Early life stress dampens stress responsiveness in adolescence: Evaluation of neuroendocrine reactivity and coping behavior

Accepted Manuscript Title: Early life stress dampens stress responsiveness in adolescence: evaluation of neuroendocrine reactivity and coping behavior Author: Young-Ming Hsiao Tsung-Chih Tsai Yu-Ting Lin Chien-Chung Chen Chiung-Chun Huang Kuei-Sen Hsu PII: DOI: Reference:

S0306-4530(16)30034-8 http://dx.doi.org/doi:10.1016/j.psyneuen.2016.02.004 PNEC 3203

To appear in: Received date: Revised date: Accepted date:

15-1-2016 3-2-2016 5-2-2016

Please cite this article as: Hsiao, Young-Ming, Tsai, Tsung-Chih, Lin, YuTing, Chen, Chien-Chung, Huang, Chiung-Chun, Hsu, Kuei-Sen, Early life stress dampens stress responsiveness in adolescence: evaluation of neuroendocrine reactivity and coping behavior.Psychoneuroendocrinology http://dx.doi.org/10.1016/j.psyneuen.2016.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ms. Ref. No.: PNEC-D-16-00031

Early life stress dampens stress responsiveness in adolescence: evaluation of neuroendocrine reactivity and coping behavior Running title:

ELS dampens stress responsiveness

Young-Ming Hsiaoa, Tsung-Chih Tsaia,b, Yu-Ting Lina,b, Chien-Chung Chena, Chiung-Chun Huanga, Kuei-Sen Hsua,b,* a

Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan b Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan *Correspondence Author: Kuei-Sen Hsu, Ph.D. Department of Pharmacology, College of Medicine, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan Tel: 886-6-2353535 ext. 5498 Fax: 886-6-2749296 E-mail: [email protected]

1  

Highlights 

 

ELS promotes the development of stress resistance in adolescence. ELS blunts acute stress-induced alterations of hippocampal synaptic plasticity. ELS-induced change in stress reactivity is related to elevated plasma corticosterone.

2  

Summary Stressful experiences during early life (ELS) can affect brain development, thereby exerting a profound and long-lasting influence on mental development and psychological health.

The stress inoculation hypothesis presupposes that individuals

who have early experienced an attenuated form of stressors may gain immunity to its more virulent forms later in life.

Increasing evidence demonstrates that ELS may

promote the development of subsequent stress resistance, but the mechanisms underlying such adaptive changes are not fully understood.

The present study

evaluated the impact of fragmented dam-pup interactions by limiting the bedding and nesting material in the cage during postnatal days 2-9, a naturalistic animal model of chronic ELS, on the physiological and behavioral responses to different stressors in adolescent mice and characterized the possible underlying mechanisms. We found that ELS mice showed less social interaction deficits after chronic social defeat stress and acute restraint-tailshock stress-induced impaired long-term potentiation (LTP) and enhanced long-term depression (LTD) in hippocampal CA1 region compared with control mice. The effects of ELS on LTP and LTD were rescued by adrenalectomy. While ELS did not cause alterations in basal emotional behaviors, it significantly enhanced stress coping behaviors in both the tail suspension and the forced swimming tests.

ELS mice exhibited a significant decrease in corticosterone response and

trafficking of glucocorticoid receptors to the nucleus in response to acute restraint stress. Altogether, our data support the hypothesis that stress inoculation training, via early exposure to manageable stress, may enhance resistance to other unrelated extreme stressors in adolescence. KEYWORDS: Early life stress; HPA-axis; Stress reactivity; Glucocorticoid receptor; Hippocampus; Mouse 3  

1. Introduction Stressful experiences during early life (ELS) can affect brain development, which could lead to a profound and long-lasting influence on cognitive and emotional functions, as well as on the susceptibility to developing psychopathology (Loman and Gunnar, 2010; Lucassen et al., 2013; Singh-Taylor et al., 2015).

To date, most

mechanistic studies have focused on determining the detrimental consequences of ELS on the development of stress-related disorders later in life (Brunson et al., 2005; Murgatroyd et al., 2009; Ivy et al., 2010), but relatively little is known about the adaptive changes in response to ELS that promote the development of stress resistance and successful psychological functioning (Lucassen et al., 2013). The idea that ELS may induce the development of subsequent stress resistance is consistent with the “stress inoculation hypothesis”, which posits that prior mildly stressful experiences can foster resilience to future stress or trauma (Eysenck, 1983). Retrospective human studies have reported that experienced survivors of floods or earthquakes exhibit lower anxiety and less depressed affect encounters with the same disasters than inexperienced counterparts (Norris and Murrell, 1988; Knight et al., 2000).

This hypothesis is also supported by prospective longitudinal studies in

nonhuman primates demonstrating that monkeys exposed to brief periods of maternal separation

stress

exhibited

hypothalamic-pituitary-adrenal

less

anxiety-related

(HPA)-axis

responses

behaviors to

and

subsequent

attenuated stressors

compared with unmanipulated control monkeys (Parker et al., 2004, 2006; Lyons et al., 2010).

Likewise, neonatal rats exposed to brief maternal separations (3 min per

day during the first three weeks of life) exhibited diminished emotionality and attenuated HPA-axis responses to stressors in adults (Levine, 1957, 2005). Other rodent studies, however, suggest a role for maternal care in the development of stress 4  

resistance, rather than stress exposure per se (Macrì and Würbel, 2006; Meaney, 2010). Indeed, diminished neuroendocrine stress responses were observed in unhandled offspring that naturally received higher levels of maternal care during developmental stages (Liu et al., 1997; Francis et al., 1999). In contrast, some studies have failed to observe a link between the adult offspring regulations and the levels of maternal care (Tang and Reeb, 2004; Tang et al., 2006; 2008). More recently, a novel mouse model for the study of stress inoculation hypothesis has been established using a modified chronic social defeat stress (CSDS) protocol (a 15-min session every other day for 21 days) (Brockhurst et al., 2015). This model has been consistently shown to stimulate corticosterone response and thereby enhance active stress coping behaviors; however, the mechanisms underlying such adaptive changes have not been resolved. Previous studies showed that dam-pup interactions in mice could be disrupted by limiting nesting and bedding material in the cage in a dose-dependent manner (Rice et al., 2008; Baram et al., 2012). Fragmentation of dam-pup interactions during the first postnatal week is sufficient to produce enduring neuroendocrine and behavioral changes (Rice et al., 2008).

Because the overall duration or quantity of dam-pup

interactions is not altered by limiting nesting and bedding material (Rice et al., 2008), this naturalistic mouse model of ELS would provide an advantage of examining the stress inoculation hypothesis without considering the impact of increased maternal care following brief intermittent separations.

Furthermore, insofar as very little is

known regarding the impact of ELS on the development of stress resistance in adolescence, we therefore used this fragmented maternal care model to investigate the influence of ELS on the physiological and behavioral responses to different stressors in the adolescent offspring and characterized the possible underlying mechanisms. 5  

Our results reveal that ELS alters HPA axis reactivity leading to diminished stress responsiveness and enhanced stress coping behavior in adolescence.

2. Material and methods 2.1. Animals Pregnant C57BL/6 mice (n =80 for control group and n = 82 for ELS group) were housed individually under controlled illumination (12-h light/dark cycle with lights off at 18:00 h) and ambient temperature (24  2C, 40% humidity), and had ad libitum access to food and water.

Pups were born on gestation days 19-21 and the

day of birth was termed postnatal day (P) 0. On P1, pups were removed from the nests and five healthy pups that included both genders (three males and two females) were randomly placed back with each dam.

All behavioral tests were performed

during the light cycle between 9:00 h and 15:00 h. 

At the day of testing,

experimental mice were transferred to the experiment room and allowed to acclimate allowed to acclimate for approximately 3 h before testing.

To avoid variability

caused by hormonal cycles in females, only male offspring were used in this study. To avoid litter effects, only one offspring per dam was used in each experiment. In addition, all mice were used only once for an individual behavioral task.

If any pup

died during the lactation period, all other offspring were sacrificed immediately and were not included in the study. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of National Cheng Kung University.

2.2. Adrenalectomy and corticosterone replacement Adrenalectomy (ADX) was performed via small bilateral dorsal flank incisions under 6  

isoflurane anesthesia as previously described (Chen et al., 2010).

ADX mice

received replacement corticosterone (10 µg/ml) in drinking water containing 0.9% saline immediately after surgery. Corticosterone was first dissolved in 100% ethanol and then diluted 1,000 times in drinking water containing 0.9% saline.

Mice were

used for experiments 1 week after surgery. Control mice underwent a sham surgery with the same surgical procedure as the ADX mice, except that the adrenal glands were not removed. corticosterone levels.

Successful ADX was verified by measurement of plasma Only mice with plasma corticosterone concentrations < 1

μg/dL were considered successful and included in data analysis.

2.3. Plasma ACTH and corticosterone assay Blood samples were collected into chilled tubes containing EDTA from the tail vein, centrifuged at 1,000 × g at 4°C for 15 min and the plasma was separated and stored at -80oC until analysis. Determination of adrenocorticotropic hormone (ACTH) and corticosterone levels in plasma was performed using commercially available enzyme immunoassay kits for the detection of mouse ACTH (catalog #EK-001-21, Phoenix Pharmaceuticals) and corticosterone (catalog #500655, Cayman Chemical) according to the manufacturer’s instructions.

All assays were carried out in duplicate and a

blind manner.

2.4. Early life stress procedure The limited nesting and bedding material paradigm was performed as previously described (Rice et al., 2008).

Briefly, two litters were randomly assigned to either

control or ELS group on P2.

Control dams with their pups were housed in

polycarbonate cages (28 × 15 × 15 cm) with standard amount of sawdust bedding and 7  

one square piece (5 × 5 cm) of felt-like nesting material (Nestlets, Indulab). For the ELS group, dams with their pups were placed in cages with limited nesting and bedding material, consisting of one half of one felt square.

The ELS cages were

fitted with a soft plastic mesh platform, layered approximately 2.5 cm above the cage floor, to allow collection of urine and dropping, as previously described (Brunson et al., 2005).

Because dams did not spend a significant amount of time in exploring the

plastic mesh platform, we suggest that equipping the cages with plastic mesh platform had no significant influence on the environmental conditions. All litters were left undisturbed during P2-P9, after which all dams were provided with normal bedding and nesting material.

Male offspring were weaned on P21 and group housed in 3-4

per cage until use.

2.5. Assessment and analysis of maternal behaviors Maternal behaviors were video recorded using a digital video camera under a low illumination (approximately 10 Lux) to minimize interference with normal cage activities and were analyzed by two independent observers  as previously described (Wang et al., 2011). Observers were trained to a high level of interrater reliability (>0.90). The observers quietly entered the room at least 5 min before the start of the recordings to allow the mice to habituate to their presence.

The duration of dam

presence within the nest area, where she was in contact with the pups, was evaluated two sessions per day in both the light (9:00 h) and dark (20:00 h) phases from P2 to P8.

Each maternal observation session consisted of 30 min and the dam-pup

interaction was scored every other minute, resulting in 15 1-min epochs per observation session. Within each epoch, the duration of dam’s licking (dam touched any part of the pup's body with her tongue) and grooming (dam handled the pup’s 8  

body with her forepaws or nose) of the pups and the number of sorties that the dam left the nest were recorded.

2.6. Elevated plus maze The elevated plus maze (EPM) test was performed as previously described (Pellow et al., 1985).

The plus-cross-shaped maze was custom-made of black Plexiglas

consisting of two open arms (25 × 5 × 0.5 cm) and two enclosed arms (25 × 5 × 16 cm) extending from a central square platform (5 × 5 cm) mounted on a wooden base raised 50 cm above the floor. Animals were placed on the center square platform facing an open arm and allowed to freely explore the maze for 5 min.

The apparatus

was illuminated with dimmed light (approximately 10 Lux). The behavior of the animals was video recorded and scoring was performed with the behavioral tracking system Ethovision (Noldus). The activity was evaluated based on the percentage of entries into the open arms, the number of total entries and the percentage of time spent in the open versus closed arms.

The apparatus was thoroughly cleaned with 70%

ethanol after each trial.

2.7. Open field test For the open field (OF) tests, mice were placed individually in the center of a test chamber to freely explore for 10 min under a low illumination (approximately 10 Lux). The test chamber consisted of a circular ground area (40 cm in diameter) with a 40 cm high wall set on a non-reflective white plastic base. The behavior of the animals was video recorded and scoring was performed with the behavioral tracking system Ethovision. The activity was evaluated based on time spent in the center zone and total distance traveled in the open field. 9  

The chamber was thoroughly

cleaned with 70% ethanol after each trial.

The percentage of time spent in the center

zone is defined as the percentage of time for the animals exploring the center 25% (20 cm in diameter) of the chamber. 2.8. Sucrose preference test The sucrose preference test (SPT) was performed as described elsewhere (Huang et al., 2012).

Briefly, mice were first habituated to consume water in the

two-bottle-choice paradigm for 3 days. At the beginning of the experiment, mice were housed individually in the test chamber identical to their home cage and water was deprived for 1 h to increase drinking behavior. In the test session, mice were provided access to two bottles with 1% sucrose solution and water, respectively, for 3 h.

The sucrose preference score was determined by dividing the volume of sucrose

consumed by the total liquid consumption.

2.9. Social defeat stress C57BL/6 mice were subjected to 10 days of CSDS as described elsewhere (Krishnan et al., 2007; Chen et al., 2015). During each defeat episode, a C57BL/6 mouse was introduced into the home cage of an unfamiliar and aggressive CD1 retired breeder mouse (4-6 months old; Charles River Laboratories) for physical encounter. Following a 10-min agonistic interaction, the aggressor and the test mouse were then housed in the same cage separated by a wire mesh partition for the next 24 h, which allowed continuous sensory contact without physical interaction.

The C57BL/6

mouse was housed in equivalent cages with members of the same strain.

The social

interaction test (SIT) was performed 24 h after the last training day by measuring the time spent in the interaction zone during the first 5-min (target absent) and second 5-min (target present) trials. Social approach-avoidance behaviors were recorded 10  

using a digital video camera and analyzed with the Ethovision tracking system. The interaction ratio was calculated as (time spent in the interaction zone in the presence of target)/(time spent in the interaction zone in the absence of target).

Because the

majority of control mice spent significantly more time to interact with a social target than with an empty target enclosure, an interaction ratio of one was set as a cutoff: mice with ratio < 1 were classified as susceptible subpopulation and those with ratio  1 were classified as resistant subpopulation (Krishnan et al., 2007; Chen et al., 2015).

2.10. Tail suspension test The tail suspension test (TST) was performed as described elsewhere (Huang et al., 2012). Mice were individually suspended by the tail to a horizontal ring-stand bar (distance from floor = 25 cm) using adhesive tape wrapped around its tail (1 cm from tip). Typically, mice displayed multiple escape-oriented behaviors interspersed with bouts of immobility as the session progressed. period of 6 min and were video recorded. individual animal was recorded.

The trials were conducted for a

The total duration of immobility for

Mice were considered immobile when they hung

passively and motionless.

2.11. Forced swimming test The forced swimming test (FST) was used to measure stress-coping behavior as described elsewhere (Varadarajulu et al., 2011). Mice were restrained in a Plexiglas tube for 15 min and then returned to their home cages for  1 h.

To investigate the

effect of acute restraint stress exposure on the coping behavior, mice were individually placed in Plexiglas cylinder (30 cm in diameter and 40 cm deep) containing 20 cm water (24.0 ± 1.0°C) and were video recorded for 5 min. Active 11  

(swimming, climbing and struggling) or passive (immobility) behaviors were scored with the behavioral tracking system Ethovision.

After the forced swim session, mice

were towel dried thoroughly and then returned to their home cages. The water was changed after each mouse.

2.12. Restraint-tailshock stress An acute unpredictable and inescapable restraint-tailshock stress paradigm was used as described elsewhere (Chen et al., 2010).

Briefly, mice were restrained in a

Plexiglas tube and exposed to 60 tailshocks (1 mA for 1 sec, 30-90 seconds apart). This stress protocol, adapted from the learned helplessness paradigm, has been shown previously to reliably induce behavioral and endocrine signs of stress (Kim et al., 1996; Yang et al., 2004). Unstressed control mice remained in their home cages. Stressed and unstressed animals did not have available food and water during the experiments.

2.13. Electrophysiological recordings For electrophysiological recordings, hippocampal slices were prepared as described  elsewhere (Chen et al., 2010). Briefly, mice were killed immediately after stress by decapitation under isoflurane anesthesia, and hippocampal slices (400 m thick) of the dorsal part were prepared using a Leica VT1200S vibrating blade microtome (Leica). The slices were placed in a storage chamber of artificial cerebrospinal fluid (ACSF) oxygenated with 95% O2/5% CO2 and kept at room temperature for at least one hour before recording. The composition of the ACSF solution was (in mM): NaCl 117, KCl 4.7, CaCl2 2.5, MgCl2 1.2, NaHCO3 25, NaH2PO4 1.2 and glucose 11. For recording, one slice was transferred to a submersion-type recording chamber 12  

continually perfused oxygenated ACSF at 32.0  0.5°C.

Extracellular field potential

recordings were performed using an Axoclamp-2B amplifier (Axon Instruments, Union City, CA). The responses were low pass filtered at 2 kHz, digitally sampled at 10 kHz, and analyzed using pCLAMP software (Version 8.0; Axon Instruments). Postsynaptic responses were evoked in CA1 stratum radiatum by stimulation of Schaffer collateral/commissural afferents at 0.033 Hz with a bipolar stimulating electrode.

The stimulation strength was set to elicit response for which the

amplitude was 30-40% of the maximum spike-free response.

Field excitatory

postsynaptic potentials (fEPSPs) were recorded with a glass pipette filled with 1 M NaCl (2-3 M resistance) and the fEPSP slope was measured from approximately 20-70% of the rising phase using a least-squares regression.

The LTP was induced

by high-frequency stimulation (HFS), at the test pulse intensity, consisting of two 1-second trains of stimuli separated by an intertrain interval of 20 sec at 100 Hz. The LTD was induced by low-frequency stimulation (LFS) delivered at 1 Hz for 15 min (900 pulses).   The magnitudes of LTP and LTD were averaged the responses recorded during the last 10 min of the recording and normalized to 10 min of baseline before LTP or LTD induction.

2.14. Assessment of neuroendocrine stress reactivity For assessing HPA-axis stress reactivity, mice were restrained in a Plexiglas tube for a period of 30 min and blood samples were collected from a tail nick immediately before and after stress exposure to determine plasma levels of ACTH and corticosterone.

To assess the effectiveness of HPA-axis negative feedback, a

dexamethasone suppression test was conducted.

Dexamethasone phosphate (0.1

mg/kg in 0.9% NaCl, D-1759; Sigma-Aldrich) or saline (0.9% NaCl) was  13  

subcutaneously injected 90 min prior to acute restraint stress for 30 min.

Low-dose

dexamethasone (0.1 mg/kg) was selected because previous studies have demonstrated that this dose effectively suppresses the HPA-axis activation in mice (Nollet et al., 2012).

The percentage suppression of plasma corticosterone levels induced by

dexamethasone injection was calculated by taking the mean plasma corticosterone levels in mice of corresponding group that received saline injections.

2.15. Western blotting Western blot analysis was used to investigate the expression of mineralocorticoid receptor (MR), glucocorticoid receptor (GR) and FK506 binding protein 51 (FKBP51) proteins.

The microdissected hippocampal CA1 tissue samples were lysed in

ice-cold Tris-HCl buffer solution (TBS; pH 7.4) containing a cocktail of protein phosphatase and proteinase inhibitors, and ground with a pellet pestle (Kontes glassware, Vineland, NJ). Samples were sonicated and spun down at 1,000 × g at 4°C for 10 min to obtain the pellet corresponding to the nuclear fraction. The supernatant obtained was centrifuged at 10,000 × g at 4°C for 15 min and the supernatant obtained correspond to the cytosolic fraction.

The purity of subcellular

fractions was confirmed by anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:10,000, catalog #GTX627408, GeneTex) or anti-histone H3 (1:10,000, catalog #GTX122148, GeneTex) antibodies for the cytoplasmic or nuclear compartments, respectively. Total protein content was measured using Bio-Rad Bradford Protein Assay Kit (Hercules).

The proteins in each sample were electrophoretically

separated in 10% SDS-PAGE gel. Following transfer onto nitrocellulose membranes, blots were blocked in TBS containing 3% bovine serum albumin and 0.1% Tween 20 for one hour and then blotted overnight at 4°C with antibodies that recognize MR 14  

(1:200, catalog #sc-11412, Santa Cruz Biotechnology), GR (1:200, catalog #sc-1004, Santa Cruz Biotechnology), FK506 binding protein 51 (FKBP51, 1:2000, catalog #sc-11518, Santa Cruz Biotechnology) and -actin (1:4,000, catalog #MAB1501, Millipore), respectively.

It was then probed with HRP-conjugated secondary

antibody for 1 h and developed using the ECL Plus™ immunoblotting detection system (Amersham

Biosciences),

according

to

manufacturer’s

instructions.

Immunoblots were analyzed by densitometry using Bio-profil BioLight PC software (Vulber Lourmat).

Only film exposures that were not saturated were used for

quantification analysis.

2.16. Corticosterone treatment Corticosterone was first dissolved in 100% ethanol and then suspended in sesame oil as described elsewhere (Huang et al., 2014). The dose of corticosterone (0.75 mg/kg) was chosen because this dose can cause a significant elevation of plasma corticosterone levels (approximately 80 g/dL) in ADX mice within 20 min after subcutaneous injection, we therefore used this dose to evaluate the role of corticosterone in mediating ELS effect.

2.17. Statistical analysis The results are presented as means  SEM. n.

Number of animals used is indicated by

For LTP experiments, statistical analysis was performed using the non-parametric

Mann-Whitney U test.

The significance of the difference between the groups was

calculated by one-way or two-way ANOVA with Bonferroni’s post hoc analyses or Student’s t-test where appropriate.   Probability values of p < 0.05 were considered to represent significant differences. 15  

3. Results 3.1. Reduced bedding and nesting material in the cage fragments dam-pup interaction We initially assessed the effects of reduced bedding and nesting material in the cage on the interaction of mouse dams with their pups.

Consistent with previous report

(Rice et al., 2008), the number of leaving the nest area (sorties) increased significantly in dams housed in ELS cages compared with those housed in standard cages (two-way repeated-measures ANOVA: F(1,70) = 154.7, p < 0.001; n = 6 mice in each group;  Fig. 1A). However, the total duration of dam’s licking and grooming pups was not affected by the altered cage environment (two-way repeated-measures ANOVA: F(1,70) = 0.04, p = 0.84 ; n = 6 mice in each group; Fig. 1B). Moreover, as shown in Fig. 1C, we found that mice exposed to ELS (n = 35) showed significantly less weight gain when compared with control mice (n = 20) over the time period examined (two-way repeated-measures ANOVA: F(1,1185) = 1180, p < 0.0001). These findings are in agreement with previous reports and support the view that, fragmented maternal care behavior is sufficient to provoke ELS in the offspring of mice (Rice et al., 2008; Baram et al., 2012).

3.2. ELS shows no effect on emotional behavior under basal conditions To assess whether our ELS paradigm has enduring effects on basal emotional state in adolescence, three classical experimental models were used to evaluate anxiety(EPM and OF) and depression-like behaviors (SPT), respectively. When examined during the adolescent period (aged between P32 and P35), we found no significant differences between control and ELS mice in all parameters assessed in the EPM (Fig. 2A-C), the OF test (Fig. 2D-F), and the preference of 1% sucrose solution (Fig. 2G). 16  

Control and ELS mice displayed comparable levels of anxiety-like behavior, exploratory drive, locomotor activity and depression-like behavior, when examined under basal conditions.

3.3. ELS mice exhibit less susceptibility to CSDS To examine whether ELS alters the vulnerability to stress in adolescence, we utilized CSDS, a mouse model of chronic stress with psychotic features of major depressive disorder and posttraumatic stress disorder (Krishnan et al., 2007). The experimental procedure is depicted in Fig. 3A. Consistent with previous report (Krishnan et al., 2007), based on the measure of social interaction ratio, control mice subjected to CSDS can be separated into susceptible and resistant subpopulations, in which susceptible (n =20) displayed significantly reduced social interaction compared with both naive (n =30) and resistant mice (n = 10) (one-way ANOVA: F(2,57) = 53.5, p < 0.0001; Fig. 3B). As shown in Fig. 3C, in ELS mice, we also found a significant effect of CSDS (one-way ANOVA: F(2,62) = 42.5, p < 0.0001; n =15 for resistant mice, n = 40 for naive mice and n = 10 for susceptible mice). Nevertheless, the proportion of susceptible mice to CSDS was significantly lower in ELS mice than in control mice (Chi-square test: 2 = 14.7, p < 0.0001; Fig. 3D).

3.4. ELS blunts acute restraint-tailshock stress-induced impairment of LTP and enhancement of LTD Stress priming was found to impair LTP but enhance LTD at hippocampal Schaffer collateral-CA1 synapses (Yang et al., 2004; Huang et al., 2005). We thus examined whether ELS may alter acute restraint-tailshock stress-induced modifications in hippocampal synaptic plasticity. The experimental procedure is depicted in Fig. 4A. 17  

In slices from nonstressed control mice, conditioning HFS induced a robust LTP of fEPSPs (50-60 min after HFS: 64.4  9.3%, n = 12 slices from 8 mice), whereas in slices from stressed control mice, the magnitude of LTP was significantly reduced (14.9  5.3%, n = 10 slices from 6 mice; p < 0.01; Fig. 4B and F). Consistent with our previous findings (Yang et al., 2005), LFS induced a reliable LTD in slices from stressed (50-60 min after the end of LFS: 21.9  4.8%, n = 12 slices from 7 mice; p < 0.05) but not in nonstressed control mice (6.4  3.4%, n = 9 slices from 6 mice; Fig. 4C and G). Notably, slices from ELS mice showed impaired LTP (t(17) = 3.3, p < 0.01) but enhanced LTD (t(11) = 2.6, p < 0.05) compared with slices from control mice (Fig. 4F and G). However, in contrast to the differences observed in control mice, there was no difference between nonstressed and stressed ELS mice in the magnitude of LTP (nonstressed mice: 32.6  4.8%, n = 15 slices from 11 mice; stressed mice: 33.4  5.9%, n = 11 slices from 7 mice; p = 0.09; Fig. 4D and G). Similarly, no significant difference was observed in LTD magnitude in slices from nonstressed and stressed ELS mice (nonstressed mice: 20.9  4.3%, n = 10 slices from 7 mice; stressed mice: 16.3  3.4%, n = 12 slices from 8 mice; p = 0.39; Fig. 4E and G). Because both ELS and acute restraint-tailshock stress paradigms used herein promote corticosterone release and elevated levels of corticosterone are an important determinant of the effects of stress on hippocampal synaptic plasticity (Xu et al., 1998; Yang et al., 2004), we therefore examined whether ELS may reset basal corticosterone to higher levels, which in turn, activate downstream signaling events, thereby blunting acute restraint-tailshock stress-induced modifications in hippocampal synaptic plasticity. To confirm the importance of corticosterone in mediating the effects of ELS on LTP and LTD, we performed the experiments in bilateral ADX mice. The experimental procedure is depicted in Fig. 5A. ADX mice had significantly reduced 18  

basal plasma corticosterone levels (0.6  0.2 g/dL, n = 11, p < 0.001) compared with sham-operated mice (7.6  1.6 g/dL, n = 11). As shown in Fig. 5B-E, ADX completely abolished the effects of ELS on LTP and LTD. Slices from ADX ELS mice showed no difference in the magnitude of LTP (49.8  5.7% of baseline, n = 12 slices from 7 mice, p = 0.82) when compared with slices from ADX control mice (48.2  4.2% of baseline, n = 6 slices from 4 mice; Fig. 5B and C). Furthermore, no significant LTD was observed in slices from both ADX ELS mice (7.6  3.5% of baseline, n = 7 slices from 5 mice, p = 0.89) and ADX control mice (8.9  4.6% of baseline, n = 6 slices from 5 mice; Fig. 5D and E). Although the results above delineate an important role for corticosterone in mediating the effect of ELS on LTP and LTD, it is not known whether ELS elevated basal corticosterone levels that may dampen responsiveness to subsequent restraint-tailshock

stress

treatment.

Because

subcutaneous

injection

of

corticosterone can mimic the effects of acute restraint stress on hippocampal synaptic plasticity (Huang et al., 2014), we replaced the restraint-tailshock stress protocol with corticosterone

injection

and

then

examined

the

effects

of

ELS

corticosterone-induced modifications of LTP and LTD in ADX mice. experimental design is shown in Fig. 6A.

on The

As shown in Fig. 6B, plasma

corticosterone levels in naive ADX mice reached a peak at 15 min and remained elevated until 45 min following subcutaneous injection of corticosterone (0.75 mg/kg). Moreover, we found that plasma corticosterone levels did not differ significantly at 20 min after injections between ADX control and ADX ELS mice received corticosterone (t(8) = 0.3, p = 0.77) or vehicle treatment (t(6) = 0.8, p = 0.46; Fig. 6C). We observed that the magnitudes of LTP were significantly lower in slices from ADX mice treated with corticosterone (ADX control: 21.5  6.5% of baseline, n = 7 slices 19  

from 5 mice; ADX ELS: 23.1  8.1% of baseline, n = 8 slices from 5 mice; p < 0.05) than from vehicle-treated ADX mice (ADX control: 49.9  6.2% of baseline, n = 6 slices from 4 mice; ADX ELS: 46.9  6.8% of baseline, n = 6 slices from 4 mice), whereas no significant differences were observed between ADX control and ADX ELS mice received corticosterone (t(8) = 0.14, p = 0.89) or vehicle treatment (t(6) = 0.33, p = 0.76; Fig. 6D and E).

Similarly, LFS induced a reliable LTD in slices from

ADX mice treated with corticosterone (ADX control: 27.5  5.7% of baseline, n = 7 slices from 5 mice; ADX ELS: 25.8  4.3% of baseline, n = 7 slices from 5 mice; p < 0.05) but not in slices from vehicle-treated ADX mice (ADX control: 4.4  3.8% of baseline, n = 6 slices from 4 mice; ADX ELS: 6.4  5.2% of baseline, n = 6 slices from 4 mice), whereas no significant differences were observed between ADX control and ADX ELS mice received corticosterone (t(8) = 0.24, p = 0.82) or vehicle treatment (t(6) = 0.29, p = 0.78; Fig. 6F and G). Taken together, these results suggest that corticosterone is the main mediator of ELS effects on the development of subsequent stress resistance.

3.5. ELS increases active coping in the TST and the FST We next examined whether ELS could lead to more active coping response when they are faced with aversive situations in adolescence.

In the first experiment, mice were

exposed to a 15-min restraint stress, returned to their home cage for 15 min, and then subjected to a 6-min TST (Fig. 7A). We found that ELS mice exhibited more active coping responses in the TST compared with control mice (t(10) = 3.0, p < 0.05). Restraint stress significantly increased immobility time in control mice (t(12) = 2.4, p < 0.05) but had no effect on ELS mice (t(8) = 0.64, p = 0.54; Fig. 7B). In another experiment, mice were exposed to a 15-min restraint stress, returned 20  

to their home cage for 15 min, and then subjected to a 6-min FST (Fig. 7C). We found that ELS mice again showed more active coping response in the FST compared with control mice.

Restraint stress significantly increased immobility time in control

mice (t(16) = 3.7, p < 0.01) but had no effect on ELS mice (t(18) = 0.52, p = 0.61; Fig. 7D).

We were somewhat surprised to find a significant decrease (p < 0.05) in

immobility time in ELS mice compared with control mice with or without restraint stress exposure. These results suggest that ELS promotes active stress coping in  adolescence.

3.6. ELS alters neuroendocrine stress reactivity Alterations in HPA-axis function and regulation are associated with stress resistance and vulnerability (de Kloet et al., 2005).

To determine whether ELS may result in

persistent altered HPA-axis function and regulation, we examined HPA-axis reactivity and corticosteroid receptor expression changes in response to restraint stress. Consistent with previous report (Rice et al., 2008), ELS resulted in persistent elevations in plasma corticosterone levels (7:00 hour: 24.8  3.3 g/dL; 19:00 hour: 52.3  4.5 g/dL, n = 8; p < 0.05) when compared with control group at the age of P32-P35 (7:00 h: 12.3  0.8 g/dL; 19:00 h: 27.9  2.1 g/dL, n = 8; Fig. 8A). As depicted in Fig. 8B, restraint stress for 30 min increased plasma ACTH levels in both control and ELS mice, and no significant difference was observed between control and ELS mice in ACTH response to restraint stress (two-way repeated-measures ANOVA: F(4,70) = 0.15, p = 0.96 ; n = 8 mice in each group; AUC inset, t(14) = 0.67, p = 0.51).

By contrast, ELS showed a trend towered attenuated corticosterone

response to restraint stress compared with control mice (two-way repeated-measures ANOVA: F(3,56) = 2.2, p = 0.10 ; n = 8 mice in each group; AUC inset, t(14) = 3.3, p < 21  

0.01).

At immediately following and 30 min after restraint stress, plasma

corticosterone levels were significantly lower in ELS mice than in control mice (Fig. 8C). The dexamethasone suppression test was also performed to evaluate the functional integrity of negative feedback control of the HPA-axis (Cole et al., 2000; Nollet et al., 2012). Dexamethasone (0.1 mg/kg) was  subcutaneously injected 90 min prior to acute restraint stress.

Both control and ELS mice responded

significantly to the injection of dexamethasone with clearly decreased plasma corticosterone levels both under basal conditions and during restraint stress (two-way repeated-measures ANOVA: F(3,56) = 0.45, p = 0.72 ; n = 8 mice in each group; AUC inset, t(14) = 0.62, p = 0.55; Fig. 8D). To isolate the degree of negative feedback inhibition, we subtracted the corticosterone responses to restraint stress in the absence from the presence of dexamethasone. We found that ELS mice had a small but significant decrease in the percentage of deduced corticosterone suppression induced by dexamethasone injection compared with control mice (t(14) = 2.5, p < 0.05; Fig. 8E). These results suggest that ELS attenuates neuroendocrine stress reactivity and the effectiveness of the negative feedback loop of the HPA-axis.

3.7. ELS blunts stress-induced nuclear trafficking of GR We next examined whether ELS could alter expression levels of GR, MR and FKBP51 [a protein that controls GR translocation to the nucleus (Schülke et al., 2010)] proteins.

As shown in Fig. 9A, we found no significant effects of ELS on GR (t(12) =

0.19, p = 0.85), MR (t(12) = 0.47, p = 0.65) and FKBP51 (t(12) = 0.87, p = 0.41) protein levels in hippocampal CA1 region. Finally, we examined whether ELS may alter stress-induced nuclear trafficking 22  

of GR. In control mice, restraint stress for 30 min elicited a significant increase in GR levels (t(10) = 3.4, p < 0.01) in the nuclear fraction.

In addition, the basal levels

of nuclear GR proteins were higher in ELS mice than in control mice (t(10) = 2.4, p < 0.05). In contrast to control mice, there were no significant effect of restraint stress on nuclear trafficking of GR (t(10) = 1.0, p = 0.33) in ELS mice (Fig. 9B), which is in line with dampened stress responsiveness observed in ELS mice.

4. Discussion The stress inoculation hypothesis posits that early life exposure to mildly stressful experiences can trigger adaptive processes that promote the development of subsequent stress resistance later in life (Eysenck, 1983).

The present study provides

novel evidence for attenuated hormonal and behavioral HPA-axis-mediated responses to subsequent stressors by ELS in mice.

We have demonstrated that reduced

bedding and nesting material in the cage during the early postnatal period, which fragments dam-pup interactions, results in profound and enduring changes in active coping behavior, HPA-axis reactivity and nuclear trafficking of GR in responses to stressors in the adolescent offspring (Table 1). In addition, we reveal for the first time that ELS blunts acute restraint-tailshock stress-induced impairment of LTP and enhancement of LTD at Schaffer collateral-CA1 synapses. Furthermore, our results suggest that ELS-induced change in stress reactivity is associated, at least in part, with the persistent elevation of basal plasma corticosterone levels, which is likely a consequence of the decrease in the negative feedback control of the HPA-axis. Our findings in mice coincide with the observations from primates indicating that brief daily maternal separation promotes stress resistance later in life (Levine and Mody, 2003; Parker et al., 2006; Lyons and Parker, 2007; Parker and Maestripieri, 2011; Lee et al., 2014).

We found that ELS decreased neuroendocrine reactivity to 23

 

acute stress and negative feedback regulation of the HPA-axis in adolescence. The same pattern of results was also observed in the pioneering work by Levine and colleagues showing that daily maternal separation for only 3 min per day during the first three weeks of life reduced stress-mediated responses in rats (Levine, 1957; Levine et al., 1957). However, since the observed early handling-induced increase in the levels of active maternal care (Liu et al., 1997), an alternative hypothesis, called the “maternal mediation hypothesis”, has been proposed to explain why neonatal manipulations reduce hormonal and behavioral HPA axis-mediated responses to stress in adult rats (Macrì and Würbel, 2006; Meaney, 2010).

Further independent

evidence for the maternal mediation hypothesis stems from a series of studies on individual differences in maternal care behavior of Long Evans rat dams and associated differences in stress responses in the adult offspring (Weaver et al., 2004, 2005; Champagne et al., 2008; Peña et al., 2013). Nonetheless, there is evidence supporting a dissociation in the effects of neonatal maternal separations on maternal care and the development of subsequent stress resistance in rats (Macrì et al., 2004), suggesting that the differences in the adult offspring’s stress responses induced by neonatal maternal separations cannot simply be explained by the variations in the levels of active maternal care alone (Macrì and Würbel, 2006).

Therefore, a more

promising way of reexamining these two hypotheses would be to induce ELS without increasing dam-pup interactions when the treated pups were returned to the dams. Unlike the commonly used early postnatal handling or maternal separation rodent models with highly artificial treatments, rearing mouse pups in stress-provoking cage environment would be a more natural way to induce long-lasting consequences of ELS without human handling. In accordance with what has been shown previously, the current mouse model of ELS exhibits a disruption in the quality of dam-pup 24  

interactions (measured by its continuity and consistency), with preserved total duration of maternal care (Rice et al., 2008; Baram et al., 2012). The lack of change in quantitative aspect of maternal care indicates that the development of stress resistance in our ELS mouse model is not associated with elevated levels of active maternal care.

These findings suggest that the quality, rather than the overall

quantity of maternal interaction, governs the generation of ELS in the pups (Rice et al., 2008). The fact that stress inoculation, rather than higher levels of maternal care, promotes the development of stress resistance in our current ELS paradigm suggests that this mouse model would be more appropriate for studying the mechanisms of stress inoculation training. Although our findings are consistent with previous work (Macrì et al., 2004; Macrì and Würbel, 2006), it should be noted that, in Macrì et al.’s study (2004), maternal behavior was scored at 3-min intervals for 1 h every third hour (a total of 160 samples per day), whereas dam-pup interaction was scored every other minute during two 30-min observation sessions (a total of 30 sample per day) in our current study.

Furthermore, we could not exclude the possibility that reduced

bending and nesting material in the cage has an influence on maternal corticosterone which may transfer to the offspring through lactation and adjust the developing phenotype (Casolini et al., 2007; Catalani et al., 2011;  Macrì et al., 2011). studies are required to test this possibility.

Further

Despite these findings, a novel

stress-inoculated mouse model has also been established recently using a modified CSDS protocol, providing further support for the stress inoculation hypothesis to build stress resistance (Brockhurst et al., 2015). Although it has long been recognized that stress can alter LTP and LTD induction in different subregions of the hippocampus (Kim and Diamond, 2002; Huang et al., 2005; Schmidt et al., 2013), much interest in the past decades has been focused on 25  

delineating cellular and molecular mechanisms underlying stress effects.

To the best

of our knowledge, this study is the first to demonstrate that ELS blunts the effects of acute stress on hippocampal CA1 LTP and LTD in the adolescent offspring. Our data support a model in which ELS leads to an enduring elevation of basal plasma corticosterone levels that occlude the responsiveness to acute stress treatment. Three lines of evidence support this conclusion.

First, ELS mice had blunted

corticosterone response to acute stress compared with control mice (Fig. 7C). Indeed, elevated level of corticosterone is an important determinant of the effects of stress on hippocampal synaptic plasticity (Xu et al., 1998; Yang et al., 2004). Second, ELS mice exhibited impaired LTP but facilitated LTD, and reducing corticosterone levels of ELS mice by ADX restored LTP and LTD to normal levels (Fig. 5). Third, corticosterone injections mimicked the effects of acute stress to impair LTP and facilitate LTD induction in both control and ELS ADX mice (Fig. 6D-G), suggesting that the mechanisms underlying the effects of corticosterone on hippocampal synaptic plasticity remain intact in ELS mice.

The action of ELS on

hippocampal synaptic plasticity is consistent with previous findings that hippocampal-dependent learning and memory (e.g. Morris water maze and novel object recognition) impairments occurred in adult ELS mice (Rice et al., 2008). We also demonstrate that ELS leads to more active coping behaviors after exposure to acute and chronic stressors.

We confirmed the existence of two animal

populations differing in their responses to CSDS (Krishinan et al., 2007) and found that ELS promoted the development of the resistant phenotype after CSDS (Fig. 3). Similarly, ELS mice exhibited more active coping behavior after acute stress exposure in both the TST and the FST (Fig. 7). Although control mice generally showed increased immobility after exposure to restraint stress, restraint stress did not further 26  

alter immobility in ELS mice.

Moreover, the lack of phenotypic differences between

control and ELS mice in anxiety- (the EPM and the OF) or depression-like behaviors (the SPT) suggest that more active coping behaviors observed in ELS mice are not related to the altered emotional-like state under basal conditions. It is intriguing that ELS led to a significant decrease in immobility in the FST compared with control mice (Fig. 7D). Although the precise underlying mechanisms remain unclear, it could be partly attributed to inadequate weight gain in ELS mice. In fact, it has been reported that body weight of animals has a significant effect on their behavior in the FST (Brenes et al., 2008; Bogdanova et al., 2013).

Because ELS did not alter

immobility in the TST or preference for sucrose solutions, it seems unlikely that ELS exerts an antidepressant-like effect in the FST. In accordance with previous findings in this ELS model (Rice et al., 2008), our data also indicate that ELS mice weighed less than control mice over the time period examined (Fig. 1C). Single prolonged maternal separation in mice has been shown to decrease plasma glucose and leptin levels (Schmidt et al., 2006). Because the total duration of maternal care was similar in the control and ELS mice, it appears unlikely that the observed decrease in weight gain in ELS mice is mainly mediated by the prolonged activation of metabolic signal such as hypoglycemia (Rice et al., 2008). Although we could not exclude the possibility that the body weight decrease is an important pathological marker and/or co-stressor factor, Rice et al. (2008) demonstrated that plasma corticosterone levels did not correlate with pup weight in this ELS model, suggesting that ELS affected stress hormone levels and body weight independently and plasma corticosterone elevations were not simply a consequence of reduced body weight.

Additional studies are needed to clarify this issue.

How can ELS promote the development of subsequent stress resistance? The 27  

literature suggested that ELS-induced persistent alteration in hippocampal GR levels, possibly via epigenetic mechanisms, is relevant for the development of stress resistance and vulnerability (Lucassen et al., 2013). In our current mouse model, ELS resulted in no significant changes in hippocampal GR protein (Fig. 9A), whereas, as reported previously, basal plasma corticosterone levels were still increased in the adolescent offspring (Fig. 8A), suggesting that the persistent elevation of corticosterone may be responsible for the effects of ELS on the development of neuroendocrine stress resistance.

Because we found no difference in either basal or

acute stress-induced  increases in plasma ACTH levels in ELS mice compared with control mice, it seems possible that a blunted corticosterone response to acute stress seen in ELS mice is partially attributed to decreased sensitivity of the adrenal to ACTH. Although chronic elevation of basal corticosterone levels may be relevant for decreased adrenal sensitivity to ACTH in ELS mice, additional investigations will be necessary to fully understand this process. In conclusion, our work supports the key prediction of the stress inoculation hypothesis that ELS promotes the development of subsequent stress resistance as well as stress coping behavior in adolescence and, more importantly, identifies specific mechanisms underlying such adaptive changes.

While this mouse model was

designed to mimic the aspect of human chronic ELS situation in which mother is present but unable to provide appropriate care (Lucassen et al., 2013), some developmental differences in rodent and human HPA-axis physiology must be considered.

Regardless of this limitation, such inoculation mechanisms may engage

stress responses in an appropriate context and endow the capacity for reducing the occurrence of allostatic load in response to environmental challenges later in life.

28  

Role of the funding source All funding sources had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Contributors Y.M.H., C.C.C., C.C.H. and K.S.H. designed experiments; Y.M.H., T.C.T. and Y.T.L. performed experiments; Y.M.H., T.C.T. and Y.T.L. analyzed the data; C.C.C., C.C.H. and K.S.H. wrote the paper.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements This work was supported by research grants from the National Health Research Institute (NHRI-EX104-10336NI), the Ministry of Science and Technology (MOST 104-2321-B-006-007 and 104-2811-B-006 -056), and the Ministry of Education (Aim for the Top University Project to the NCKU), Taiwan. We thank members of the Hsu’s laboratory and Drs. Hsueh-Cheng Chinag and Alice Y. W. Chang for helpful discussion and suggestions.

29  

References Baram, T.Z., Davis, E.P., Obenaus, A., Sandman, C.A., Small, S.L., Solodkin, A., Stern, H. 2012. Fragmentation and unpredictability of early-life experience in mental disorders. Am. J. Psychiatry 169, 907-915. Bogdanova, O.V., Kanekar, S., D'Anci, K.E., Renshaw, P.F. 2013. Factors influencing behavior in the forced swim test. Physiol. Behav. 118, 227-239. Brenes, J.C., Rodríguez, O., Fornaguera, J. 2008. Differential effect of environment enrichment and social isolation on depressive-like behavior, spontaneous activity and serotonin and norepinephrine concentration in prefrontal cortex and ventral striatum. Pharmacol. Biochem. Behav. 89, 85-93. Brockhurst, J., Cheleuitte-Nieves, C., Buckmaster, C.L., Schatzberg, A.F., Lyons, D.M. 2015. Stress inoculation modeled in mice. Transl. Psychiatry 5, e537. Brunson, K.L., Kramár, E., Lin, B., Chen, Y., Colgin, L.L., Yanagihara, T.K., Lynch, G., Baram, T.Z. 2005. Mechanisms of late-onset cognitive decline after early-life stress. J. Neurosci. 25, 9328-9338. Catalani, A., Alemà, G.S., Cinque, C., Zuena, A.R., Casolini, P. 2011. Maternal corticosterone effects on hypothalamus-pituitary-adrenal axis regulation and behavior of the offspring in rodents. Neurosci. Biobehav. Rev. 35, 1502-1517. Casolini, P., Domenici, M.R., Cinque, C., Alemà, G.S., Chiodi, V., Galluzzo, M., Musumeci, M., Mairesse, J., Zuena, A.R., Matteucci, P., Marano, G., Maccari, S., Nicoletti, F., Catalani, A. 2007. Maternal exposure to low levels of corticosterone during lactation protects the adult offspring against ischemic brain damage. J. Neurosci. 27, 7041-7046. Champagne, D.L., Bagot, R.C., van Hasselt, F., Ramakers, G., Meaney, M.J., de Kloet, E.R., Joëls, M., Krugers, H. 2008. Maternal care and hippocampal plasticity: 30  

evidence for experience-dependent structural plasticity, altered synaptic functioning, and differential responsiveness to glucocorticoids and stress. J. Neurosci. 28, 6037-6045. Chen, C.C., Yang, C.H., Huang, C.C., Hsu, K.S. 2010. Acute stress impairs hippocampal

mossy

fiber-CA3

long-term

potentiation

by

enhancing

cAMP-specific phosphodiesterase 4 activity. Neuropsychopharmacology 35, 1605-1617. Chen, C.C., Huang, C.C., Hsu, K.S. 2015. Chronic social stress affects synaptic maturation of newly generated neurons in the adult mouse dentate gyrus. Int. J. Neuropsychopharmacol. doi: 10.1093/ijnp/pyv097. Cole, M.A., Kim, P.J., Kalman, B.A., Spencer, R.L. 2000. Dexamethasone suppression of corticosteroid secretion: evaluation of the site of action by receptor measures and functional studies. Psychoneuroendocrinology 25, 151-67. De Kloet, C.S., Vermetten, E., Geuze, E., Kavelaars, A., Heijnen, C.J., Westenberg, H.G. 2005. Assessment of HPA-axis function in posttraumatic stress disorder: pharmacological and non-pharmacological challenge tests, a review. J. Psychiatr. Res. 40, 550-567. Eysenck, H.J. 1983. Stress, disease, and personality: The inoculation effect. In: Cooper CL, editor. Stress research. Wiley, New York, pp. 121-146. Francis, D.D., Caldji, C., Champagne, F., Plotsky, P.M., Meaney, M.J. 1999. The role of corticotropin-releasing factor--norepinephrine systems in mediating the effects of early experience on the development of behavioral and endocrine responses to stress. Biol. Psychiatry 46, 1153-1166. Huang, C.C., Yang, C.H., Hsu, K.S. 2005. Do stress and long-term potentiation share 31  

the same molecular mechanisms? Mol. Neurobiol. 32, 223-235. Huang, C.C., Chu, C.Y., Yeh, C.M., Hsu, K.S. 2014. Acute hypernatremia dampens stress-induced enhancement of long-term potentiation in the dentate gyrus of rat hippocampus. Psychoneuroendocrinology 46, 129-140. Huang, Y.F., Yang, C.H., Huang, C.C., Hsu, K.S. 2012. Vascular endothelial growth factor-dependent spinogenesis underlies antidepressant-like effects of enriched environment. J. Biol. Chem. 287, 40938-40955. Ivy, A.S., Rex, C.S., Chen, Y., Dubé, C., Maras, P.M., Grigoriadis, D.E., Gall, C.M., Lynch, G., Baram, T.Z. 2010. Hippocampal dysfunction and cognitive impairments provoked by chronic early-life stress involve excessive activation of CRH receptors. J. Neurosci. 30, 13005-13015. Kim, J.J., Diamond, D.M. 2002. The stressed hippocampus, synaptic plasticity and lost memories. Nat. Rev. Neurosci. 3, 453-462. Kim, J.J., Foy, M.R., Thompson, R.F. 1996. Behavioral stress modifies hippocampal plasticity through N-methyl-D-aspartate receptor activation. Proc. Natl. Acad. Sci. USA 93, 4750-4753. Knight, B.G., Gatz, M., Heller, K., Bengtson, V.L. 2000. Age and emotional response to the Northridge earthquake: a longitudinal analysis. Psychol. Aging 15, 627-634. Krishnan, V., Han, M.H., Graham, D.L., Berton, O., Renthal, W., Russo, S.J., Laplant, Q., Graham, A., Lutter, M., Lagace, D.C., Ghose, S., Reister, R., Tannous, P., Green, T.A., Neve, R.L., Chakravarty, S., Kumar, A., Eisch, A..J, Self, D..W, Lee, F.S., Tamminga, C.A., Cooper, D.C., Gershenfeld, H.K., Nestler, E.J. 2007. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131, 391-404. 32  

Lee, A.G., Buckmaster, C.L., Yi, E., Schatzberg, A.F., Lyons, D.M. 2014. Coping and glucocorticoid

receptor

regulation

by

stress

inoculation.

Psychoneuroendocrinology 49, 272-279. Levine, S. 1957. Infantile experience and resistance to physiological stress. Science 126, 405. Levine, S. 2005. Developmental determinants of sensitivity and resistance to stress. Psychoneuroendocrinology 30, 939-946. Levine, S., Alpert, M., Lewis, G.W. 1957. Infantile experience and the maturation of the pituitary adrenal axis. Science 126, 1347. Levine, S., Mody, T. 2003. The long-term psychobiological consequences of intermittent postnatal separation in the squirrel monkey. Neurosci. Biobehav. Rev. 27, 83-89. Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M., Meaney, M.J. 1997. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science 277, 1659-1662. Loman, M.M., Gunnar, M.R. 2010. Early experience and the development of stress reactivity and regulation in children. Neurosci. Biobehav. Rev. 34, 867-876. Lucassen, P.J., Naninck, E.F., van Goudoever, J.B., Fitzsimons, C., Joels, M., Korosi, A. 2013. Perinatal programming of adult hippocampal structure and function; emerging roles of stress, nutrition and epigenetics. Trends Neurosci. 36, 621-631. Lyons, D.M., Parker, K.J. 2007. Stress inoculation-induced indications of resilience in monkeys. J. Trauma Stress 20, 423-433. Lyons, D.M., Parker, K.J., Schatzberg, A.F. 2010. Animal models of early life stress: 33  

implications for understanding resilience. Dev. Psychobiol. 52, 616-624. Macrí, S., Mason, G.J., Würbel, H. 2004. Dissociation in the effects of neonatal maternal separations on maternal care and the offspring's HPA and fear responses in rats. Eur. J. Neurosci. 20, 1017-1024. Macrì, S., Würbel, H. 2006. Developmental plasticity of HPA and fear responses in rats: a critical review of the maternal mediation hypothesis. Horm. Behav. 50, 667-680. Macrì, S., Zoratto, F., Laviola, G. 2011. Early-stress regulates resilience, vulnerability and experimental validity in laboratory rodents through mother-offspring hormonal transfer. Neurosci. Biobehav. Rev. 35, 1534-1543. Meaney, M.J. 2010. Epigenetics and the biological definition of gene x environment interactions. Child. Dev. 81, 41-79. Murgatroyd, C., Patchev, A.V., Wu, Y., Micale, V., Bockmühl, Y., Fischer, D., Holsboer, F., Wotjak, C.T., Almeida, O.F., Spengler, D. 2009. Dynamic DNA methylation programs persistent adverse effects of early-life stress. Nat. Neurosci. 12, 1559-1566. Nollet, M., Gaillard, P., Tanti, A., Girault, V., Belzung, C., Leman, S. 2012. Neurogenesis-independent antidepressant-like effects on behavior and stress axis response of a dual orexin receptor antagonist in a rodent model of depression. Neuropsychopharmacology 37, 2210-22211. Norris, F.H., Murrell, S.A. 1988. Prior experience as a moderator of disaster impact on anxiety symptoms in older adults. Am. J. Community Psychol. 16, 665-683. Parker, K.J., Buckmaster, C.L., Schatzberg, A.F., Lyons, D.M. 2004. Prospective investigation of stress inoculation in young monkeys. Arch. Gen. Psychiatry 61, 933-941. 34  

Parker, K.J., Buckmaster, C.L., Sundlass, K., Schatzberg, A.F., Lyons, D.M. 2006. Maternal mediation, stress inoculation, and the development of neuroendocrine stress resistance in primates. Proc. Natl. Acad. Sci. USA. 103, 3000-3005. Parker, K.J., Maestripieri, D. 2011. Identifying key features of early stressful experiences that produce stress vulnerability and resilience in primates. Neurosci. Biobehav. Rev. 35, 1466-1483. Pellow, S., Chopin, P., File, S.E., Briley, M. 1985. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods 14, 149-167. Peña, C.J., Neugut, Y.D., Champagne, F.A. 2013. Developmental timing of the effects of maternal care on gene expression and epigenetic regulation of hormone receptor levels in female rats. Endocrinology 154, 4340-4351. Rice, C.J., Sandman, C.A., Lenjavi, M.R., Baram, T.Z. 2008. A novel mouse model for acute and long-lasting consequences of early life stress. Endocrinology 149, 4892-4900. Schmidt, M.V., Levine, S., Alam, S., Harbich, D., Sterlemann, V., Ganea, K., de Kloet, E.R., Holsboer, F., Müller, M.B. 2006. Metabolic signals modulate hypothalamic-pituitary-adrenal axis activation during maternal separation of the neonatal mouse. J. Neuroendocrinol. 18, 865-874. Schmidt, M.V., Abraham, W.C., Maroun, M., Stork, O., Richter-Levin, G. 2013. Stress-induced metaplasticity: from synapses to behavior. Neuroscience 250, 112-120. Schülke, J.P., Wochnik, G.M., Lang-Rollin, I., Gassen, N.C., Knapp, R.T., Berning, B., Yassouridis, A., Rein, T. (2010). Differential impact of tetratricopeptide repeat proteins on the steroid hormone receptors. PLoS One 5, e11717. 35  

Singh-Taylor, A., Korosi, A., Molet, J., Gunn, B.G., Baram, T.Z. 2015. Synaptic rewiring of stress-sensitive neurons by early-life experience: a mechanism for resilience? Neurobiol. Stress 1, 109-115. Tang, A.C., Reeb, B.C. 2004. Neonatal novelty exposure, dynamics of brain asymmetry, and social recognition memory. Dev. Psychobiol. 44, 84-93. Tang, A.C., Akers, K.G., Reeb, B.C., Romeo, R.D., McEwen, B.S. 2006. Programming social, cognitive, and neuroendocrine development by early exposure to novelty. Proc. Natl. Acad. Sci. USA. 103, 15716-15721. Tang, A.C., Zou, B., Reeb, B.C., Connor, J.A. 2008. An epigenetic induction of a right-shift in hippocampal asymmetry: selectivity for short- and long-term potentiation but not post-tetanic potentiation. Hippocampus 18, 5-10. Varadarajulu, J., Lebar, M., Krishnamoorthy, G., Habelt, S., Lu, J., Bernard Weinstein, I., Li, H., Holsboer, F., Turck, C.W., Touma, C. 2011. Increased anxiety-related behavior in Hint1 knockout mice. Behav. Brain Res. 220, 305-311. Wang, Y.C., Huang, C.C., Hsu, K.S. 2010. The role of growth retardation in lasting effects of neonatal dexamethasone treatment on hippocampal synaptic function. PLoS One 5, e12806. Weaver, I.C., Cervoni, N., Champagne, F.A., D'Alessio, A.C., Sharma, S., Seckl, J.R., Dymov, S., Szyf, M., Meaney, M.J. 2004. Epigenetic programming by maternal behavior. Nat. Neurosci. 7, 847-854. Xu, L., Holscher, C., Anwyl, R., Rowan, M.J. 1998. Glucocorticoid receptor and protein/RNA synthesis-dependent mechanisms underlie the control of synaptic plasticity by stress. Proc. Natl. Acad. Sci. US A. 95, 3204-3208. Yang, C.H., Huang, C.C., Hsu, K.S. 2004. Behavioral stress modifies hippocampal synaptic plasticity through corticosterone-induced sustained extracellular 36  

signal-regulated kinase/mitogen-activated protein kinase activation. J. Neurosci. 24, 11029-11034. Yang, C.H., Huang, C.C., Hsu, K.S. 2005. Behavioral stress enhances hippocampal CA1 long-term depression through the blockade of the glutamate uptake. J. Neurosci. 25, 4288-4293.

37  

Figure Legends

Figure 1

Reduced bedding and nesting material in the cage influences the interactions mouse dams with their pups. (A) Summary graph showing the average number of dam off pups from control (Con) and ELS dams through P2-P8.

(B) Summary graph showing the percentage of time

spent in maternal licking/grooming behavior from Con and ELS dams through P2-P8.

(C) Summary graph showing the body weight of Con

and ELS mice during P1-P35. Data are represented as mean  SEM. ***p < 0.001 compared with Con group.

Figure 2

Effects of ELS on anxiety- and depression-like behaviors in adolescence. (A-C) Bar graph comparing the effect of ELS on the percentage of entrances into the open arms (A), the number of total entries (B) or the time spent in the open and closed arms or center (C) in the elevated plus maze (EPM). (D-F) Bar graph comparing the effects of ELS on the total distance traveled (D), the time spent in the center (E) and the velocity (F) in the open field (OF). (G) Bar graph comparing the effect of ELS on the 1% sucrose preference test (SPT).

The numbers in

parentheses indicate the number of animals examined.

Data are

presented as means  SEM.

Figure 3

Effects of ELS on vulnerability to CSDS. representation of the experimental design.

(A) Schematic

The ELS paradigm was

applied from P2 to P9. Control (Con) and ELS mice were subjected to 38  

CSDS for 10 days and then separated into susceptible and resistant subpopulations based on a measure of SIT 24 h after the last defeat.

(B)

Summary graph showing the distribution of interaction ratios for naive, susceptible and resistant mice over CSDS in control group.

(C)

Summary graph showing the distribution of interaction ratios for naive, susceptible and resistant mice over CSDS in ELS group. (D) Bar graph showing

the

relative

distribution

of

susceptible

and

resistant

subpopulations after CSDS in control and ELS mice in adolescence. Data are represented as mean  SEM. ***p < 0.001 compared with Con group.

Figure 4

ELS blunts acute restraint-tailshock stress-induced impairment of LTP and enhancement of LTD. experimental design. Control

(Con)

and

(A) Schematic representation of the

The ELS paradigm was applied from P2 to P9. ELS

mice

were

subjected

to

an

acute

restraint-tailshock stress paradigm for 60 min and then sacrificed for electrophysiological recordings.

(B) Slices from stressed control

(Con-S) mice displayed a deficit in HFS-induced LTP compared with nonstressed control (Con-NS) mice at Schaffer collateral-CA1 synapses. (C) LFS induced a reliable LTD in slices from stressed Con-S mice, but not in slices from Con-NS mice.

(D) The magnitudes of LTP were

almost identical in slices from ELS-NS and ELS-S mice.

(E) No

significant difference was observed in LTD magnitude in slices from ELS-NS and ELS-S mice. (F) Bar graph comparing the effect of acute restraint-tailshock stress on LTP in slices from Con and ELS mice. 39  

The magnitude of LTP was measured 50-60 min after HFS. (G) Bar graph comparing the effect of acute restraint-tailshock stress on LTD in slices from Con and ELS mice. The magnitude of LTD was measured 50-60 min after the end of LFS. The superimposed fEPSPs in the inset illustrate respective recordings from example experiments taken at the time indicated by number. Dash lines show level of baseline. The numbers in parentheses indicate the number of animals examined. Data are presented as means  SEM.

*p < 0.05 compared with NS

group.

Figure 5

ADX prevents the effects of ELS on LTP and LTD. representation of the experimental design.

(A) Schematic

The ELS paradigm was

applied from P2 to P9. Control (Con) and ELS mice were bilaterally adrenalectomized or given sham surgery at P28 and then sacrificed for electrophysiological recordings at P35. (B) The magnitudes of LTP were almost identical in slices from Con-ADX and ELS-ADX mice. (C) Bar graph comparing the effects of ELS on LTP in slices from sham and ADX mice. The magnitude of LTP was measured 50-60 min after HFS. (D) No reliable LTD was induced in slices from Con-ADX and ELS-ADX mice.

(E) Bar graph comparing the effects of ELS on LTD

in slices from sham and ADX mice.

The magnitude of LTD was

measured 50-60 min after the end of LFS. The superimposed fEPSPs in the inset illustrate respective recordings from example experiments taken at the time indicated by number. baseline.

The numbers in parentheses indicate the number of animals 40

 

Dash lines show level of

Data are presented as means  SEM.

examined.

*p < 0.05 compared

with Con group.

Figure 6

Effects of ELS on corticosterone-induced modifications of LTP and LTD in ADX mice. (A) Schematic representation of the experimental design. The ELS paradigm was applied from P2 to P9. Control (Con) and ELS mice were bilaterally adrenalectomized or given sham surgery at P28 and then sacrificed for electrophysiological recordings at P35. Vehicle

(Veh)

or

corticosterone

(0.75

mg/kg)

subcutaneously into mice 20 min before sacrifice.

was

injected

(B) Summary of

experiments showing that plasma corticosterone levels were increased following intraperitoneal corticosterone (0.75 mg/kg) injections in ADX mice.

(C) Summary of experiments showing the elevations of plasma

corticosterone levels in Con- or ELS-ADX mice received either Veh or corticosterone injections. (D) Summary of experiments showing the induction of LTP in slices from Con- or ELS-ADX mice received either Veh or corticosterone injections. (E) Bar graph comparing the effects of ELS on corticosterone-induced impairment of LTP in slices from ADX mice.

The magnitude of LTP was measured 50-60 min after HFS.

(F) Summary of experiments showing the induction of LTD in slices from Con- or ELS-ADX mice received either Veh or corticosterone injections.

(E) Bar graph comparing the effects of ELS on

corticosterone-induced enhancement of LTD in slices from ADX mice. The magnitude of LTD was measured 50-60 min after the end of LFS. The superimposed fEPSPs in the inset illustrate respective recordings 41  

from example experiments taken at the time indicated by number. Dash lines show level of baseline.

The numbers in parentheses indicate

the number of animals examined.

Data are presented as means  SEM.

*p < 0.05 and ***p < 0.001 compared with basal corticosterone levels.

Figure 7

ELS increases active coping behaviors after stress exposure. Schematic representation of the experimental design.

(A)

The ELS

paradigm was applied from P2 to P9. Coping behavior of control (Con) and ELS mice was assessed in the TST performed 15 min after exposure a 15-min restraint stress at P35.

(B) Bar graph comparing the effect of

ELS on stress-induced enhancement of immobility in the TST. Schematic representation of the experimental design.

(C)

The FST

paradigm was applied from P2 to P9. Coping behavior of control (Con) and ELS mice was assessed in the FST performed 15 min after exposure a 15-min restraint stress at P35. (D) Bar graph comparing the effect of ELS on stress-induced enhancement of immobility in the FST. *p < 0.05 compared with NS group.

Figure 8

ELS alters neuroendocrine stress reactivity. (A) Bar graph comparing the basal plasma corticosterone levels for 7:00 h and 17:00 h in control (Con) and ELS mice.

(B) Summary graph showing the plasma ACTH

responses to 30 min of restraint stress in control (Con) and ELS mice. Insert in graph panel indicates values of areas under the response curve (AUC) of ACTH.

(C) Summary graph showing the plasma 42

 

corticosterone responses to 30 min of restraint stress in Con and ELS mice. Insert in graph panel indicates values of AUC of corticosterone. (D) Summary graph showing the dexamethasone-induced suppression of corticosterone release to 30 min of restraint stress in Con and ELS mice. Insert in graph panel indicates values of AUC of corticosterone. (E) Bar graph

comparing

the

percentage

of

dexamethasone-induced

corticosterone suppression to 30 min of restraint stress (the subtraction of the AUC of corticosterone in the absence from the presence of dexamethasone) in Con and ELS mice. The numbers in parentheses indicate the number of animals examined. Data are presented as means  SEM. *p < 0.05 compared with Con group.

Figure 9

ELS

blunts

stress-induced

nuclear

trafficking

of

GR.

(A)

Representative immunoblots and corresponding densitometric analysis showing the expression of GR, MR and FKBP51 proteins in hippocampal CA1 region of Control (Con) and ELS mice.

(B)

Representative immunoblots and corresponding densitometric analysis showing the cytosolic and nuclear expression of GR protein in hippocampal CA1 region of Con and ELS mice after a 30-min restraint stress.

The purity of subcellular fractions was confirmed by

anti-GAPDH or anti-histone H3 antibodies for the cytoplasmic or nuclear compartments, respectively.

The numbers in parentheses

indicate the number of animals examined. Data are presented as means  SEM.

*p < 0.05 and **p < 0.01 compared with Con-NS group.

43  

44  

45  

46  

47  

48  

49  

Table 1. ELS mice display distinct phenotypes compared to littermate controls. Phenotype

Effect

Source

Body weight



Fig. 1C

Elevated plus maze (EPM) Open field (OF) test Sucrose preference test (SPT) Chronic social defeat stress (CSDS)

   

Fig. 2A-C Fig. 2D-F Fig. 2G Fig. 3

Hippocampal CA1 LTP Hippocampal CA1 LTD Stress-induced impairment of LTP Stress-induced enhancement of LTD

   

Fig. 4D, F Fig. 4E, G Fig. 4D, F Fig. 4E, G

Active coping in the tail suspension test (TST) Active coping in the forced swimming test (FST) Basal plasma corticosterone levels Stress-induced ACTH response

   

Fig. 7B Fig. 7B Fig. 8A Fig. 8B

  

Fig. 8C Fig. 8E Fig. 9B

Stress-induced corticosterone response Dexamethasone-induced HPA-axis suppression Stress-induced nuclear trafficking of GR

・

, , and  represent no change, significantly greater than, or less than control group (p < 0.05), respectively.

50