of sacrifice, the kidney and surrounding tissues were visually examined to verify a complete removal of the adrenal

ACCEPTED MANUSCRIPT gland in ADX rats. Rats with any trace of adrenal tissue at the site were excluded from

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the study.

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Behavior tests

Sucrose perference test (SPT) was conducted as previously described (Taliaz et al.,

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2010; Gaelle et al., 2011). Briefly, rats were habituated to 1% sucrose solution for 48 h,

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followed by 4 h of water deprivation and 1 h exposure to two identical bottles filled with either sucrose solution or water. Sucrose preference was defined as the ratio of the

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volume of sucrose vs total volume of sucrose and water consumed during the 1 h test.

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Novelty suppressed feeding test (NSFT) was performed after 24 h of food

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deprivation in an open field (76.5 cm×76.5 cm×40 cm) with a small amount of food in the center, and latency to feed was determined as previously described (Warner-Schmidt

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et al., 2007). Home cage food intake was also measured as a control. For the forced swim test (FST), rats were introduced into a plastic cylinder (40 cm deep, 20 cm in diameter) filled with water at 23-25 ℃ up to a height of 25 cm from the base, and forced to swim for 15 minutes in the first session. Approximately 24 h after the first session, the animals were reintroduced into the same cylinder, and their 5-minute swim session was recorded on a video recorder. After each swim session, the rat was removed from the cylinder, dried with paper towels, and returned to its home cage. Water in the cylinder was renewed between subjects (Shuichi et al., 2012). The elevated plus maze (EPM) has two closed arms that have 50 cm-high walls

ACCEPTED MANUSCRIPT around the arms (10 cm wide, 50 cm long; east and west) and two open arms that have 0.5 cm ridges around the arms (10 cm wide, 50 cm long; north and south). The maze

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was raised 40 cm above the floor. Each animal was positioned to the center square with

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a nose aiming to one of the closed arms. The performance of rats in the EPM was video recorded for five minutes. The surface of the maze was cleaned with 20% ethyl alcohol and dried between the individual testing (Pometlová et al., 2012).

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In the weight-loaded forced swimming (WLFS) test, the rats swam with a load of

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steel rings that weighed approximately 8% of their body weight and were attached to their tails. The swimming time from the beginning of swimming with weight until the

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point at which rats could not again return to the surface of the water 10 seconds after

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(Masaaki et al., 2003).

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sinking was measured. Then, rats were returned to their home cage for recovery

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Electron microscopy (EM)

Processing and EM were conducted as previously described (Jasinska et al., 2006). Briefly, anesthetized animals were perfused and the brains were trimmed to produce sections. They were postfixed with 2.5% glutaraldehyde for two hours, washed with 0.1 M PBS, and then exposed to 1% osmium tetraoxide for two hours. After washing for several times, the tissues were dehydrated with gradient alcohol and embedded in Epon resin. Randomly selected ultrathin sections were stained with uranyl acetate and lead citrate and observed using a transmission electron microscope (H-7650, HITACHI,

ACCEPTED MANUSCRIPT Tokyo, Japan). Astrocyte gap junctions are observed at the interface between neighboring astrocytes.

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Therefore, two adjacent astrocytes were first located and pictures of the two close

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membranes were taken. Then, at a magnification of 100000×, the integrity of the structure was evaluated and the width of gap was randomly measured at six points with

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MetaMorph software. Four pairs of astrocytes in each rat were analyzed (n = 6).

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Western blotting

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The PFC region and hippocampus were dissected and homogenized in lysis buffer.

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Protein concentration was determined by bicinchoninic acid protein assay. A total of 15

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μg of proteins for each sample was separated by SDS-PAGE, and then transferred to PVDF membrane (Millipore, Temecula, CA). The membrane was blocked with 3%

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BSA and incubated in primary antibody overnight at 4 ℃ (anti-Cx43, CST, Danvers, MA, 1:1000; anti-β-actin, sigma, 1:10000; anti-GR, santa-cruz, 1:500; anti-BDNF, santa-cruz, 1:1000; anti-FKBP5, abcam, 1:1000) followed by horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 5000; KPL, Gaithersburg, MD). The protein bands were detected using enhanced chemi-luminescence. Densitometric analysis of immunoreactivity for each protein was conducted using Gel-Pro Analyzer software (Media Cybernetics).

Assessment of corticosterone levels in blood plasma

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After the behavior tests, blood collection was taken under chloral hydrate anesthesia

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(400 mg/kg, i.p.) from orbital blood as quickly as possible, normally, it took within 10

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seconds for each animal. Then the animals were sacrificed and specific brain regions were obtained. Blood was centrifuged at 5000 g for 10 minutes. The supernatant was collected and kept at -80℃. This assay was performed using the corticosterone EIA Kit

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(Cayman chemical, Ann Arbor, MI). Samples were diluted 1:1000 in EIA buffer and

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incubated in triplicate with antiserum and AchE tracer for two hours at room temperature. After washing, Ellman's reagent was added to the plate and incubated in

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the dark for 1.5 hours at room temperature. The absorbance was measured at 412 nm on

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a Mul-tiskan JX (Thermo Labsystem, W altham, MA). Plasma concentrations of

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corticosterone were calculated using a linear equation, derived from logit transformation of the absorbance and log concentrations of the standards. Intra-assay

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variation was less than 15% for all samples.

Statistical analysis

Data are expressed as mean±SEM. Differences among experimental groups were determined by two-way ANOVA followed by Bonferroni post-hoc test or a Student‟s t-test. The level of statistical significance was set at p < 0.05.

Results

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Behavioral changes after CMS

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In the present study, CMS-stressed animals gained less weight than their control rats. The weight of ADX rats decreased significantly after CMS exposure. Similarly, CMS also significantly decreased the weight of rats in the sham group (CMS, F(1, 44) =

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84.13, p < 0.001, η2 = 0.73; ADX, F(1, 44) = 58.04, p < 0.001, η2 =0.65; interaction, F(1,

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44) = 7.09, p = 0.01, η2 =0.19; see Fig. 2e).

In sucrose preference test, we found that CMS-exposed sham rats, when compared

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with unstressed sham rats, exhibited a reduction in the ratio of sucrose solution to total

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liquid consumed, an indication of anhedonia behavior. However, CMS-exposed ADX

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rats, when compared with ADX rats, did not show significant difference in the ratio of sucrose solution to total liquid consumed. We also found that CMS had a different effect

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on CMS-exposed sham and CMS-exposed ADX group (CMS, F(1, 44) = 15.67, p < 0.001, η2 = 0.38; ADX, F(1, 44) = 0.0006, p = 0.98, η2 < 0.01; interaction, F(1, 44) = 18.51, p < 0.001, η2 = 0.42; see Fig. 2a). In addition, there is no significant difference among groups in total fluid consumption for the 1h test (data not show). In the FST, the duration of immobility was significantly longer in the CMS-exposed sham rats than that in the sham group. However the difference between ADX and CMS-exposed ADX rats was not significant. CMS-exposed sham rats spent more time in an immobile state compared with CMS-exposed ADX rats (CMS, F(1, 44) = 37.23, p < 0.001, η2 = 0.47; ADX, F(1, 44) = 95.69, p < 0.001, η2 = 0.70; interaction, F(1, 44) =

ACCEPTED MANUSCRIPT 103, p < 0.001, η2 = 0.71; see Fig.2b). The activities in the open field were not significantly changed, which were indicated

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by parameters in locomotor activity (CMS, F(1, 44) = 1.56, p = 0.22, η2 = 0.04; ADX,

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F(1, 44) = 0.01, p = 0.93, η2 < 0.01; interaction, F(1, 44) = 0.20, p = 0.66, η2 < 0.01; see Fig. 2c) or rearing activity (CMS, F(1, 44) = 1.8, p = 0.19, η2 = 0.04; ADX, F(1, 44) = 3.62, p = 0.06, η2 = 0.09; interaction, F(1, 44)= 0.01, p = 0.94, η2 < 0.01; see Fig. 2d).

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Weight-loaded forced swimming was conducted to test the ability of antifatigue of

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rats to exclude the influence of ADX surgery. Also it is a useful method to evaluate the extent of fatigue and energy metabolism in rats (Masaaki et al, 2003). The strength of

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rats from groups exposed to CMS decreased significantly, but no difference between

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CMS-exposed sham and CMS-exposed ADX was shown (CMS, F(1, 44) = 12.21, p <

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0.01, η2 = 0.29; ADX, F(1, 44) = 0.10, p = 0.75, η2 < 0.01; interaction, F(1, 44) = 0.10, p = 0.75, η2 < 0.01; see Fig. 2f). This data indicated that the antifatigue ability decreased

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to the same level in rats from CMS-exposed sham and CMS-exposed ADX groups and the surgery had no effect on the rat‟s strength. Meanwhile, the extent of fatigue in the stressed rats was higher than that in the non stressed correspondings. This result indicated that CMS caused energy metabolism dysfunction in rats. Previous reports (Warner-Schmidt et al, 2007) have confirmed that CMS exposure increased the latency to feed in a novel environment, an indication of increased anxiety levels. Intruigingly, both CMS exposed groups showed anxiety-like behaviors by demonstrating that the latency to feed increased significantly, when compared with their corresponding control groups, respectively (CMS, F(1, 44) = 11.86, p < 0.01, η2 = 0.37;

ACCEPTED MANUSCRIPT ADX, F(1, 44) = 0.29, p = 0.60, η2 = 0.01; interaction, F(1, 44) = 0.25, p = 0.62, η2 = 0.01; see Fig. 3a), and food consumption was not significantly different (see Fig. 3b).

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For further confirming, we conducted EPM test to observe the anxiety-like behaviors.

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The data indicated decreased time in the open arms (CMS, F(1, 44) = 403.6, p < 0.001, η2 = 0.92; ADX, F(1, 44) = 0.62, p = 0.44, η2 =0.02; interaction, F(1,44) = 0.37, p = 0.55, η2 = 0.01; Fig. 3c), as well as the entry number in CMS-exposed sham and

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CMS-exposed ADX rats as shown in Figure 2 (CMS, F(1, 44) = 202.9, p < 0.001, η2 =

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0.85; ADX, F(1, 44) = 3.32, p = 0.08, η2 = 0.08; interaction, F(1, 44) = 1.33, p = 0.26, η2 = 0.04; see Fig. 3d). Also, food consumption and survival rate were measured during

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the weeks, which suggest that this manipulation could be used in this experiment (see

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Fig. 3e; 3f).

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Plasma CORT concentration of rats after exposure to CMS

To elucidate the molecular basis of these behavioral changes, we first examined the CORT concentration in plasma of all groups. As expected, there was a significant increase in plasma CORT concentration in the CMS+sham group versus the sham, while there was no such phenomenon seen in two ADX groups (CMS, F(1, 20) = 308.8, p < 0.001, η2 = 0.97; ADX, F(1, 20) = 407.8, p < 0.001, η2 = 0.97; interaction, F(1, 20) = 314.3, p < 0.001, η2 = 0.97; see Fig. 4). This data indicated that the surgery is successful to control the level of corticosterone .

ACCEPTED MANUSCRIPT Protein expression in the PFC and hippocampus after CMS

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Next, we measured the expression of GR、FKBP5 and BDNF in the PFC and

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hippocampus, respectively. Representative western blots of these molecules were shown in Fig. 5, 6, 7. The expression of GR was decreased in both the hippocampus and PFC of CMS-exposed group（Fig. 5a, CMS, F(1, 20 ) = 16.45, p < 0.01, η2 = 0.67; ADX, F(1,

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20) = 2.49, p = 0.15, η2 = 0.24; interaction, F(1, 20) = 0.46, p = 0.52, η2 = 0.05; Fig. 5b,

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CMS, F(1, 20 )= 12.93, p < 0.01, η2 = 0.62; ADX, F(1, 20) = 2.23, p = 0.17, η2 = 0.22; interaction, F(1, 20) = 0.30, p = 0.60, η2 =0.04）, while ADX rescued the GR expression

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in CMS-exposed ADX group compared with CMS-exposed sham group (Fig. 5a, p <

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0.05, Cohen‟d = 2.84; Fig. 5b, p < 0.01, Cohen‟d = 4.3, by t-test). FKBP5 expression

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was increased in both the PFC and hippocampus of CMS-exposed sham groups. Similarly, no such changes was found in CMS-exposed ADX group (Fig. 6a, CMS, F(1,

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20) = 10.08, p < 0.05, η2 = 0.56; ADX, F(1, 20) = 5.80, p < 0.05, η2 = 0.42; interaction, F(1,20) = 17.13, p < 0.01, η2 = 0.68; Fig. 6b, CMS, F(1, 20 ) = 12.05, p < 0.01, η2 = 0.6; ADX, F(1, 20) = 0.084, p = 0.78, η2 = 0.01; interaction, F(1, 20) = 6.184, p < 0.05, η2 = 0.56). CMS had a down-regulation effect on BDNF expression of both ADX+CMS and sham+CMS in the hippocampus (Fig. 7a; CMS, F(1, 20 ) = 26.71, p < 0.001, η2 = 0.77; ADX, F(1, 20) = 0.06, p = 0.81, η2 < 0.01; interaction, F(1,20) = 0.64, p = 0.45, η2 = 0.07), and PFC (Fig. 7b; CMS, F(1, 20 ) = 39.12, p < 0.001, η2 = 0.6; ADX, F(1, 20) = 0.32, p = 0.59, η2 = 0.03; interaction, F(1, 20) = 0.22, p = 0.65, η2 = 0.04). The results indicated that CMS impaired the expression of GR and FKBP5, and that these

ACCEPTED MANUSCRIPT disabilities were blocked by manipulating CORT level through ADX. However, CMS induced dysfunction on BDNF was not reversed by ADX. These data suggest that (1)

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decreased GR expression and increased FKBP5 expression were involved in depression

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induced by hypersecretion of corticorsterone, (2) BDNF down-regulation in the hippocampus and PFC in both stressed groups indicated that other mechanisms independent of corticosterone induced by stress exist in neuropsychiatric illnesses like

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anxiety disorder.

Ultrastructural alteration of astrocyte gap junction and changes of Cx43 induced by

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CMS in the PFC

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Growing evidence indicates that the pathophysiology of glia, especially astrocytes, contributes to the pathophysiology of mood and anxiety disorders (Banasr, 2010).

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Astrocyte is a supplier of metabolites for neurons to sustain their oxidative metabolism and also astrocytes act as glutamate producers for neurons (Leif Hertz et al., 1999). It is reported that astrocytes are organized as networks and communicate through specialized channels, the so-called gap junctions (Giaume et al., 2010). Thus, damage in these channels might contribute to the overall decline of astrocytic function. Ultrastructural study has provided strong support for the generally accepted view that astrocytes are extensively coupled to form what are defined as „functional syncytia‟ (Nagy et al., 2000). Detailed ultrastructural examination by electron microscopy could clearly identify the cell types linked by gap junctions as well as astrocyte gap junctions

ACCEPTED MANUSCRIPT at the interface between neighboring astrocytes. It was observed that the cell body of astrocyte was swollen and that the integrity of the structure between adjacent astrocytes

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was damaged in both groups exposed to CMS (Fig. 8 ). The width of gaps was taken as

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an index. The gap between neighboring astrocytes in the PFC from both groups exposed to CMS was wider than that from corresponding non stressed groups (CMS, F(1, 20 ) = 684.6, p < 0.001, η2 = 0.97; ADX, F(1, 20) = 2.37, p = 0.14, η2 = 0.97; interaction, F(1,

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20) =1.95, p = 0.18, η2 = 0.97, see Fig. 8c). Meanwhile, significant decreases of total

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Cx43 protein levels were found in the PFC in CMS exposed groups (Fig. 8e, CMS, F(1, 20 ) = 17.82, p < 0.01, η2 = 0.69; ADX, F(1, 20) = 1.80, p = 0.22, η2 = 0.18; interaction,

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F(1, 20) =1.80, p = 0.32, η2 = 0.13). These results indicated that CMS impaired the

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structure of astrocyte gap junction were independent of the presence of circulating

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Discussion

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CORT.

Chronic stress has been implicated in the pathogenesis of depression. In the present study we demonstrated that sham animals exposed to CMS exhibited behavioral deficits in the tests measuring anhedonia, anxiety and helpless behaviors, as well as ultrastructural deficits of astrocytic gap junction and reductions of Cx43 in the PFC and BDNF expression in the hippocampus. Moreover, the expression of GR reduced significantly while FKBP5 increased in both the PFC and hippocampus. Intriguingly, the anhedonia, helpless behavioral disability, molecular alterations of GR and FKBP5

ACCEPTED MANUSCRIPT were blocked by surgically manipulating glucocorticoid level via ADX, but the deficits in anxiety behaviors, astrocytic gap junction and Cx43 persisted under chronic mild

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stress in the absence of adrenal activation.

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The CMS paradigm is an animal model of depression with high predictive and construct validity (Willner et al.,2005) and is thought to simulate stressful life events that promote the development of human depression, which produces behavioral and

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neuroendocrine outcomes similar to that observed in depressed patients (Willner et al.,

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1997). We confirmed that this stress sequence reduces sucrose preference, assessing the hedonic state of stressed animals (Willner et al., 1992). Moreover, we demonstrated that

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the same procedure induces anxiety-like behaviors, supported by increased latency to

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feed in NSFT and less time spent in the open arms in EPM test as well as the helpless

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behavior showing increased immobility time in the forced swim test (Jian et al., 2013). Major depression is a common psychiatric disorder with a complex and multifactor

include

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aetiology. Potential mechanisms associated with the pathogenesis of this disorder monoamine

deficits,

HPA

axis

dysfunctions,

inflammatory

and

neurodegenerative alterations (Zunszain et al., 2011). Except behavioral abnormalities with depression, moderately elevated CORT for a prolonged period is sufficient to induce cellular changes in the hippocampus that are prevented by chronic administration of antidepressants (Chaouloff et al., 2008; Lee et al., 2009; Murray et al., 2008). In line with these studies we demonstrated that the depression-like behaviors were blocked by manipulating glucocorticoid level by ADX. And this phenomenon confirmed that increased CORT after CMS exposure may regulate depression-like

ACCEPTED MANUSCRIPT behaviors including anhedonia and helpless behaviors. Interestingly, the anxiety-like behaviors were not blocked by ADX, which reminded us that anxiety-like behaviors

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were not directly linked with CORT exposure. Recent studies have provided the

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evidence that CORT injection induced helpless behavior in the forced swim test and anhedonia behavior in the sucrose preference test (Wu et al., 2013), but had no significant effect on anxiety in the open-field or social interaction tests (Gregus et al.,

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2005; Cathy et al., 1997) which is consistent with our results. However, some have

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reported that repeated CORT injection could induce anxiety-like behaviors (Denis et al., 2009). How to explain these contrary data? The possible explanation is that the

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relationship between glucocorticoid and anxiety is dependent on the methods, doses and

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period of the corticosterone administration. CMS with or without CORT elevation may

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trigger the same mechanism that lead to anxiety (Anacker, 2013; James, 1997). Since depression and anxiety share an underlying element of stress, it has a high rate of

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co-morbidity (Stephanie, 2007). While our data suggested differences between depression and anxiety in response to corticosterone. In order to clarify the molecular mechanisms underlying these phenomena, we also demonstrated the attenuated GR protein expression in both the PFC and hippocampus of CMS-exposed sham rats, and that this CMS-induced alteration was reversed by ADX. The involvement of GR down-regulation in the pathophysiology of depression has been documented in clinical and preclinical studies (Florian, 2000; Ridder et al., 2005; Ridder et al., 2012; Silverman and Sternberg, 2012). In our study another key molecule is FKBP5 that is important for HPA axis function and GR activity (Jakob et al., 2012).

ACCEPTED MANUSCRIPT The data showed alteration of FKBP5 after sham animals exposed to CMS. FKBP5 is a co-chaperone of hsp90, which regulates GR sensitivity, lowers the affinity for its ligand

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and reduces the efficiency of the nuclear translocation. Polymorphisms of the human

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FKBP5 gene that are associated with enhanced expression of the co-chaperone protein affect GR sensitivity, leading to increased GR resistance and decreased efficiency of the negative feedback control, resulting in a prolongation of the hormonal response

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following stress exposure (Binder et al., 2008; Binder et al., 2009; Tatro et al., 2009;

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Thompson and Katona, 2001; Kang et al., 2012). In line with these findings, we found that FKBP5 expression increased after CMS, which may disrupt HPA axis, and this

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increase was blocked by ADX in both the PFC and hippocampus. Thus, this evidence

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suggested that enhancement of FKBP5 resulting from the increased CORT exposure

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might mediate stress-induced anhedonia and helpless behaviors. However, our results revealed that CMS-induced CORT increase only regulated

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depression-like behaviors and manipulation of CORT level could not normalize behaviors of anxiety. Because the anxiety-like behavior, ultrastructural deficits of astrocytic gap junction, the reductions in Cx43 in the PFC or BDNF expression in the hippocampus was not reversed after ADX. Thus the anxiety-like behavior induced by CMS might be associated with astrocytic dysfunction and BDNF levels (Steven et al., 2013). Also, several studies linked metabolism disorder induced by astrocytic dysfunction as a mechanism underlying the pathophysiology of some psychiatric disorders (Dost, 1998). Since glia affect several processes including regulation of extracellular potassium, glucose storage and metabolism, and glutamate uptake, all of

ACCEPTED MANUSCRIPT which are crucial for normal neuronal activity (Dost, 1998). Aberrations in central metabolic function is a key component of anxiety disorder (Haim, 2005), and astrocytes

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are an important cell population contributing to brain metabolism and brain energy

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suply (Franziska, 2011). So the dysfunction of astrocytes could render brain vulnerable to psychiatric disorders including depression and anxiety. We also presented here that animals exposed to CMS showed higher level of fatigue and anxiety-like behaviors

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which suggested similarities in depression and anxiety. Furthermore, a damaged

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astrocytic ultrastructure, the so-called gap junction, was observed in the PFC, and it is reported that gap junctions have a vital action in the buffering of ions, long-range

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signaling, and exchange of small molecules in astroglial networks (Jian et al., 2011;

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Theis et al., 2005). Furthermore, astroglial gap junctionnal communications play an

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important role in allowing information processing and integration from a large number of neurons, as well as in providing metabolites to remote sites during high neuronal

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demand (Giaume et al., 2010), thus, stress could lead initially to the pathology of glial cells and consequently to the pathology of neurons. Our previous work has demonstrated that gap junction dysfunction has a role in the pathophysiology of depression (Jian et al., 2011), our results here indicate that it is not only associated with depression but also take a role in anxiety. Besides, we found that BDNF expression in the hippocampus and PFC was involved in anxiety-like behaviors. BDNF has been suggested to occupy a key place in regulation of neurogenesis and synaptic plasticity (Ronald et al., 2012; Cirulli et al., 2010). Increasing evidence supports a role for BDNF and neurogenesis in development

ACCEPTED MANUSCRIPT of anxiety (Steven et al., 2013; Roth and Sweatt, 2011; Gomez-Pinilla and Vayn-man, 2005). Changes in BDNF appear to be associated with increased anxiety behaviors

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(Berry et al., 2012). In line with these data, our results showed anxiety-like behaviors in

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ADX rats with the down-regulation of BDNF in the hippocampus and PFC after CMS, which revealed the stress-induced impairment in these brain structures. However, the significance that CMS reduced BDNF protein expression was independent of CORT in

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our study.

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How to explain this phenomenon? Glutamate excitotoxicity is likely involves in the mechanism of astrocytic dysfunction under chronic mild stress without a manipulation

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of glucocorticoid (Kenji, 2009). Glutamate is released from presynaptic neurons and

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taken up by surrounding astrocytes. And an increased levels of glutamate could be

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found after exposure to stress (Popoli et al., 2011; Mitani et al., 2006). Prolonged high levels of glutamate could exert great burden on astrocytes and consequently result in

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pathology to the astrocytes. Stress could lead initially to the pathology of astrocytes, and consequently to the reduction of BDNF as it is released by astrocytes (Bosch and Robberecht, 2008). However, other factors including neuroinflammation, oxidative stress, altered energy budget, and mitochondrial dysfunction cannot be excluded (Haim et al., 2005; Zunszain et al., 2011). What is more, since neither anxiety-like behaviors nor astrocytic dysfunction was dependent on glucocorticoid action, it is reasonable to posit the link between the two features. More direct work need to be done to validate this hypothesis. Our study is the first to demonstrate that anxiety-like behaviors but not

ACCEPTED MANUSCRIPT depression-like behaviors can be induced under a condition of CMS with glucocorticoid-controlled. This study suggests that corticosterone may impair proteins

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which regulates HPA axis that, in turn, impairs HPA axis function and consequently

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induce depression-like behaviors. On the other hand, CMS may damage astrocytic morphology and function as well as BDNF function which may trigger anxiety-like behaviors. However, astrocytic dysfunction and BDNF impairment seem to be common

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in psychiatric disorders, they are involved in many psychiatric diseases including

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depression and anxiety. Since depression disorder often has a high rate of comorbility with anxiety, it is reported that the onset of anxiety occurred before the onset of

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depression, and the fact that temporally primary anxiety significantly predicts the

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subsequently onset of depression (Kessler et al., 2001). Further, it is estimated that high

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concentration levels of cortisol are shown in 40-60% of severe depressive patients (Mitchel et al., 2009), but this phenomenon is less common in anxiety (Hesham et al.,

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2014). Our data has demonstrated that the glucocorticoid may not have direct relation with anxiety.

Conclusion

To link corticosterone exposure to the pathophysiology of major depression, we used CMS model in glucocorticoid-controlled rats to test depression-like and anxiety-like behaviors and related molecular changes. We have demonstrated that CMS induced

anxiety-like

behaviors

but

not

depression-like

behaviors

with

ACCEPTED MANUSCRIPT glucocorticoid-controlled. Stress may enhance FKBP5 expression and decrease GR expression via glucocorticoid exposure. Gap junction dysfunction and change in BDNF

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might be associated with anxiety-like behaviors. Therefore, our experiment illustrated

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that the depression-like behaviors were induced by the elevation of glucocorticoid through GR and FKBP5, while astrocytic dysfunction and BDNF impairment, which were related to depression, were the consequences of stress. Further work need to be

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Conflict of interest statement

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done to elucidate the mechanism underlying anxiety disorder.

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There is not any potential conflict of interest that we should disclose.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China Grants (No. 81274122, 81202507, , 81373998, U1402221), the National Mega-project for Innovative Drugs (2012ZX09301002-004, 2012ZX09301002-001), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (No. IRT1007), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20121106130001), Beijing Natural Science Foundation (7131013, 7142115), Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study ( BZ0150).

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Fig. 1. Diagram depicts time course and flow of experimental procedures, including

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initial surgeries (ADX or sham), initiation of CMS, behavioral testing and sampling. SPT: sucrose preference test; NSFT: novelty suppressed feeding test; FST: forced swim

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test; EPM: elevated plus maze; WLFS: weight-loaded forced swim.

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Fig. 2. Adrenalectomy (ADX) reversed the depressive-like behaviors induced by CMS in sucrose preference test (SPT) and forced swim test (FST) (n = 12). (a) Sucrose

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preference was decreased by 4 weeks of CMS exposure and was reversed by ADX. (b)

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Immobility time was increased in sham-exposed CMS and was decreased significantly

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in ADX-exposed CMS. (c) (d) there was no behavioral changes in locomotor activity or rearing activity in the open field test. (e) The body weight of rats in both groups

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decreased significantly after exposure to CMS. (f) CMS animals showed a decreased swimming time to the same level in the weight-loaded swim test. Error bars represent SEM. All values plotted are mean ± SEM. * denotes significance compared with control. ** p < 0.01, *** p < 0.001, ### p < 0.001, two-way analysis of the variance (ANOVA), Bonferroni post-hoc analysis.

Fig. 3. Adrenalectomy (ADX) can not normalize the anxiety-like behaviors after exposure to CMS in novelty suppressed feeding test (NSFT) and elevated plus maze (EPM) (n = 12). (a) Both ADX and sham showed a significant increase of latency to

ACCEPTED MANUSCRIPT feed after exposure to 4 weeks of CMS; (b) Food consumption of the rats during 5 minutes in all groups immediately after NSFT was shown. (c) (d) showed decreased

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time in open arms and fewer entry numbers into open arms respectively after exposure

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to CMS. (e) Food consumption was measured in home cage once a week during 4 weeks of CMS. (f) Survival curve was depicted during the period of CMS. Error bars represent SEM. All values plotted are mean ± SEM. * denotes significance compared

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with control. ** p < 0.01, *** p < 0.001, ### p < 0.001, two-way analysis of the

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variance (ANOVA), Bonferroni post-hoc analysis.

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Fig. 4. Plasma corticosterone (CORT) levels of all groups in rats after exposure to CMS.

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Each column is the mean±SEM of 6 rats per group. * denotes significance compared

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ANOVA).

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with their corresponding control (*** p < 0.001, ### p < 0.001，following two-way

Fig. 5. Modulation of GR protein levels by CMS and ADX in the rat brain (n = 6/group). ADX blocked the trend of attenuation in GR expression induced by 4 weeks of CMS in hippocampus (a) and PFC (b). The data expressed as a percentage of sham (set at 1). Columns and error bars represent mean±SEM. * denotes significance compared with their corresponding control. ** p < 0.01, # p < 0.05, ## p < 0.01, following two-way ANOVA.

Fig. 6. The protein levels of FKBP5 were determined by western blot. Upregulation of

ACCEPTED MANUSCRIPT FKBP5 after exposure to CMS and it was reversed by ADX. (a) Representative images of western blot of FKBP5 in hippocampus and quantification was performed underneath. (b)

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Representative images of western blot of FKBP5 in PFC and quantification was

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performed underneath. Columns and bars represent mean ± SEM, respectively (n = 6 for each condition ). * denotes significance compared with their corresponding control. * p <

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0.05, ** p < 0.01, # p < 0.05, ## p < 0.01, following two-way ANOVA.

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Fig. 7. The protein levels of BDNF were determined by western blot. Downregulation of BDNF both in sham and ADX after exposure to CMS. (a) Representative images of

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western blot of BDNF in hippocampus, quantification was performed underneath. (b)

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Representative images of western blot of BDNF in PFC, quantification was performed

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underneath. Columns and bars represent mean ± SEM, respectively (n = 6 for each condition ). * denotes significance compared with their corresponding control. *** p <

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0.001, following two-way ANOVA.

Fig. 8. Ultrastructural alterations of astrocyte gap junction and changes in Cx43 protein expression induced by chronic mild stress (CMS; n = 6/group). (a) The sections shown in the black square were cut from the brains of all groups. (b) The astrocyte of PFC showed swollen-like change after CMS. Scale bar = 5um. (c) The width of gap was significantly enlarged in both ADX and sham after CMS exposure. (d) Electron micrographs showing astrocytic gap junction in the PFC of each group. Astrocytic gap junctions are indicated by gray arrowheads. Scale bar = 500nm. (e) Western blot

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ACCEPTED MANUSCRIPT Highlight 1. Stress did not lead to depression without glucocorticoids elevation.

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2. Stress led to anxiety with glucocorticoid-controlled.

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3. BDNF reduction and astrocyte dysfunction were associated with anxiety.