Corticosterone mitigates the stress response in an animal model of PTSD

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Journal of Psychiatric Research xxx (2014) 1e11

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Journal of Psychiatric Research

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Corticosterone mitigates the stress response in an animal model ofPTSD

Min Jia, Stanley E. Smerin, Lei Zhang, Guoqiang Xing, Xiaoxia Li, David Benedek,Robert Ursano, He Li*

Department of Psychiatry, Center for the Study of Traumatic Stress, Uniformed Service University of Health Sciences (USUHS), 4301 Jones Bridge Rd.,Bethesda, MD 20814, USA

a r t i c l e i n f o

Article history:Received 18 July 2014Received in revised form16 September 2014Accepted 18 September 2014

Keywords:StressTraumatic stress responseCorticosteroneGlucocorticoidsAmygdalaHypothalamus

* Corresponding author. Tel.: þ1 301 295 3295; faxE-mail addresses: [email protected], [email protected]

http://dx.doi.org/10.1016/j.jpsychires.2014.09.0200022-3956/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Jia M, et aResearch (2014), http://dx.doi.org/10.1016/j.

a b s t r a c t

Activation of glucocorticoid receptor signaling in the stress response to traumatic events has beenimplicated in the pathogenesis of stress-associated psychiatric disorders such as post-traumatic stressdisorder (PTSD). Elevated startle response and hyperarousal are hallmarks of PTSD, and are generallyconsidered to evince fear (DSM V). To further examine the efficacy of corticosterone in treating hyper-arousal and elevated fear, the present study utilized a learned helplessness stress model in which rats arerestrained and subjected to tail shock for three days. These stressed rats develop a delayed long-lastingexaggeration of the acoustic startle response (ASR) and retarded body weight growth, similar tosymptoms of PTSD patients (Myers et al., 2005; Speed et al., 1989). We demonstrate that both pre-stressand post-stress administration of corticosterone (3 mg/kg/day) mitigates a subsequent exaggeration ofthe ASR measured 14 days after cessation of the stress protocol. Furthermore, the mitigating efficacy ofpre-stress administration of corticosterone (3 mg/kg/day for three days) appeared to last significantlylonger, up to 21 days after the cessation of the stress protocol, in comparison to that of post-stressadministration of corticosterone. However, pre-stress administration of corticosterone at 0.3 mg/kg/day for three days did not mitigate stress-induced exaggeration of the ASR measured at both 14 and 21days after the cessation of the stress protocol. In addition, pre-stress administration of corticosterone(3 mg/kg/day for three days) mitigates the retardation of body weight growth otherwise resulting fromthe stress protocol. Congruently, co-administration of the corticosterone antagonist RU486 (40 mg/kg/day for three days) with corticosterone (3 mg/kg/day) prior to stress diminished the mitigating efficacy ofthe exogenous corticosterone on exaggerated ASR and stress-retarded body weight. The relative efficacyof pre versus post administration of corticosterone and high versus low dose of corticosterone on stress-induced exaggeration of innate fear response and stress-retarded body weight growth indicate thatexogenous corticosterone administration within an appropriate time window and dosage are efficaciousin diminishing traumatic stress induced pathophysiological processes. Clinical implications associatedwith the efficacy of prophylactic and therapeutic corticosterone therapy for mitigating symptoms ofPTSD are discussed, particularly in relation to diminishing hyperarousal and exaggerated innate fearresponse.

© 2014 Published by Elsevier Ltd.

1. Objectives

Exposure to traumatic events alters the function of neuronalcircuitry in the prefrontal cortex, amygdala, hippocampus, and,particularly, the hypothalamic-pituitary-adrenal axis (HPA)(Adamec et al., 2005; Belda et al., 2008; Osterlund and Spencer,2011; Weiss, 2007; Yehuda, 1997). Enhanced plasma

: þ1 301 295 1536.(H. Li).

l., Corticosterone mitigates thjpsychires.2014.09.020

glucocorticoid concentrations have been observed in human sub-jects exposed to traumatic events (Resnick et al., 1997; Yehuda,2009). The extent and time course of plasma glucocorticoidelevation is dependent on the intensity and duration of the trau-matic stressor (Servatius et al., 1995, 2001). The elevation of plasmaglucocorticoid concentration may be salutary since lower baselinecortisol levels have been associated with a higher incidence of PTSD(Hauer et al., 2009).

In fact, several studies of PTSD patients do suggest that exoge-nously administered glucocorticoids diminish fear memoryretrieval and other traumatic stress associated behaviors and

e stress response in an animal model of PTSD, Journal of Psychiatric

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M. Jia et al. / Journal of Psychiatric Research xxx (2014) 1e112

symptoms (Aerni et al., 2004; Schelling et al., 2001; Zohar et al.,2011; Miller et al., 2011) and in animal models of PTSD (Cohenet al., 2008; de Quervain et al., 1998). The benifical effects of glu-cocorticoids in reducing PTSD associated symptoms have beenobserved in patients who received high doses of hydrocortisonefollowing treatment for septic shock and major surgery (Schellinget al., 2003, 2006). The efficacy of glucocorticoids in psychiatricconditions has been further observed in clinic trials (Aerni et al.,2004; Schelling et al., 2006; Weis et al., 2006). Further studiesdemonstrate that exogenous glucocorticoids can interfere with theretrieval of traumatic memories (de Quervain et al., 1998; deQuervain, 2008). In two double-blind, placebo-controlled studies,pre-administered glucocorticoids reduced phobic fear in subjectswith social phobia and spider phobia (Soravia et al., 2006).Furthermore, administration of hydrocortisone has been reportedin some studies to decrease re-experiencing and avoidance symp-toms in patients with PTSD or impaired retrieval of declarativememory (Aerni et al., 2004; de Quervain et al., 2000; de Quervainet al., 2003). Likewise, memory retrieval is diminished in a water-maze spatial task in corticosterone treated rats (de Quervainet al., 1998).

Patients with PTSD are a heterogeneous population withdifferent levels of trauma. PTSD symptoms may develop duringvarious time frames post exposure and patients may present fortreatment in various time frames after symptoms develop. Todetermine the efficacy of glucocorticoid in PTSD requires a popu-lation that is similar in its time prior to or post exposure and in thesame stage of disease development.

Experimentally, such a population can be found in the restraintand tail shock animal stress model which demonstrates both anexaggerated fear response e hyperarousal e one of the mostprominent symptoms of PTSD (Tomb, 1994) and HPA-axisdysfunction. Hyperarousal and HPA-axis dysfunction togethercomprise the closest model we have for simulating PTSD (Servatiuset al., 1995). In this animal model of PTSD, the onset of enhancedASR is not immediately observed but is delayed twoweeks from thestressor, similar to the delay of onset of some symptoms of PTSD(Andrews et al., 2007; Jia et al., 2012; Solomon et al., 1989). In priorstudies we addressed pharmacotherapy for PTSD in this stressmodel by examining the efficacy of a1-adrenoceptor and 5-HT2Areceptor antagonists in mitigating exaggeration of the ASR (Jianget al., 2009; Manion et al., 2007; Zhang et al., 2005). In the pre-sent study we continue along this line by evaluating the efficacy ofcorticosterone before and immediately after exposure to restraintand tail-shock. As before, exaggeration of the ASR is monitored. Incomparison with non-stressed control or stress-alone subjects,current results from this study demonstrate a differential efficacy ofcorticosterone (3 mg/kg/day and 0.3 mg/kg/day) administrationbefore versus immediately after three stress-exposures on themeasurements of innate fear (acoustic startle) response and gain ofbody weight 14 and 21 days after the stress protocol.

The neuronal mechanisms associated with the differential ef-fects of corticosterone administration pre and post-stress ondelayed, exaggerated fear response and bodyweight gain, as well asthe potential clinical implications for diminishing symptoms ofPTSD, are discussed.

2. Materials and methods

2.1. Experimental animals

Male Sprague-Dawley rats initially weighing between 80 and100 g (Taconic Farms, Derwood, MD, USA) were used. The animalswere equally assigned to each group based on their body weightand baseline startle response. Animals were housed two per cage in

Please cite this article in press as: Jia M, et al., Corticosterone mitigates thResearch (2014), http://dx.doi.org/10.1016/j.jpsychires.2014.09.020

a climate controlled environment with free access to food andwater, and were maintained on a 12 h reverse light/dark cycle(lights on 18:00) at 22 �C. All experimental procedures wereapproved by the Institutional Animal Care and Use Committee ofthe Uniformed Services University of the Health Sciences andconducted in accordance with their Guidelines and Regulations.

2.2. Acclimation

Animals were acclimated for three days to both the animal fa-cility and to the acoustic startle chamber. Three consecutive daysprior to the initial measurements animals were briefly handled inthe acoustic startle chamber for 5 min each day to acclimate them.

2.3. A baseline measurement

Body weight and acoustic startle response measurements weretaken one day before stress and/or other procedures as baselinemeasurements. Baseline body weights were 138 ± 7.3 g on average.Daily food consumption was measured. Since body weights be-tween control group and stress group were significantly differentafter stress, to exclude the effect of bodymass on food consumptionresults were expressed as food consumption (in mg) per gram ofbody mass.

2.4. Acoustic startle measurement

Acoustic startle response (ASR) measurement (Blaszczyk, 2003)was conducted with a Startle Response Acoustic Test System(Coulbourn Instruments, Columbus, Ohio, USA). This system con-sists of weight-sensitive platforms in a sound-attenuated chamber.The pressure against the platform due to the animal's movement inresponse to sound stimuli was measured as a voltage change by astrain gauge inside each platform and recorded as the maximumresponse occurring within 200 ms of the onset of the startle-eliciting stimulus (Jiang et al., 2011a). There were six types ofstimulus trials: 100 dB alone, 100 dB with pre-pulse, 110 dB alone,110 dB with pre-pulse, pre-pulse alone and no stimulus control.Each trial type was presented eight times. Trial types were pre-sented in random order to avoid order effects and habituation.Inter-trial intervals ranged randomly from 15 to 25 s. In the currentstudy the responses to 100 dB sound stimuli are presented. Amongthe eight trials only the maximum values were collected in theresults and finally adjusted with the animal body weight of thesame day to avoid the force difference due to different animal bodyweights on the platform, and adjusted with baseline. Animals weretested one day before stress or corticosterone as baseline readingand 0, 7, 14 and 21 days following the final day of the consecutive 3days of the stress or corticosterone.

2.5. Stress

Stress exposure consisted of a 2-h per day session of immobili-zationand tail-shocks for three consecutivedays. Stressingwasdonein the morning (between 0800 and 1200). Animals were restrainedby being immobilized in a ventilated plexiglass tube. Forty electricshocks (2e3 mA, 3s duration; Animal Shocker, Coulbourn In-struments, USA) were delivered to their tails at semi-random in-tervals of 150e210 s (Graphic State Notation software, HabitestUniversal Link, Coulbourn Instruments, USA) (Jiang et al., 2011a).

2.6. Chemicals

Corticosterone (Sigma) dissolved in 10% ethanol (3mg/kg/day or0.3 mg/kg/day) 30 min before or after stress was injected intra-


M. Jia et al. / Journal of Psychiatric Research xxx (2014) 1e11 3

peritoneally for each of three consecutive days with stress (stressplus corticosterone groups) or without stress (corticosterone alonegroup). The doses we used (3 mg/kg/day or 0.3 mg/kg/day) were inthe range of the plasma corticosterone concentrations produced bystress (Cohen et al., 2006; Kaouane et al., 2012; Resnick et al., 1997).

Ethanol (10%) as vehicle was injected intra-peritoneally 30 minbefore stress for each of three days of stress (in the stress plusvehicle group) or injected intra-peritoneally alone without stress(in the vehicle alone group). The volume and the concentration ofthe vehicle were kept the same at different doses of corticosterone.The corticosterone receptor antagonist Mifepristone (RU486)(Cayman Chemical Co.) (40 mg/kg/day) was dissolved in 20%ethanol and injected intra-peritoneally with corticosterone (3 mg/kg/day) for each day of three consecutive days 30 min prior tostress.

2.7. Data analysis

Analysis of variance (ANOVA) for repeated measures was per-formed on net weight gain and ASR with the factors of days, stressstatus and drug dosage. The one-way ANOVA Dunnett test andstudent t test (indicated in results) were used to assess significantpost-hoc differences in individual groups. Statistical analysis wasperformed using SPSS software (SPSS Inc., Chicago, IL, USA). Thedata were represented as mean ± S.E.M.

3. Results

3.1. Stress of restraint and inescapable tail-shock for 3 days induceda delayed and exaggerated acoustic startle response

Consistent with previous reports, stress induced a delayed andexaggerated ASR to the 100 dB sound stimulus measured on day 14and day 21 after cessation of the stress protocol. Compared withcontrol, the maximum value of the ASR to 100 dB sound stimuli,adjusted for body weight and baseline value, is significantlyenhanced in the stress group on day 14 (p < 0.01, t test) and day 21(p < 0.01, t test) but not on Day 7. The ASR value in the stress groupwas significantly reduced (p < 0.01, t test) (Fig. 1) immediately aftercessation of the stress protocol.

Fig. 1. Percentage of control (mean ± S.E.M.) of peak startle amplitude (100 dB, bodyweight adjusted, represented as % of the baseline ASR) for groups of stress and controlmeasured immediately after stress, 7, 14 and 21 days following stress. Asterisks (**)indicate significant differences between groups p < 0.01 (t test). Day-3: n ¼ 32 incontrol group and n ¼ 39 in stress group. Day 0: n ¼ 24 in both control and stressgroups. Day 7, 14 and 21: n ¼ 32 in control group and n ¼ 39 in stress group. Horizontalbar indicates the days of stress.


3.2. Corticosterone attenuated the stress induced exaggeration ofthe ASR to 100 dB sound stimuli

Corticosterone (3 mg/kg/day), whether injected 30 min beforestress or 30 min after stress, attenuated the exaggerated anddelayed ASR on day 14 after stress (p < 0.05, Fig. 2a), indicating theprophylactic efficacy of corticosterone administration at higherconcentration. On day 21, a significant reduction of the exaggera-tion of the ASR was still observed in the group of stress pluscorticosterone (3 mg/kg/day) injected 30 min before stress(p < 0.01, compared with stress alone) but not when injected afterstress, indicating that pre-stress treatment with 3 mg/kg cortico-sterone has a longer efficacy than post stress treatment. Thus, thetime frame for pharmacologic intervention is a critical determinantfor optimal efficacy. Corticosterone (0.3mg/kg/day) injected 30minbefore stress did not show the reduction in exaggeration of the ASRon day 21 after stress (p > 0.05) (Fig. 2b). These results indicate thatboth time window for the administration of corticosterone anddosage are critical determinants. Clinically, multiple applications oflower dosage have been demonstrated to reduce the symptoms ofPTSD (Aerni et al., 2004).

In an attempt to address whether the efficacy of corticosteroneis mediated through activation of the glucocorticoid receptor, theglucocorticoid receptor antagonist RU486 (40 mg/kg/day) wasadministrated with 3 mg/kg/day corticosterone prior to the stressprotocol. While the effect of corticosterone on the exaggerated ASRwas attenuated in the presence of RU486 on day 14 and 21, theattenuation was not significant (p > 0.05, compare either with thestress alone group or with the stress group receiving 3 mg/kg/daycorticosterone prior to stress).

3.3. Stress of restraint and inescapable tail-shock for 3 days resultedin a reduction of body weight

Animals during the three days of restraint and inescapable tail-shock ceased to gain body weight. Although post stress the weightgain of the stress group paralleled that of the control group andcorresponded to the pre-stress rate, the three day stress-inducedbody weight retardation was never compensated, as measured at21 days following cessation of the stress protocol (Fig. 3a). Student'st-test showed significant differences at each data point from day0 to day 21 after stress protocol (p < 0.01).

In the stress group during the days of stress, food consumptionwas significantly reduced (p < 0.01, t test) compared with that inthe control group (Fig. 3b), and remained lower after stress. In orderto exclude the impact of the difference of body mass in food con-sumption, we divided the food consumption by the individual an-imal's body weight. Fig. 3c shows that the calculated foodconsumption per unit body mass in the stress group was signifi-cantly lower than that in the control group during the 3 days ofstress (n ¼ 8 in each group, p < 0.01, t-test). After stress no signif-icant difference in food consumption per unit body mass wasobserved between the two groups (n ¼ 8 in each group, p > 0.05, t-test).

3.4. The effect of corticosterone on stress reduced gain in bodyweight

Measured at 21 days after the cessation of stress, the retardationof body weight by the stress protocol was mitigated in the grouppre-treated with corticosterone at 3 mg/kg/day (Fig. 4d). Bodyweight of groups injected with corticosterone alone or vehicle wasnot significantly different from the naïve control group over thedays coeval with the 21 days post stress of the stress group.(p > 0.05, Fig. 4a, b, c, d). Since body weight of groups treated with


Fig. 2. Mean ± S.E.M. of peak startle amplitude (body weight adjusted, represented as % of the baseline ASR) for groups of control, stress, stress plus corticosterone (3 mg/kg, 30 minbefore stress), stress plus corticosterone (3 mg/kg, 30 min after stress), stress plus corticosterone (0.3 mg/kg, 30 min before stress), stress plus RU486 and corticosterone (3 mg/kg,before stress), corticosterone (3 mg/kg), vehicle alone and stress plus vehicle, measured 14 (a) and 21 (b) days following treatments. n ¼ 32 control, n ¼ 39 stress, n ¼ 24stress þ corticosterone (3 mg/kg, before stress), n ¼ 16 stress þ corticosterone (3 mg/kg, after stress), n ¼ 8 stress þ corticosterone (0.3 mg/kg, before stress), n ¼ 8stress þ RU486 þ corticosterone, n ¼ 24 corticosterone, n ¼ 20 vehicle alone, n ¼ 16 stress þ vehicle. Asterisks (*) indicate significant differences between groups (p < 0.05) and (**)indicate p < 0.01 (versus stress).


high dose corticosterone (3 mg/kg/day) 30 min either before orafter stress, or low dose corticosterone (0.3 mg/kg/day) 30 minprior to stress was still retarded at day 1 and day 7 after treatmentcomparedwith naïve control (Fig. 4a and b, p < 0.01), themitigatingeffect of corticosterone on the stress retarded body weight had notappeared at these times and doses. In addition, body weight gainwas also not affected in the stress group pre-treated with RU486(40 mg/kg/day) and corticosterone (3 mg/kg/day) on day 1 and day7 after stress, indicating that pharmacological manipulation witheither agonist or antagonist of corticosterone has no immediateeffect on the body weight gain within the first 7 days after stress.

However, 21 days after stress the body weight gain of the groupsinjected with corticosterone 3 mg/kg/day, 0.3 mg/kg/day, or 3 mg/kg/day þ RU486 before stress was no longer differentiable fromnaïve controls. By day 14 after stress the body weight of thestressed animals receiving even the lower dose of corticosterone(0.3 mg/kg/day) prior to stress was no longer differentiable(p > 0.05) from that of naïve controls (Fig. 4c). Meanwhile, attwenty-one days after stress (Fig. 4d), a significant reduction inbody weight compared to naïve controls was still seen in the stressalone group (p < 0.01) and in the stressed groups injected withcorticosterone after stress.

Furthermore, while the body weight gains between the stressedgroup and stressed group pre-treated with high dose corticoste-rone (3 mg/kg/day) are significantly different (p < 0.05) on day 21,


the group pretreatedwith the lower dose of corticosterone (0.3 mg/kg/day) showed no significant difference compared to the stressgroup measured on day 14 and day 21 after the cessation of stress(p > 0.05). This lack of effect suggests that a low dose of cortico-sterone prior to stress treatment does not mitigate growth retar-dation after stress. Furthermore, in the stressed group pre-treatedwith RU486 and 3 mg/kg/day corticosterone no significant differ-ence appeared in comparison with the control group, stress alonegroup, and stress treated with corticosterone group (p > 0.05),indicating that mechanisms other than glucocorticoid mediatedmechanisms may be also involved in mitigating the retardation ofbody weight gain induced by the stress protocol.

Food consumption per unit body weight showed a significantreduction during stress in the stress group and the stress pluscorticosterone (3 mg/kg/day) administered 30 min prior to stressgroup compared with the control group (p < 0.01, ANOVA, Tukey),but did not show a significant difference either one week or twoweeks after stress (Fig. 5).

4. Discussion

The efficacy of administration of high concentration cortico-sterone on mitigating the enhanced fear response and reducedbody weight could be attributed to the signaling activation of bothmineralocorticoid (MR) and glucocorticoid receptors (GR), which


Fig. 3. (a) Effect of the stress protocol on net body weight gain. Means ± S.E.M of netweight gains of both control and stress groups of Sprague-Dawley rats in three days ofrestraint-tail shock (measured before stress, immediately following stress and 1, 7, 14and 21 days following stress). Asterisk (**) indicates significant differences betweengroups (**p < 0.01), (day e 7 control group n ¼ 25, stress group n ¼ 24; day e 3 controln ¼ 61, stress n ¼ 60; day 0 control n ¼ 53, stress n ¼ 52 stress; day 1 control n ¼ 52,stress n ¼ 52, day 7, day 14 and day 21 control n ¼ 61, stress n ¼ 60). Arrows indicatethe days of stress. (b) Average daily food consumption of control and stress animalsbefore stress, during stress, one week after stress and two weeks after stress (n ¼ 8 instress group, n ¼ 8 in control group, *p < 0.05 and **p < 0.01 compared to controlgroup, t test). (c) Unit body weight food consumption of stressed animals relative tothat in control animals (as 100%) (n ¼ 8 in stress group, n ¼ 8 in control group,**p < 0.01 compared to control group, t test).


mediate rapid non-genomic as well as slow genomic mechanisms.Neuronal tissue binding studies demonstrate that MR has a highaffinity binding site for glucocorticoids ranging from 0.1 to 10 nMconcentrations (Reul et al., 1990). As indicated in Figs. 2 and 4,corticosterone pre-stress administration at 0.3 mg/kg (lower dose),


which certainly results in corticosterone blood levels above 10 nM,has no significant long term impact on the exaggerated fearresponse measured by acoustic startle response and reduced bodyweight 14 days after stress. A blood level of CORT in even our lowconcentration corticosterone rats is certainly above 10 nM. Thus,activation of membrane and/or cytosolic MR alone is not sufficientto mitigate stress-induced enhanced fear and reduced body weight.In addition, in a current microarray study examining the geneexpression profile in predator-scents-stress exposure, corticoste-rone (25 mg/kg) post-stress treatment prevented anxiety and hy-perarousal measured by startle and elevated plus maze 7 day laterin both sexes of mice, supporting the observation (shown in Figs. 2and 4) in our rat model of PTSD that pre- and post-administrationof corticosterone are able to mitigate stress-induced anxiety ab-normality (Daskalakis et al., 2014).

4.1. Fear and arousal

Most of the current literature supports a therapeutic efficacy ofactivation of the glucocorticoid receptor in the relief of fearresponse in PTSD patients (de Quervain and Margraf, 2008; deQuervain, 2008; Hauer et al., 2014). Administration of cortisol re-sults in a significant reduction of PTSD symptoms in critically ill orinjured patients. From a clinical and animal morphologic studyZohar et al. demonstrated that early application of high dose hy-drocortisone significantly reduced the risk of the development ofPTSD (Zohar et al., 2011). In agreement, when we injected corti-costerone in rats before traumatic stress exposure the incidence ofdelayed and exaggerated fear response was reduced and there waseven an additional beneficial effect after stress e normalization ofweight gain.

In apparent contradiction with the above beneficial effect ofcorticosterone are a number of studies that have shown anincreased levels of hydrocortisone in plasma in humans with PTSD(Lemieux and Coe, 1995; Resnick et al., 1997) and in animal stressmodels (Richardson Morton et al., 1990; Servatius et al., 1995). Thatthe effect of corticosterone on stress symptomatology seems pos-itive under some conditions and negative in others may be at leastpartially explained by differences between studies in four di-mensions e (1) intensity of stress, (2) time of evaluation, (3)memory versus arousal, and (4) other agents possibly co-releasedwith corticosterone. We consider these points in order:

4.1.1. Intensity of stressAnimal experiments so far reported employed only foot shocks,

in small number (de Quervain et al., 1998, for instance, used threefoot shocks), or a relatively short period of restraint (Kaouane et al.,2012, for instance, used 15 min of restraint), with such stressincreasing the level of corticosterone in the plasma in about 30 min(de Quervain et al., 1998). However, the stress protocol used in ourcurrent experiment consists of 40 shocks delivered to restrainedrats for 120 min each day for three days. The stress protocol weemploy is clearly more repetitive, prolonged and intense than thatof either de Quervain's or Kaouane's group (de Quervain et al., 1998;Kaouane et al., 2012) and therefore expected to produce longerlasting neurobiologic alterations. Our findings to date (Braga et al.,2004; Jiang et al., 2011a, 2011b; Manion et al., 2007; Xing et al.,2011; Jiang et al., 2009) show that with this more intense stressparadigm the neuronal circuitry engaged in exaggerated fear isaltered for a period of at least weeks. In the current study our stressprotocol substantially altered both innate fear response and bodyweight growth. As shown in Fig. 1, the amplitude of the acousticstartle response is significantly reduced when measured immedi-ately after the termination of the stress protocol.


Fig. 4. The comparison of net body weight gains for groups of control, stress, stressplus corticosterone (3 mg/kg, 30 min before stress), stress plus corticosterone (3 mg/kg, 30 min after stress), stress plus corticosterone (0.3 mg/kg, 30 min before stress) andstress plus RU486 and corticosterone (3 mg/kg, before stress), corticosterone (3 mg/kg), vehicle alone, stress plus vehicle, 1(a), 7(b), 14(c) and 21(d) days following



4.1.2. Time of evaluationPTSD animal model studies in the literature evaluated the effect

of stress soon after the stress while the level of corticosterone re-mains high (de Quervain et al., 1998; Kaouane et al., 2012). Kaouaneet al., especially, found that either foot shock stress, systemic in-jection of corticosterone, or infusion of corticosterone in the hip-pocampus can induce PTSD-like memory impairments associatedwith neuronal activation within the hippocampal-amygdala cir-cuitry in a time frame of 24 h. Since the neurobiologic consequenceof traumatic stress can be delayed by weeks or more, we evaluatedASR two and three weeks after stress, at which point the level ofcorticosterone returns to baseline (Servatius et al., 1995). As shownin our current study (Fig. 2), administration of corticosteroneattenuated stress-induced startle exaggeration two weeks aftercorticosterone treatment and the beneficial effect remainedevident up to three weeks, indicating a delayed and long lastingefficacy of such therapy. So it is not unreasonable to expect that ourmeasurements of fear memory and innate fear response would bedifferent from those evaluated immediately after stress. Further-more, the exogenous pre-treatment and post-treatment withcorticosterone within the appropriate time window associatedwith traumatic events can mitigate the development of patho-physiological processes underlying the symptoms of PTSD. On theother hand, activation of the glucocorticoid receptor might exac-erbate the symptoms of already established PTSD under certainconditions. For example, the efficacy of bolus administration ofhydrocortisone (4 mg/kg) on combat-related PTSD symptoms wasexamined using a traumatic memory reactivation therapeuticregime (Suris et al., 2010). No significant improvement was found inthe ‘impact of event’ score report (IES-R) and symptom subgroupscores immediately after the therapy. In addition, the patients inthe group treated with hydrocortisone had a significantly higherquick inventory depression score (QIDS) immediately following thetherapy. But QIDS scores one week after the visit returned to thebaseline level with a significantly lower IES-R avoidance/numbingscores. These results show that while exogenous glucocorticoidinduced activation of the glucocorticoid receptor might transientlyexacerbate certain symptom of already established PTSD in thepresence of a higher concentration of cortisol (Suris et al., 2010),one week later a beneficial effect of hydrocortisone treatment canmanifest.

4.1.3. Memory versus arousalIt is the fear memory component rather than the arousal

component of PTSD that has mainly been dealt with in the litera-ture. For instance, rate of forgetting is generally a function of timesince the stress event (Wixted, 2004). In PTSD patients, traumaticmemories remain and are often recalled vividly even years after thetraumatic incident indicating that there is a failure of forgetting inPTSD patients. Systemic administration of corticosterone, evenbefore a traumatic event, could promote the forgetting of (deQuervain et al., 1998) or impair formation of (Kaouane et al.,2012) those traumatic memories underlying PTSD. Our test, ASR,is not so much a test of memory, but rather a test for hyperarousal

treatments. (a) n ¼ 52 control group, n ¼ 52 stress group, n ¼ 22stress þ corticosterone (3 mg/kg, before stress), n ¼ 16 stress þ corticosterone (3 mg/kg, after stress), n ¼ 8 stress þ corticosterone (0.3 mg/kg, before stress), n ¼ 8stress þ RU486 þ corticosterone (3 mg/kg, before stress), n ¼ 20 corticosterone (3 mg/kg), n ¼ 12 vehicle, n ¼ 8 stress þ vehicle. (b, c, d) n ¼ 61 control, n ¼ 60 stress, n ¼ 30stress þ corticosterone (3 mg/kg, before stress), n ¼ 24 stress þ corticosterone (3 mg/kg, after stress), n ¼ 8 stress þ corticosterone (0.3 mg/kg, before stress), n ¼ 8stress þ RU486 þ corticosterone (3 mg/kg, before stress), n ¼ 36 corticosterone, n ¼ 20vehicle, n ¼ 16 stress þ vehicle. Asterisks (*) indicate significant differences betweengroups (p < 0.05) and (**) indicate p < 0.01 (versus control group).


Fig. 5. Body weight adjusted daily food consumption (mg/gm body weight) in control,stress, stress plus corticosterone (3 mg/kg) administered 30 min prior to stress,corticosterone (3 mg/kg) groups during stress, one week after stress and two weeksafter stress (n ¼ 8 in all 4 groups) (*p < 0.05, **p < 0.01).


(Koch, 1999; Tomb, 1994) e a test of state. That de Quervain et al.and Kaouane et al. found exogenous corticosterone given poststress to be detrimental, while we found it to be beneficial, couldhave resulted from our looking at state of arousal while they lookedat fear memory (de Quervain et al., 1998; Kaouane et al., 2012).

4.1.4. Agents co-released with corticosteroneThat exogenous corticosterone mitigates the stress response

even though endogenous corticosterone is elevated by stress di-rects us to the realization that agents other than corticosterone risein response to stress. It may be that these co-released agents andnot corticosterone elevate fear and arousal. One such co-releasedagent is norepinephrine (Quirarte et al., 1998; Tanaka et al., 1991).In a memory paradigm based animal model studies of stress,glucocorticoid effects on stress memory have been demonstrated toinvolve noradrenergic activity (de Quervain et al., 2007; deQuervain et al., 2009; McGaugh, 2013; Roozendaal, 2002). Inagreement with our ASR results, several authors have found in theiranimal models that beta-receptor mediated facilitation of memoryby norepinephrine is suppressed by glucocorticoids (Borrell et al.,1984; de Quervain et al., 1998; Roozendaal et al., 2006). Suppres-sion of facilitation of memory requires that corticosterone risesbefore the enhancement of memory by norepinephrine (Joels et al.,2011; Schwabe et al., 2012). The importance of timing during thecooperation of these agents during stress is further discussed inSection 4.2.

In attempting to address whether the efficacy of corticosteronein attenuating exaggerated fear is mediated through glucocorticoidreceptor activation, the commonly used glucocorticoid receptorantagonist Mifepristone (RU486) (40mg/kg/day for three days) wasco-administrated with corticosterone (3 mg/kg/day for three days)in the stressed group. Although corticosterone significantly atten-uated stress-induced exaggeration of ASR when measured 14 daysand 21 days after the cessation of our stress protocol (Fig. 2a), co-administered RU486 diminished but did not significantly reversethis attenuation (p > 0.05, compared to either stress or stress pluscorticosterone 3 mg/kg/day).

It has been reported that RU486 can be a partial agonist ratherthan pure antagonist of glucocorticoid receptors in certain condi-tions, as it is when administered at a high dose (30mg/kg/day) for along period (14 days) in a genetically obese, diabetic rat (Havelet al., 1996). Although RU486 has no agonistic effect observed inhealthy human tissues, RU486 has been shown to have a partialagonist activity in human cancer cell lines (Fryer et al., 2000) and inprimary adrenal insufficiency subjects in vivo (Laue et al., 1988). In


addition, Golier et al. (2012) demonstrated that administration ofRU486 (600 mg/day) in treating combat related PTSD is associatedwith acute increases in cortisol and ACTH level. These results sug-gest that therapeutic administration of RU486 (600 mg/day) mayalso induce a secondary effect on the HPA axis to enhance gluco-corticoid release in addition to its primary antagonistic action onthe glucocorticoid receptor.

In summary most of the current literature supports a thera-peutic efficacy of activation of the glucocorticoid receptor in therelief of fear response in PTSD patients (Aerni et al., 2004; deQuervain and Margraf, 2008; de Quervain, 2008) and our resultsin animals concur. Corticosterone must be administered at the righttime window.

4.2. The timing of cortisol administration during processing of stress

Corticosterone/cortisol administration modulates memoryretention and retrieval and traumatic memory in animal and inhuman studies (de Quervain et al., 2000; de Quervain et al., 1998).The impaired retentionwas evident in the groupwith pretreatment30 min before retention testing or after footshock stress but noimpaired performance was observed when footshock stress wasgiven 2 min or 4 h before testing (de Quervain et al., 1998). Thesetime-dependent effects of stress on impairing retention perfor-mance is associated with circulating corticosterone levels at thetime of testing (de Quervain et al., 1998). In addition, repeated oraladministration of cortisol (10 mg) 1 h before stressor exposurereduces spider-induced phobic fear response (Soravia et al., 2006).In agreement with our ASR results shown in Figs. 2 and 4, corti-costerone administration 30 min before restraint and tail shock haslong lasting efficacy in mitigating stress-induced exaggerated fearon day 14 and retarded body weight gain on day 21 after thetermination of the stress protocol in comparison to the cortico-sterone treatment 30 min after the stress (Figs. 2 and 4). In a morerecent study using the predator-scent-stress exposure model, 1 hpost-stress corticosterone administration prevented anxiety andhyperarousal 7 days later, confirming that pharmacological inter-vention in a proper time window associated with a traumatic eventis the key to therapeutic efficacy (Daskalakis et al., 2014).

Using microdialysis, norepinephrine concentration wasobserved to increase up to three fold in the basolateral amygdaladuring footshock stress as well as during the restraint and tail shockstress, which is the same stress protocol as used in our currentstudy (Quirarte et al., 1998; Tanaka et al., 1991). Recent studies alsodemonstrated that glucocorticoids can interact with beta norad-renergic mechanisms inmemory retrieval in vivo (Roozendaal et al.,2004). The characteristics of timing related to the efficacy of thecorticosterone mediated effect on glutamatergic transmission wasfurther investigated at the cellular level in the in vitro brain slicepreparation demonstrating that evoked AMPA receptor mediatedEPSCs as well as miniature EPSCs were enhanced after corticoste-rone exposure approximately 3e4 h after treatment (Karst andJoels, 2005). In addition, that such corticosterone exposureinduced enhancement of glutamatergic synaptic transmission re-veals a gradual suppression of the amplitude of the EPSC after 4 htreatment (Joels et al., 2012b; Karst and Joels, 2005). Furthermore,both evoked NMDA and AMPA receptor-mediated synaptic trans-missionwere enhanced in a period of 1e4 h after stress (Yuen et al.,2011; Joels et al., 2012a). Viewing stress induced elevation of ASR asa measure of delayed fear memory, we hypothesize that in ourexperiments corticosterone acted by diminishing the action ofnorepinephrine. One locus of action is evidenced to be the baso-lateral amygdala. Pu et al. (2009), recording excitatory postsynapticfield potentials, and Liebmann et al. (2009), recording amplitude ofexcitatory postsynaptic currents from basolateral amygdala



neurons, found that the facilitation of glutamatergic synaptictransmission by the noradrenergic beta agonist isoproteranol wasdiminished by corticosterone, but only if the corticosterone wasapplied ahead (2e4 h) of the noradrenergic agonist (Pu et al., 2009).This 2e4 h time course corresponds to the half hour that we gavecorticosterone prior to stress, plus the two hours of the stressperiod. A plausible cellular mechanism underlying the interactionof corticosterone with noradrenergic signaling recently was alsodemonstrated by the enhancement of AMPA receptor currents bynorepinephrine via the cAMP signaling pathway in hippocampalneurons (Zhou et al., 2012). Corticosterone applied prior to theactivation of noradrenergic enhancement of memory consolidationin the amygdala circuitry may thus interfere with the traumaticstress-induced fear response in the current model of PTSD byaffecting noradrenergic mediation of glutamatergic transmission.

In addition, Osterlund and Spencer (2011) found that threehours of corticosterone pre-treatment reduced restraint-stressinduced c-fos gene expression in the anterior pituitary (Osterlundand Spencer, 2011). Their work also suggests that corticosteronepretreatment could modulate HPA axis stress reactivity via a pro-tein synthesis-dependent mechanism (Osterlund and Spencer,2011). Although the current stress paradigm is more severe thanthat described in Osterlund and Spencer's study, pre-activation ofglucocorticoid receptors in the brain is considered to dampen astress-induced hyperactivation of the HPA axis (Herman et al.,2005). In addition to the activation of glucocorticoid receptorsignaling, administration of corticosterone at 15 mg/kg and trau-matic stress can induce distinct changes in inflammatory andmetabolic pathway related gene expression profiles, including theNFkb, Bcl2, and TNF signaling pathways, which may preventanxiety-like behaviors through direct binding to glucocorticoidresponse elements (GREs) in the nucleus (Li et al., in press; Datsonet al., 2011; Gray et al., 2013). Therefore, corticosterone mediatedshort term and long term effects on neuronal functions appear to benecessary for optimal neurobiological adaptation to stressfulevents.

4.3. Multiple mechanisms in body weight loss by stress

In the current experiments, the growth of the rats exposed tothe three-day restraint/tail shock stress protocol ceased during thethree day stress period and their body weight remained at a lowerlevel than that of unstressed controls for at least 21 days followingstress. This is consistent with previous reports including ours(Braga et al., 2004; Manion et al., 2007; Jiang et al., 2009). Suchdecrement in growth has also been observed in animals experi-encing milder stress protocols (Chotiwat et al., 2010; Harris et al.,2002a; Smagin et al., 1999). Decrease in gain of body weight ispresent in patients with depression, which is highly comorbid withPTSD and may share some neurobiological mechanisms (Doniniet al., 2003; Eberly and Engdahl, 1991; Gazewood and Mehr,1998; Myers et al., 2005; Speed et al., 1989). Since lower bodyweight persists for weeks after cessation of stress (Harris et al.,2002b; Jiang et al., 2009), reduction in the decrement in growthconsequent to stress, as found in our animal model, could be usedas a sensitive indicator for evaluating the therapeutic efficacy ofpharmacological agents. In Fig. 3a, gain of body weight ceasedduring the stress period and remained lowafter the period of stress,without any trend towards restoration of normal body weight. Thebody weight of stressed animals remained low even up to day 21(Fig. 3a). According to “set-point theory”, after stress, food con-sumption should rebound and body weight should catch up tonormal (Harris, 1990; Harris et al., 2002a; Wilson and Osbourn,1960). In our experiments, stress seems to lower the “set-point”after stress for a long period of time. This long term depression of


the set point suggests that neuronal mechanisms are involved inthe long term depression of body weight by stress.

These neuronal mechanisms are proposed to be related toincreased energy expenditure and reduced food intake duringstress (Harris et al., 2002a, 2002b). Food consumption was, in fact,significantly decreased in our experiments during the days ofstress, both absolutely (Fig. 3b) and when normalized relative tobody mass (Fig. 3c). Food consumption in the stressed groupremained below controls after stress too (Fig. 3b). At that point thedecreased eating may be due to the decreased energy required tomaintain the lower body mass in the stressed group, since afterstress no significant difference was observed between stress andcontrol groups when food consumption was normalized to bodymass (Fig. 3c). The stressed rats immediately resumed food andwater intake after stress. Since food consumed per body mass wasnormal in the stress group post stress, metabolic rate maynormalize after stress. However, no additional food was consumedto make up the weight loss in the stressed group after stress.

Body weight loss during stress and the sustained lower bodyweight after stress may have different mechanisms. In mild stresssuch as restraint only, there is a small loss of body weight duringrestraint, but after restraint body weight soon rebounds (Harriset al., 1998, 2002a). But, as described above in more severe stressconditions, the body weight of stressed animals may never berestored to that of control animals. Therefore, in severe stress, alowering of the set point for body mass may take place and endure.

4.4. Corticosterone effects on stress induced body weight loss

Elevation of endogenous corticosterone during and after stressblocks the rectification of body weight. It is well established that ahigh level of plasma corticosterone in the periphery is catabolic,with an increase in glycogenolysis, conversion of protein and fat toglucose, and metabolism of the resulting free glucose. This catab-olism both releases energy for coping with stress and likely un-derlies the halting in the growth of the stressed rats. The plasmalevel of corticosterone was greatly enhanced immediately after thethree day stress protocol in the stress alone group (Jia et al., 2012),which indicates that the elevation of endogenous corticosteroneduring and after stress blocks the rectification of body weight thatwould otherwise occur after a period of hypophagia. Similarly,stressed animals that were administered corticosterone after eachof the three periods of stress did not normalize their body weight,even 21 days after stress (Fig. 4).

A different mechanism emerges when corticosterone isadministered before stress. When corticosterone is administered30 min before stress at 3 mg/kg/day, no significant difference wasfound in the body weight compared to control group whenobserved on day 21 after the termination of the three day stress.Thus while corticosterone injection prior to stress did not preventcessation of body weight gain during stress, it did block thelowering of the homeostatic set point for body weight by stress,ultimately leading to the normalization of body weight.

Both hormonal and neuronal mechanisms may be involved inthe body weight loss during stress and the post-stress reinitiationof weight gain. The neuropeptide corticotrophin-releasing factor(CRF), secreted from the parvocellular neurons of the para-ventricular nucleus (PVN) suppresses food intake and result in bodyweight loss (Smagin et al., 1999). In addition, third ventricle CRFreceptor antagonist administration prevents stress-induced weightloss (Smagin et al., 1999). Thus, homeostasis in food intake andenergy consumption is controlled at least in part by CRF mediatedneuroendocrine systems. Recently, stress-induced cessation ofbody weight gain and retarded body weight growth after stresswere found to relate with demethylation of the CRF gene in the



hypothalamus thus, increase CRF production (Elliott et al., 2010). Inparticularly, chronic social stress induced long-term demethylationof CRF genomic transcriptome sites in a subset of defeated micethat displayed social avoidance (Elliott et al., 2010). Futhermore,site-specific knockdown of CRF gene products attenuated stress-induced social avoidance indicating that chronic hyperactivationof the CRF system linked to stress-related emotional disorders suchas anxiety, anorexia nervosa and depression via epigenetic medi-ated limbic hyperthalamus adrenalin (LHPA) axis regulation. (Elliottet al., 2010).

Systemic injection of corticosterone in animal models results ina rapid rise of corticosterone concentration in brain (Pariante et al.,2004), as well as in blood (Dallman and Yates, 1969; Yehuda et al.,2006) which may induce the central release of CRF. Enhancedcentral CRF release would reduce food intake, corresponding to ourresults shown in Fig. 3b and c, as well as enhance catabolism in theperiphery. As shown in Fig. 3c, food intake resumed to the controllevel after the three day stress and the daily weight gain returned tocontrol level, suggesting that central CRF level and basal meta-bolism recovered after the three day stress. However post-stress, asshown in Fig. 3a, the stressed animals showed no tendency to makeup for the weight not gained during stress. One plausible mecha-nism explaining why the stressed animals did not make up weightis that the stress protocol altered the homeostatic modulation inthe central circuitry (Falkenstein et al., 2000; Heim et al., 2000;Liberzon et al., 1997; Yehuda et al., 2006). In particular, disruptionof the canonical CRF ��>ACTH ��> glucocorticoid �j�> CRFfeedback loop may prevent, as shown in Fig. 3c, the hyperphagiarequired to normalize bodyweight (Heim et al., 2000; Smagin et al.,1999).

Besides CRF, a player entirely outside of the glucocorticoidsystem, serotonin, may be involved in the set point for bodyweight. It has been demonstrated that the 5-HT2A receptor in-hibitor MDL 11,939 (Jiang et al., 2011a), like corticosterone, hasno acute effect on body weight during three day stress, buteventually normalizes body weight gain 21 days later. That theglucocorticoid antagonist RU486 diminished but did not signifi-cantly reverse the effect of corticosterone on stress inducedexaggerated ASR and body weight loss, and that low dose ofcorticosterone did not significantly reverse stress induced exag-gerated ASR, suggests that besides the glucocorticoid system,other mechanisms such as the previously reported 5-HT2A andCRF mediated systems may also be involved in stress inducedbody weight regulation and exaggerated ASR (Jiang et al., 2011a;Smagin et al., 1999; Jiang et al., 2009). The interactions amongthe different systems involved in stress induced pathologicalalterations require further investigation.

In conclusion our experiments show that corticosteroneadministration (3 mg/kg/day) 30 min prior to or after stress at-tenuates the enhanced ASR induced by three days of restraint andinescapable tail-shock. Administration of corticosterone (3 mg/kg/day) prior to stress also results in normalization of body weightmeasured 21 days after stress, although such a regime does notacutely affect the cessation of bodyweight gain during three days ofstress. Neuronal mechanisms may play an important role in thereversal of both enhanced ASR and body weight gain reductionobservedwhen corticosterone is administered immediately prior toor after stress. The possibility exists that corticosterone adminis-tration elevates brain as well as plasma concentrations of cortico-sterone, thereby alleviating the cascade triggered by stress thatotherwise results in hyperarousal. Corticosterone administrationmay thereby impede the development of key symptoms of PTSDand should be further considered as a possible preventive as well astherapeutic agent for traumatic stress-associated psychiatric dis-orders such as PTSD.


Role of funding source

This work was supported by Congressionally Directed MedicalResearch Programs (CDMRP), Award #W81XWH-08-02-006 to Dr.He Li and in part by the Center for the Study of Traumatic Stress(CSTS) at the Uniformed Services University of the Health Sciences(USUHS).

Contributors

HL was responsible for the design of the study, interpretation ofthe data, and editing the manuscript.

MJ did the experiments, participated in the data analysis andinterpretation, and the manuscript preparation.

SS, RU and DB revised the manuscript critically and addedimportant intellectual content.

GX, LZ, XL made contributions to biochemical measurement ofstress response and revised the manuscript critically for importantintellectual content.

Conflict of interest

All authors report no biomedical financial interests or potentialconflicts of interest.

Acknowledgments

The authors gratefully thank Dr. Cara Olsen, a professionalbiostatistician for the assistance on statistical analysis and Ms.Eleanore H. Gamble for technical help. The opinions or assertionscontained herein are the private ones of the authors and are not tobe construed as official or reflecting the views of the Department ofDefense or the Uniformed Services University of The HealthSciences.

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