Environmental enrichment protects against stress-induced anxiety: Role of glucocorticoid receptor, ERK, and CREB signaling in the basolateral amygdala

Neuropharmacology 113 (2017) 457e466

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Environmental enrichment protects against stress-induced anxiety: Role of glucocorticoid receptor, ERK, and CREB signaling in the basolateral amygdala Leonardo S. Novaes, Nilton Barreto dos Santos, Rafaela F.P. Batalhote, Marília Brinati Malta, Rosana Camarini, Cristoforo Scavone, Carolina Demarchi Munhoz* ~o Paulo, Sa ~o Paulo, 05508-000, Brazil Department of Pharmacology, Institute of Biomedical Science, University of Sa

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 April 2016 Received in revised form 23 October 2016 Accepted 24 October 2016 Available online 1 November 2016

Environmental enrichment (EE) is an experimental animal model that enhances an animal's opportunity to interact with sensory, motor, and social stimuli, compared to standard laboratory conditions. A prominent benefit of EE is the reduction of stress-induced anxiety. The relationship between stress and the onset of anxiety-like behavior has been widely investigated in experimental research, showing a clear correlation with structural changes in the hippocampus and basolateral amygdala (BLA). However, the mechanisms by which EE exerts its protective roles in stress and anxiety remain unclear, and it is not known whether EE reduces the effects of acute stress on animal behavior shortly following the cessation of stress. We found that EE can prevent the emergence of anxiety-like symptoms in rats measured immediately after acute restraint stress (1 h) and this effect is not due to changes in systemic release of corticosterone. Rather, we found that stress promotes a rapid increase in the nuclear translocation of glucocorticoid receptor (GR) in the BLA, an effect prevented by previous EE exposure. Furthermore, we observed a reduction of ERK (a MAPK protein) and CREB activity in the BLA promoted by both EE and acute stress. Finally, we found that EE decreases the expression of the immediate-early gene EGR-1 in the BLA, indicating a possible reduction of neuronal activity in this region. Hyperactivity of BLA neurons has been reported to accompany anxiety-like behavior and changes in this process may be one of the mechanism by which EE exerts its protective effects against stress-induced anxiety. © 2016 Published by Elsevier Ltd.

Keywords: Environment enrichment Stress Anxiety Glucocorticoids Basolateral amygdala ERK CREB

1. Introduction Environmental enrichment (EE) is an experimental procedure in which animals are housed in cages with greater opportunities for interaction with sensory, motor, and social stimuli (Rosenzweig et al., 1978). In general, EE consists of housing the animals in groups and in large cages in which objects and structures, such as ladders, running wheels, and toys, are periodically introduced. This facilitates not only social interaction but also exploratory and cognitive activities (Rosenzweig et al., 1978; Rosenzweig and Bennett, 1996; van Praag et al., 2000; Mohammed et al., 2002). It is well known that EE can improve learning and memory (Nilsson

* Corresponding author. Department of Pharmacology, Av. Prof. Lineu Prestes, 1524, room 323, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo - SP, 05508-000, Brazil. E-mail address: [email protected] (C.D. Munhoz). http://dx.doi.org/10.1016/j.neuropharm.2016.10.026 0028-3908/© 2016 Published by Elsevier Ltd.

et al., 1999; Rampon et al., 2000; Lee et al., 2003) and has beneficial effects in a variety of models of affective disorders, particularly in depressive- and anxiety-like behaviors (Fox et al., 2006; Brenes et al., 2008; Laviola et al., 2008; Pang et al., 2009). Specifically, it has been reported that EE can protect rodents against emotional disturbance triggered by psychological stress (Klein et al., 1994; Francis et al., 2002; Larsson et al., 2002; Benaroya-Milshtein et al., 2004). Many studies focusing on the causal relationship between stress and anxiety have identified the amygdala as the key structure in this interaction, since this brain area has crucial role in the generation and development of fear and anxiety (Davis, 1992; Rosen and Schulkin, 1998; LeDoux, 2000; Gross and Hen, 2004; Rodrigues et al., 2009). Some of this work has shown that acute stress or increased serum levels of glucocorticoid (GC) stress hormones induce not only long-lasting anxiety but also remodeling of dendritic branches in basolateral amygdala complex (BLA) of rats

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(Mitra et al., 2005; Mitra and Sapolsky, 2008). Moreover, acute stress or systemic injection of the GC corticosterone (CORT) in rats increases baseline activity of the BLA (Rodriguez Manzanares et al., 2005; Kavushansky and Richter-Levin, 2006), while blockade of excitatory synapses or augmenting GABAergic signaling, in this nucleus, prevents the onset of stress-induced anxiety (Adamec et al., 1999; Bignante et al., 2010). Although several studies have described the protective effect of EE against affective disorders triggered by different types of stress (for a review, see Fox et al., 2006), in general, they have focused on the prolonged effects of stress (spanning days to weeks). Many studies also suggest that the protective role of EE is from its capacity to buffer the hypothalamic-pituitary-adrenal (HPA) axis stress response (Roy et al., 2001; Francis et al., 2002; Belz et al., 2003; Morley-Fletcher et al., 2003; Moncek et al., 2004; Welberg et al., 2006). However, it is not known whether EE can protect against anxiety triggered immediately by a single stress session. Nonetheless, it is widely known that corticosterone signaling increases the activity of BLA neurons (Rodriguez Manzanares et al., 2005; Kavushansky and Richter-Levin, 2006; Duvarci and Pare, 2007) and also that the increase of neuronal activity of this nucleus has a direct relation to anxiety-like behavior (Rainnie et al., 2004; Wang et al., 2011). In the present study, we investigated the effect of EE on anxietylike behavior triggered immediately after acute restraint stress and the effects of EE on stress-related changes in CORT release and in its signaling in the BLA through glucocorticoid receptor (GR). Moreover, we investigated some other molecular changes promoted by both stress and EE such as ERK phosphorylation and CREB activation. Finally, we evaluated the expression of the immediately early gene EGR-1 in the BLA, a molecular marker of neuronal activity.

2. Materials and methods 2.1. Experimental animals A total of 85 adult male Wistar rats (60e65 days old at the beginning of the experiment) were used for this study. All animals were randomly pair-housed in standard polypropylene cages (30
37
18 cm) throughout the first ten days (habituation period), after which half of them were transferred to EE (see below) and the other half were maintained in pair-housing until the end of the experiment. Animals were kept in a controlled temperature room (21 ± 2  C) on a 12-h light/dark cycle (lights on at 07:00 h) with access to food and water ad libitum. Stress and behavioral testing were performed during the light cycle between 08:00 a.m. and 12:00 p.m. Animal care and all experimental protocols were approved by the local Animal Research Committee and were in accordance with the U.S. National Institute of Health Guidelines for the Care and Use of Laboratory Animals.

2.2. Environmental enrichment After the habituation period, half of the animals were then transferred to EE, where they remained for 14 consecutive days. EE consisted of housing rats in a bigger polypropylene cage (30
45
25 cm) compared to the standard one (30
37
18 cm). In addition, several types of objects, such as plastic toys and balls, wood objects, wood boxes, PVC tunnels, plastic and iron stairs, elevated platforms, and nesting materials were introduced. The animals were handled, the objects were cleaned, and the environmental stimuli were changed every two days.

2.3. Acute restraint stress On the 15th day, EE and control animals were distributed into four groups: EE-stressed (ES, n ¼ 28), control-stressed (CS, n ¼ 28), EE-unstressed (E0, n ¼ 14), and control-unstressed (C0, n ¼ 15). For each pair of rats, one was exposed to acute restraint stress, while the cagemate was unstressed. For acute restraint stress, the rats were transferred at 09:00 a.m. (during the circadian trough of corticosterone secretion) to the experimental room and were placed in ventilated PVC restraint tubes (1 h). Immediately after the stress session, the animals were subjected to the elevated plus maze test (EPM). To avoid isolation stress, the unstressed cagemates (E0 and C0) were run on the EPM test, at the same time its cage-mate were subjected to stress. All rats were euthanized immediately after completion of the EPM test. 2.4. Behavioral tests 2.4.1. Elevated plus maze The EPM apparatus consisted of two open arms (50
10 cm) and two enclosed arms (50
10 cm, surrounded by a 40-cm-high wall), forming two perpendicular and intersecting runways. The arms were connected by a central area (10
10 cm), and the maze was elevated 50 cm from the floor. Animals were tested for 5 min. Anxiety-like behavior was assessed as a function of decreased open arm exploration, measured by the total percentage of entries into the open arms (number of entries in the open arms/number of entries in all arms x 100) and the total percentage of time spent on the open arms (time in open arms/total time in the maze, excluding the central area, x 100). Based on Cohen et al. (2008), an index that integrates the EPM behavioral measures (anxiety index) was also calculated as follows: anxiety index ¼ 1 e [(time spent in open arms/total time on the maze) þ (number of entries to the open arms/Total exploration on the maze)/2 ]. Anxiety index values range from 0 to 1 where an increase in the index express increased anxiety-like behavior. At the beginning of each trial, animals were placed in the central area, facing one of the open arms. General locomotor activity was assessed by measuring the total distance traveled in the maze, using EthoVision software (Noldus, the Netherlands). Fifty-eight rats (C0, n ¼ 15; CS, n ¼ 14; E0, n ¼ 14; ES, n ¼ 15) were used for this test. 2.4.2. Light-dark box The light-dark box was made of white and black Plexiglas (45
41
21 cm light chamber, 35
41
21 cm dark chamber). The chambers were connected by a 7
7 cm door in the middle of the wall separating the two chambers. Ambient illumination was 550 lx in the light chamber and 6 lx in the dark chamber. At the beginning of each trial, animals were placed in the middle of the light chamber facing the opposite side of the door. Each trial lasted 5 min and the duration of time spent in the light chamber and the latency to re-enter the light chamber after the first bout spent in the dark chamber (non-responders were assigned a latency of 300s) were used as parameters to address anxiety-like behavior. Fifteen animals (CS, n ¼ 8; ES, n ¼ 7) were used for this test. All trials were videotaped and the apparatuses chambers were cleaned with 5% (vol/vol) ethanol after each trial. The experimental procedure is shown in Figure suppl. 1. 2.5. Euthanasia and samples preparation Immediately after the EPM test, animals were euthanatized by decapitation and the brains were removed and immersed in cold phosphate-buffered saline (0.01M PBS, pH 7.4). The BLA was rapidly dissected (using a brain matrix; 1 mm, Zivic Laboratories,

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Portesville), immersed in liquid nitrogen, and stored at - 80  C. Nuclear and cytosolic protein extracts were prepared using the CelLytic™ NuCLEAR™ Extraction Kit (Sigma-Aldrch Co.), as briefly described below. Brain structures were homogenized in cold lysis buffer (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 1 mm DTT, 0.5 mm PMSF) using a Dounce homogenizer and centrifuged at 4  C for 20 min at 11,000
g. The supernatants, used as cytosolic fraction in the Western blot assays, were stored at - 80  C. The pellets, containing the nuclear fraction, were resuspended in extraction buffer (20 mm HEPES, pH 7.9, 25% glycerol, 1.5 mm MgCl2, 300 mm NaCl, 0.25 mm EDTA, 0.5 mm DTT, 0.5 mm PMSF), kept on ice for 30 min, and centrifuged at 4  C for 5 min at 20,000
g. The buffers were complemented with the Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Inc). The resulting supernatants containing nuclear proteins were stored at 80  C. Protein concentration was determined using the Bradford method (Bio-Rad Laboratories, Inc) (Bradford, 1976). 2.6. Western blot assay Electrophoresis was performed using a 10% polyacrylamide gel and the Mini-Protean® Tetra Cell apparatus (Bio-Rad Laboratories, Inc). The proteins present in the cytosolic and nuclear fractions were combined with an equal or a quarter part of the supernatant volume with Laemmli's buffer (Bio-Rad Laboratories, Inc; complemented with 5% mercaptoethanol) and boiled at 95  C for 5 min. Protein samples (5 mg/lane) were size-separated in 10% SDS-PAGE gel (90 V), and then blotted onto Immobilon® PVDF membrane (EMD Millipore Corporation). Ponceau method to immunoblot was used to ensure equal protein loading (Salinovich and Montelaro, 1986). Blots were blocked with 5% bovine serum albumin (BSA) diluted in TBS-T buffer (50 mM Tris-HCL, 150 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h at room temperature, and subsequently incubated overnight at 4  C with specific antibodies: GR, EGR-1 (Santa Cruz Biotechnology) at a dilution of 1:1000, Thr202/tyr204phosphorERK1/2, ERK1/2 (Cell signaling Technology) at a dilution of 1:2000. After incubation with the primary antibodies, the membrane was then probed with a secondary antibody conjugated to horseradish peroxidase (dilution of 1:2000e1:4000, Kirkegaard & Perry Laboratories) for 2 h at room temperature and developed by ECLImmobilon® reagent (EMD Millipore Corporation) on X-ray films. Several exposure times were analyzed to ensure the linearity of the band intensities. The resulting film samples were scanned (600 dpi of resolution) and analyzed with ImageJ® software (National Institutes of Health, Bethesda, MD). The relative density of each band was normalized to the value of a-Tubulin (dilution of 1:5000, Santa Cruz Biotechnology). The ratio between phopho-ERK-1/2 and total ERK-1/2 expression was used as phosphorylation index and the ratio between nuclear GR and cytosolic GR expression was computed as the nuclear translocation index, and therefore, GR activity. 2.7. Electrophoretic mobility shift assay Electrophoretic mobility shift assay (EMSA) to CREB was performed by using the gel shift assay kit from Promega, as described previously (Rong and Baudry, 1996). In brief, 32P-CREB doublestranded consensus oligonucleotide probe (50 -AGAGATTGCCTGACGTCAGAGAGCTAG-30 ) (25,000 cpm) and nuclear extracts (5 mg) were used. DNA-protein complexes were separated by electrophoresis through a 5.5% nondenaturing acrylamide:bisacrylamide (37.5:1) gel in 0.5
Tris-borate/EDTA (TBE: Tris 90 mM, boric acid 90 mM, EDTA 1 mM) for 2 h at 150 V. Gels were vacuum-dried, and exposed to X-ray film. For competition experiments, CREB and TFIID (Transcription factor IID, 50 -

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GCAGAGCATATAAGGTGAGGTAGGA-30 ) unlabeled double stranded consensus oligonucleotide was included in 10-fold molar excess over the amount of 32P-CREB probe to detect specific and nonspecific DNA-protein interactions, respectively. Supershift assays, using antibodies against pCREB, CREM, and CREB-1 (1:20 dilution, Santa Cruz Biotechnology) were also conducted according to manufacturer's protocol (Santa Cruz Biotechnology). The autoradiography analyses were made as performed to Western bolt assay. 2.8. Serum CORT concentration Concentrations of CORT serum were quantified using the Corticosterone EIA Kit® (Enzo Life Sciences). Two groups of rats were used for this assay; one group underwent EPM test and blood was sampled from the body trunk after decapitation (C0, n ¼ 15, CS, n ¼ 14; E0, n ¼ 14, ES, n ¼ 14), and the other group had blood sampled from the tail during the stress experiments (CS, n ¼ 6; ES, n ¼ 6). In this second group of rats, samples were collected from a small incision along the side of the tail by milking blood from a lateral vein. Tail blood samples were collected at 0, 15, 30, 45, and 60 min after restraint initiation. To obtain serum, blood was collected in non-heparinized 2 ml tube and allowed to clot at room temperature for 30 min. The blood was then centrifuged at 4000 rpm for 10 min and the serum portion was collected and stored at 80  C until CORT analysis was conducted. Serum samples were diluted 1:30 and processed following manufacturer's instructions. 2.9. Statistical analysis For serum corticosterone levels during the stress, the statistical analysis was performed using two-way analysis of variance (ANOVA) with housing condition (standard cage or EE) and time after stress (0, 15, 30, and 60 min) as between-group sources of variance. Statistical differences were determined by repeated measures. For results with more than 2 experimental groups, the statistical analyses were performed using two-way ANOVA, in which housing conditions (standard or EE) and stress (unexposed or exposed) were factors. Pos hoc Newman-Keuls multiple comparison test examined differences between individual groups. The light-dark box test was analyzed with unpaired Student's t-test. GraphPad Prism software 5.0 was used for the statistical analyses and the level of statistical significance was set up at p < 0.05. Data are presented as mean ± SEM. The F values and experimental degrees of freedom are presented in the Results session. 3. Results 3.1. EE protects against anxiety triggered by acute stress In terms of percentage of entries into open-arms, a two-way ANOVA revealed significant main effects of stress exposure [F (1, 54) ¼ 12.49, p ¼ 0.0009], housing [F (1, 54) ¼ 12.31, p ¼ 0.0009], and their interaction [F (1, 54) ¼ 4.119, p ¼ 0.0473]. Time spent in open-arms also showed a significant main effect for stress [F (1, 54) ¼ 4.884, p ¼ 0.0314] and for housing [F (1, 54) ¼ 5.545, p ¼ 0.0222] though no effects were observed for stress-housing interaction [F (1, 54) ¼ 3.164, p ¼ 0.08]. Newman-Keuls test confirmed that rats exposed to an acute restraint stress (CS) exhibited higher anxiety-like behavior than controls (C0), as evidenced by reduced percentage of entries into and time spent on the open-arms (p < 0.001 and p < 0.01, respectively; Fig. 1A and B). On the other hand, EE exposure blunted anxiety-like behavior in stressed animals, since Newman-keuls test confirmed that EEstressed animals (ES) displayed higher levels of open-arm

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Fig. 1. EE prevents anxiety-like behavior triggered immediately after 1 h of restraint stress. Stressed animals housed in standard cages (CS) exhibited in the elevated plus-maze test reduced open arms exploration in terms of both open arm entries (A) and open arm time (B) compared with unstressed animals housed in the same type of cage (C0). Both stressed (ES) and unstressed (E0) EE-housed animals exhibited similar open arm exploration that C0 animals and showed an increase in open arm exploration compared with CS animals (A, B). The anxiety index also showed an increase in anxiety-like behavior in CS animals compared to C0, E0, and ES animals (C). Neither stress nor housing in the EE affected the locomotor activity of the animals (D). Light-dark box test also showed that prior exposure to EE prevented restraint stress-induced anxiety-like behavior. Stressed animals housed in standard cages (CS) spent less time in the light chamber compared to stressed animals housed in EE (ES, A). CS animals exhibited significantly more time to re-enter the light chamber than ES animals (B). Results are represented as mean ± SEM (n ¼ 14e15 animals for each group in A-D; n ¼ 7e8 in E-F). Two-way ANOVA (Newman-Keuls multiple comparison test, A-D) and Student's t-test (E, F). Significant difference between groups are indicated as * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

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exploration when compared to control, non-EE, stressed animals (CS) but not to control, non-EE, non-stressed animals (C0; Fig. 1A and B). Furthermore, EE itself was not able to modify the exploratory behavior in control animals (E0) since E0 animals did not show differences in percentage entries and time spent in open-arm when compared to C0 animals (Fig. 1A and B). For the anxiety index, two-way ANOVA revealed a significant main effects of stress exposure [F (1, 53) ¼ 11.41, p ¼ 0.0014], of housing condition [F (1, 53) ¼ 11.92, p ¼ 0.0011] and their interaction [F (1, 53) ¼ 5.222, p ¼ 0.0263] (Fig. 1C). Additionally, the change in exploration either by stress or EE was not due to an alteration in locomotor activity, inasmuch two-way ANOVA showed no differences in the overall distance travelled in the EPM among all experimental groups [F (1, 49) ¼ 0.5669, p ¼ 0.4551 for stress exposure; F (1, 49) ¼ 0.3813, p ¼ 0.5398 for housing condition; Fig. 2D]. Based on EPM results demonstrating that EE prevented the anxiety-like behavior triggered by acute restraint stress, we next compared the effect of stress on light-dark box test between control and EE animals. Onetailed t-test showed that the time spent in the light chamber was

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shorter [t (13) ¼ 1.891, p ¼ 0.0405] (Fig. 1E) and the latency to reenter the light chamber after entering in the dark chamber was higher [t (13) ¼ 2.061, p ¼ 0.0299] (Fig. 1F) in non-EE, stressed animals (CS) compared to EE, stressed animals (ES). 3.2. EE does not inhibit the release of corticosterone in acute stressed rats To verify whether the protective effect of EE on acute stressinduced anxiety could be due to an alteration in the release of corticosterone, we evaluated the serum levels of this hormone in both under basal and stress conditions. Two-way ANOVA for repeated measures revealed a significant main effect of restraint stress in CORT serum levels at each time point [F (4, 20) ¼ 36.87, p < 0.0001; Fig. 2A]. Indeed, housing condition showed no significant main effect in CORT serum levels [F (1, 5) ¼ 1.46, p ¼ 0.2810] and its interaction with the stress [F (4, 20) ¼ 0.3964, p ¼ 0.8088]. Newman-Keuls test confirmed that there were no significant differences between control and enriched rats under basal conditions

Fig. 2. EE does not affect both basal and the raise in serum corticosterone levels induced by acute stress although decreases its signaling through changes in the GR nuclear translocation in the BLA. Enriched and non-enriched (control) animals showed the same serum corticosterone levels at basal conditions (sampling time 0) as well as 15, 30, 45, and 60 min after onset of stress exposure (A). Stressed animals (CS and ES) showed, immediately after EPM test, serum corticosterone levels significantly higher than the unstressed animals (C0 and E0; B). CS animals showed, in the BLA, higher GR nuclear translocation index than C0 animals, while EE animals (E0 and ES) showed the same GR nuclear translocation index of C0. Right, representative autoradiography of Western blot. Left, histogram showing densitometry analysis of the specific bands of the experimental group represented in the right panel (C). Results are represented as mean ± SEM (n ¼ 6 for each group in A, n ¼ 14e15 in B, and n ¼ 4e5 in C). Two-way repeated measures ANOVA, (Newman-Keuls multiple comparison test, A) and two-way ANOVA followed by Newman-Keuls multiple comparison test (B, C). Significant difference between groups are indicated as ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

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and that exposure to restraint stress in control and enriched animals similarly increased serum CORT levels, which peaked 45 min after stress onset (sampling time 0 min vs 45 min; p < 0.01 for both control and enriched) Fig. 2A). Next, to assess the accurate levels of serum CORT under which the animals performed the EPM test, we measured the hormone levels immediately after the behavior task. As depicted in Fig. 2(B), two-way ANOVA revealed significant main effect of stress in CORT serum levels [F (1, 53) ¼ 42.35, p < 0.0001]. No significant main effect of housing [F (1, 53) ¼ 0.03357, p ¼ 0.8553] or stress-housing interaction [F (1, 53) ¼ 0.01335, p ¼ 0.9085] was found. Post hoc Newman-Keuls test confirmed that EE-stressed (ES) and control, non-EE, stressed (CS) animals showed the same CORT serum levels but higher than non-stressed animal (C0 and E0, p < 0.001 and p < 0.0001; Fig. 2B). In addition, there were no differences between C0 and E0 animals. These results suggest that the protective effect of EE on anxiety-like behavior triggered immediately by acute restraint stress was independent of corticosterone release modulation. 3.3. EE inhibits the acute stress-induced GR nuclear translocation in BLA Although there were no differences in CORT serum levels between EE-stressed and non-EE-stressed animals immediately after the EPM test, we decided to investigate whether the protective effect of EE in the stress-induced anxiety-like behavior was due to changes in the GR signaling in the BLA, addressed by GR nuclear translocation. Experimentally, GR nuclear translocation, measured as a ratio of GR expression in the nuclear compartment and GR expression in the cytosolic fraction, could be considered an index for GR activation (Tronche et al., 1998). In this way, we could infer the proportion of ligand-bound GR that migrated from the cytoplasm to the nucleus of BLA cells. Two-way ANOVA revealed a significant main effect of housing in terms of GR nuclear translocation [F (1, 13) ¼ 16.94, p ¼ 0.0012] and its interaction with stress [F (1, 13) ¼ 16.66, p ¼ 0.0013], even though there were no main effect of stress [F (1, 13) ¼ 2.096, p ¼ 0.1714]. However, NewmanKeuls test showed a substantial increase in GR nuclear translocation in the BLA of CS animals when compared to C0 animals (p < 0.01; Fig. 2C), suggesting that stress-induced increase in CORT serum levels involves GR signaling pathway in the BLA. On the other hand, EE hindered this translocation in stressed animals (ES), which showed similar serum CORT levels to CS animals (Fig. 2A and B). No differences in GR translocation was observed in both E0 and ES when compared to C0 animals (Fig. 3C). Even with no differences in CORT serum level between ES and CS animals at the time of testing, the modulation exerted by EE on GR activity (nuclear translocation) in the BLA cells suggests a likely mechanism by which EE renders animals insusceptible to stress-induced anxiety. 3.4. EE and acute stress reduce ERK-1/2 and CREB basal activity in the BLA We next addressed, through EMSA, the status of CREB activation in the BLA of these animals. Two-way ANOVA revealed significant main effect of stress on CREB binding activity [F (1, 12) ¼ 6.596, p ¼ 0.0246]. No significant main effect was found for housing [F (1, 12) ¼ 4.592, p ¼ 0.0533] and stress-housing interaction [F (1, 12) ¼ 1.458, p ¼ 0.2505]. Newman-Keuls test showed that both EE (E0 and ES) and acute stress (CS) decreased CREB binding activity when compared to control, non-EE, non-stressed animals (C0; p < 0.05; Fig. 3A). No difference was observed in CREB binding activity of E0 compared to ES animals, suggesting that EE, per se, can diminish CREB binding activity at the same level than acute stress. Also, EE effects were not synergic to those of acute restraint

stress on CREB activity in the BLA. Moreover, supershift experiment confirmed the presence of the activated form of CREB (pCREB) and CREM (a CREB family member) bounded to the radiolabeled probe (Fig. 3B). Considering the modulatory role of MAPK pathway over CREB (Shaywitz and Greenberg, 1999), we verified how EE and stress would impact this kinase activity. The results, illustrated in Fig. 4 (A, B), were analyzed by two-way ANOVA. In terms of ERK-1/2 phosphorylation, there was a significant main effect of housing [F (1, 19) ¼ 5.878, p ¼ 0.0255 for ERK1; F (1, 18) ¼ 5.629, p ¼ 0.0290 for ERK-2]. There was a main effect of stress only for ERK-2 [F (1, 18) ¼ 5.211, p ¼ 0.348 for ERK-2; F (1, 19) ¼ 2.984, p ¼ 0.1003 for ERK-1) and no effect was observed for stress-housing interaction [F (1, 19) ¼ 3.872, p ¼ 0.0639 for ERK-1; F (1, 18) ¼ 0.9457, p ¼ 0.3438]. Newman-Keuls test showed that EE (E0 and ES) and stress (CS) reduced ERK-1 and ERK-2 (in this case, statistical significance observed only in comparison between ES and C0) phosphorylation levels when compared to C0 animals (p < 0.05, for both ERK-1, Fig. 4A; and ERK-2, Fig. 4B). These results were in accordance with those of CREB activity and, collectively, they could point to the deactivation of the MAPK-CREB pathway in the BLA, since the decrease in CREB binding activity in the BLA could be due to a decreased ERK -1/2 activation. 3.5. EGR-1 expression is elevated in stressed control animals comparing to EE animals To assess recent neuronal activity, we quantified expression of the immediately early gene EGR-1 expression in the BLA of animals following EPM testing. Two-Way Anova revealed a strong main effect of housing in the EGR-1 expression [F (1, 17) ¼ 16.11, p ¼ 0.0009; Fig. 4C]. For the stress exposition [F (1, 17) ¼ 0.4377, p ¼ 0.5171] and its interaction with housing [F (1, 17) ¼ 2.344, p ¼ 0.1442] no significant effect was found. Newman-Keuls test indicates that EGR-1 expression in CS animals was elevated compared to both non-stressed and stressed EE animals (p < 0.05 vs E0 and p < 0.01 vs ES; Fig. 4C). Even though there was no difference in EGR-1 expression between C0 and CS animals, suggesting stress did no induce neuronal activity, we observed a quite significant increase at the CS group (Fig. 4C; p ¼ 0.0612, one-tailed unpaired ttest). Furthermore, the robust difference between CS and ES animals indicates that the augment of BLA neuronal activity due stress stimulus is higher in control animals than in enriched ones. Moreover, the inexistence of difference between E0 and ES suggests that EE makes the BLA neurons less responsive to acute stress in terms of activity. 4. Discussion The present study showed that EE housing prevents anxiety-like behavior triggered by acute restraint stress, assessed by means of the EPM. It is widely known that EE exerts a protective effect in several stress-induced mood disorders, including anxiety, however we showed here for the first time that the protective effect of EE on anxiety emerged immediately after the stressor cessation. This approach was designed to assess the CORT serum levels under which the animals performed the task and allowed us to verify that EE did not prevent the acute stress-induced rise in corticosterone serum levels. This finding opens new avenues to investigate the causal relationship between stress and anxiety, and the protective role of EE in this scenario, since most of the works has been attributed the beneficial role of EE to its capacity to modulate HPA axis response to stress (Fox et al., 2006). Although the protective effect of EE on anxiety triggered immediately after acute stress does not seem to result from its

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Fig. 3. EE and acute stress decrease CREB activation in the BLA of rats. Left, representative EMSA autoradiography image indicating, by arrow, CREB-specific band. Right, densitometric analysis of the specific bands showing that CS, E0 and ES animals presented reduction of DNA CREB binding in the rat BLA comparing to C0 animals (A). Results are represented as mean ± SEM (n ¼ 3e5 for each group). Two-way ANOVA followed by Newman-Keuls multiple comparison test. Significant difference between groups is indicated by * (p < 0.05). B, competition and supershift assays performed on samples from CS, E0 and ES of representative subject in the absence or presence of unlabeled specific (CREB consensus sequence 10-fold molar excess) or nonspecific (TFIID consensus sequence, 10-fold molar excess) oligonucleotide, as indicated. Supershift assays were performed with the same samples incubated with the presence of antibodies against subunits pCREB, CREB-1, and CREM (dilution, 1:20), as indicated.

capacity to changes the CORT release by the stressor, we found a substantial evidence of EE modulating GR signaling in the presence of stress in the BLA. Because this brain region is known as a key area involved in anxiety-like behavior, with a wide distribution of GR (Reul and de Kloet, 1985), the BLA has been studied in several researches devoted to elucidate the behavioral consequences of chronic and acute stress (Leuner and Shors, 2013) and also the anxiolytic effect of EE (Sztainberg et al., 2010). We showed here that 1 h of acute restraint stress promotes a substantial increase of GR nuclear translocation and that previous exposition to EE prevents this effect. Given their lipophilic characteristic, it is important to note that CORT released into the blood is able to cross the BloodBrain-Barrier, reaching the central nervous system, including BLA. Thus, it is possible to conclude that EE prevented nuclear translocation of GR apart of high CORT concentration saw in EE-stressed animals immediately after EPM task. Some of the intracellular mechanisms associated with GR responsiveness that could explain the effect of EE on GR translocation are changes in the expression of

the chaperone FKBP5 and in the state of receptor phosphorylation. Both alterations are associated with changes in subcellular GR localization (Cattaneo and Riva, 2016). Previous work has shown that both acute psychological stress and intraperitoneal infusion of CORT are accompanied by high BLA neuronal excitability (Rodriguez Manzanares PA; Kavushansky and Richter-Levin, 2006). Duvarci and Pare (2007), in turn, showed in ex vivo BLA slices a direct correlation between the increase of CORT signaling through GR nuclear activity and the enhanced neuronal excitability in the BLA (Duvarci and Pare, 2007). Furthermore, it was reported that anxiety-like behavior triggered by acute restraint stress is related to increase of BLA excitability (Guo et al., 2012), whilst electrical stimulation of BLA promotes anxiety-like behavior in rats (Nieminen et al., 1992; Saldivar-Gonzalez et al., 2003). The role of CREB activity in the neuronal excitability has been widely described, as well. Sustained basal overexpression of active CREB in several brain areas, including amygdala, increases the intrinsic neuronal excitability (Han et al., 2006; Lopez de Armentia

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Fig. 4. Western blot analysis of ERK-1/2 phosphorylation and EGR-1 expression in the BLA of rats. CS, E0 and ES animals showed reduction in the ratio between phosphorylated state and total form of ERK-1 (A) and ERK-2 (only ES, B) in the BLA comparing to C0 animals. EE-housed rats (E0 and ES) presented decrease of nuclear EGR-1 expression in comparison to CS animals, whilst there was no difference among E0, ES and C0 animals (C). Top (A) and right (C), representative autoradiography image of Western blot. Results are represented as mean ± SEM (n ¼ 5e6 for each group in A and B; n ¼ 4e5 in C). Two-way ANOVA followed by Newman-Keuls multiple comparison test. Significant difference between groups are indicated as * (p < 0.05) and **(p < 0.01).

et al., 2007; Viosca et al., 2009; Zhou et al., 2009), whereas sustained inhibition leads to the opposite effect (Jancic et al., 2009). Given the importance of BLA neuronal excitability to the emergency of anxiety-like behavior, and taking into account evidence showing the direct relationship between basal CREB overexpression in the BLA with the anxiety-like behavior (Wallace et al., 2004), we should consider the effect of EE over CREB activity in the BLA as one of the feasible protective mechanism against stress-induced anxiety. Although there was no augment in CREB signaling in the BLA of CS animals, as a signature of anxiety, we suggest that the behavioral

consequences resulting from both stress and EE were different from each other due to the specific time course of CREB activity modulation. If CREB activity reduction seen in EE animals would be due to chronic exposition to the environmental stimulus, thus the CREB activity modulation could be considered sustained. Accordingly, viral vector-mediated gene transfer approach, as used by Wallace et al. (2004), in addition of promoting sustained CREB overactivity in the BLA, induced anxiety-like behavior, whereas acute reduction of CREB activity in the BLA (as seen in CS animals), by inhibition of PKA, did not change the behavior in terms of anxiety

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(Pandey et al., 2003; Wallace et al., 2004). Our data regarding CREB activity in BLA are supported by the results relative to ERK-1/2 phosphorylation, one of the upstream regulators of CREB signaling (see Shaywitz and Greenberg, 1999). Although we did not see increase of ERK activity in CS animals, our results about EGR-1 indicate an augment in neuronal activity in these animals comparing to control. On the other hand, previous exposition to EE prevented the increase of EGR-1, suggesting that the final effect of EE on protection of the animals against anxiety triggered by stress could be by modulating the BLA neuronal excitability. Previous work showed that EGR-1 expression in cell culture treated with 1 h of CORT is dependent of nuclear GR translocation and independent of ERK activity (Revest et al., 2005). In addition, increased of ERK phosphorylation occurred only 3 h after CORT treatment. These data agree with our results as we have shown the increase of EGR-1 expression only in stressed animals, the same experimental group that showed increase in nuclear GR translocation. Moreover, even though increase of ERK activity in the BLA has been demonstrated after acute restraint stress (Maldonado et al., 2014), it is possible that the time point at which the animals were euthanized did not overlap the critical period of ERK phosphorylation. In agreement with these possibilities, Grisson and Bhatnagar (2011) also showed a decrement in ERK phosphorylation in the BLA immediately after acute restraint stress (Grissom and Bhatnagar, 2011). 5. Conclusion The present work provides important contribution toward unraveling the molecular mechanism by which EE prevents the anxiety-like behavior triggered immediately after 1 h of acute restraint stress. The findings suggest that the protective effect of EE could be independent of a possible action in prevent the acute stress-induced rise in corticosterone serum levels, drawing attention to its effect on hindering the GR nuclear translocation in the BLA caused by acute stress. In addition, we found that acute decrease of ERK-1/2 and CREB activity does not seem to exert influences in terms of anxiety-like behavior, but we argue that sustained decrease of those molecules activity could be a way that EE exert its protective role. The present results further substantiate the BLA neuronal hyperactivity as a cellular phenotype of anxiety and underscore the need for understand the mechanism that correlates the molecular changes in the BLA promoted by EE with changes in the neuronal responsiveness to stress. Acknowledgments We gratefully thank Dr. Ki Ann Goosens (Department of Brain and Cognitive Sciences, McGovern Institute for Brain Research, MIT, Cambridge, MA USA) for helpful discussions and English edition; Guiomar Wiesel for technical assistance and Dielly Lopes and Juliano Perfetti for helpful discussions. This work was supported by ~o de Amparo  research grants to C.D.M. from Fundaça a Pesquia do ~o Paulo (FAPESP, 2008/55178-0, 2012/24727-4, and Estado de Sa 2016/03572-3) and Conselho Nacional de Desenvolvimento Cien gico (CNPq: 479153/2009-4). L.S.N. and R.F$P.B. were tífico e Tecnolo supported by FAPESP (2010/13843-8 and 2011/50119-9, respectively); N$B$S was supported by CNPq(160570/2012-3); M.B.M. was supported by CAPES; C.D.M.; R.C.; and C.S. are research fellows from CNPq. The authors declare no competing financial interests. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuropharm.2016.10.026.

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