Enriched Environment Ameliorates Dexamethasone

Stress
The International Journal on the Biology of Stress
ISSN: 1025-3890 (Print) 1607-8888 (Online) Journal homepage: https://www.tandfonline.com/loi/ists20
Enriched environment ameliorates

dexamethasone effects on emotional reactivity
and metabolic parameters in mice
Eslen Delanogare, Raul Marin de Souza, Giovana Karoline Rosa, Fernando

Garcia Guanabara, Alex Rafacho & Eduardo Luiz Gasnhar Moreira
To cite this article: Eslen Delanogare, Raul Marin de Souza, Giovana Karoline Rosa, Fernando
Garcia Guanabara, Alex Rafacho & Eduardo Luiz Gasnhar Moreira (2020): Enriched environment
ameliorates dexamethasone effects on emotional reactivity and metabolic parameters in mice,
Stress
To link to this article: https://doi.org/10.1080/10253890.2020.1735344
Accepted author version posted online: 28

Feb 2020.
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Full title: Enriched environment ameliorates dexamethasone effects on emotional
reactivity and metabolic parameters in mice.
Running title: EE prevents dexamethasone behavior outcomes.
Eslen Delanogare2, Raul Marin de Souza2, Giovana Karoline Rosa1, Fernando Garcia
Guanabara3, Alex Rafacho1,4, Eduardo Luiz Gasnhar Moreira1,2,4*.
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1 2
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Departamento de Ciências Fisiológicas, Programa de Pós-Graduação em Neurociências,
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Hospital Universitário Polydoro Ernani de São Thiago, Programa de Pós-Graduaão Multicêntrico
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em Ciências Fisiolóicas, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC,
Brasil.
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*Corresponding author:
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Eduardo Luiz Gasnhar Moreira, PhD (eduardo.luiz@ufsc.br); Departamento de Ciências
Fisiológicas, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brasil.

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Phone: +55 48 3721 4617.

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Abstract
Convincing evidence shows that stress is associated with the development and course of
psychiatric and metabolic disorders. The hypothalamic-pituitary-adrenal (HPA) axis mediates the
stress response, a cascade of events that culminate in the release of glucocorticoids from the
adrenal cortex. Chronic hypercortisolism typically characterizes stress-related illnesses, such as
depression, anxiety, and metabolic syndrome. Considering previous studies pointing that
environmental enrichment (EE) mitigates the deleterious effects of stress on neurobiological
systems, we hypothesized that EE can confer resiliency against prolonged glucocorticoid
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administration-induced behavioral and metabolic alterations in mice. In this regard, three-month-
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old male Swiss mice were exposed to a four-week period of standard environment (SE) or EE.
After this period, still in the respective environments, dexamethasone was administered
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intraperitoneally (i.p.) at a dose of 4 mg/kg, for 21 consecutive days, in order to generate the
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emotional-related behavioral outcomes, as previously described. It is demonstrated herein that EE
prevents the dexamethasone-induced anxiety-like and passive stress-coping behaviors, as observed

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in the open field and tail suspension tests. Moreover, EE mitigated the hyperproteinemia and body
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weight loss induced by excess dexamethasone and decreased basal glucose levels. Taken together,
these results support the hypothesis that EE attenuates the effects of chronic administration of
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synthetic glucocorticoids in mice, a strategy that may be translated to the clinical perspective.
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Keywords: dexamethasone; environmental enrichment; behavior dysfunctions, metabolic

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dysfunctions.

Introduction
The stress-responsive hypothalamic-pituitary-adrenal (HPA) axis is causally implicated in the
development and course of major depressive disorder (MDD) (Keller et al., 2017), being described
that patients with depression present HPA-axis overactivity (De Kloet, Joëls & Holsboer, 2005).
The untoward consequences of excess cortisol are manifold, ranging from peripheral effects (e.g.,
glucose intolerance) to adverse effects on brain function. In this regard, hypercortisolemia, as seen
in Cushing’s disease or after prolonged glucocorticoid administration, can result in depression or
emotional lability (Brown et al., 2006; Newell-Price et al., 2006). For instance, chronic
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dexamethasone administration in adult life (Skupio et al., 2015) or during intrauterine
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development (Conti et al., 2017) induces a range of depressive-like symptoms in rodents, such as
anhedonia, behavioral despair, weight loss, and anxiety-like behavior.
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The ability to cope with stress is influenced by multiple factors such as early life experiences,
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gender, or personality traits (Chen & Baram, 2016). In fact, many environmental factors seem to
influence both the physiological functions of the central nervous system and its ability to
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counteract pathological changes. For instance, negative life events, such as the death of a parent,
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maternal deprivation, parental abandonment and separation of parents or divorce, can influence
the development of mental disorders, such as depression (Juruena, 2013). In laboratory animals,
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stress reduction may be achieved by enriching the animal’s environment with devices that
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promote its normal instinctive tendencies (Belz et al., 2003). In this regard, environmental
enrichment (EE) is an animal housing technique composed of increased space, physical activity,
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and social interactions, which in turn increases sensory, cognitive, motor, and social stimulation
(Nithianantharajah & Hannan, 2006). EE leads to improved cerebral health as defined by
increased neurogenesis, enhanced learning and memory and resistance to external cerebral insults
(Young et al., 1999). Moreover, living in an EE with physical and social stimulation also leads to
improved metabolic health (Cao et al., 2011). Considering previous studies pointing that EE

mitigates the deleterious effects of stress on neurobiological systems and endocrine profiles (Mitra
& Sapolsky, 2009; Lehmann & Herkenham, 2011), the present study hypothesized that EE would
prevent the development of behavioral (e.g., depressive and anxiogenic-like effects) and metabolic
alterations induced by chronic dexamethasone administration in mice. Thus, in order to evaluate
the preventative effects of EE, mice were reared in this condition prior to the dexamethasone
exposure.
Methods
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Animals
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Experiments were carried out on 2-month-old male Swiss albino mice from the animal
facility of Universidade Federal de Santa Catarina (UFSC, Florianópolis, Brazil), weighing
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around 45-55 g at the time of testing. They were kept in groups of 10 animals per cage in a room
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under controlled temperature (23 ± 1 °C) and subjected to a 12-h light cycle (lights on 6:00 a.m.)
with free access to food and water. All procedures comply with the guidelines on animal care of
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the local Ethics Committee on the Use of Animals (protocol 5497310718).

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Drug administration
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The synthetic glucocorticoid agonist dexamethasone (Laboratório Teuto, Brazil) was dissolved
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in saline and administered intraperitoneally (i.p.) at a dose of 4 mg/kg of body weight for 21
consecutive days. The dose of dexamethasone was chosen based on the study by Skupio and
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colleagues (2015), which demonstrated that chronic dexamethasone administration induces both a
range of depressive-like symptoms, such as behavior despair, weight loss, and anxiety-like
behavior. The control group received saline injection (0.1 mL per 10 grams of body weight). The
treatments occurred between 7:00 and 9:00 a.m.

Housing Conditions
The standard environment (SE) mice were housed in wire-topped clear plastic cages (38 cm ×
32 cm × 17 cm). The EE mice were housed in larger plastic boxes (44 cm × 32 cm × 18 cm),
containing a variety of stimuli, e.g., a running wheel, plastic tubing, ladders, rubber balls and a
wood shelter, as previously established in or laboratory (de Souza et al., 2019a). These items were
moved to different spatial locations of the cage every 3-4 days. The animals were maintained 24 h
per day in groups of 10 animals for both SE and EE conditions until the beginning and throughout
the duration of the behavioral experiments. Although in several studies the mice designated to the
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EE cohort were housed in larger groups to allow more social interactions, we kept the same
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number of animals per cage, as in previous studies of our research group (de Souza et al., 2019a).
Experimental design
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Forty male Swiss adult mice were divided into four experimental groups (10 mice per group):
standard environment plus saline (SE Saline), standard environment plus dexamethasone (SE
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Dexa), environmental enrichment plus saline (EE Saline) and environmental enrichment plus
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dexamethasone (EE Dexa). Animals were exposed to a 28-days period of SE or EE. After this
period, still in the respective environments, the animals received daily saline solution or
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dexamethasone, as previously described, for 21 consecutive days. Body mass was measured
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daily. On days 47 and 48, still on the influence of the above-mentioned treatments, the animals
were submitted to two behavioral tests: tail suspension test (day 47) and open field test (day 48).
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One day after the last behavioral test, mice were food-deprived for six hours (starting 7 a.m.)
and the glucose tolerance test was performed (day 49). A day after (day 50), mice were again
food-deprived for six hours, anesthetized with a mixture of ketamine (80 mg/kg, i.p.) and
xylazine (10 mg/kg, i.p.), and the blood was collected from the heart to determination of plasma

triglycerides, cholesterol, total protein, and corticosterone levels. On day 50, no dexamethasone
was administered (Figure 1).
Behavioral tasks
All experiments were conducted during the light phase (1:00–6:00 p.m.). Experiments were
conducted in a controlled-temperature room (23°C, the humidity was between 40% - 60%), with
low-light intensity (12 lx). Moreover, they were recorded (webcam Microsoft VX 3000) and the
videos were analyzed using the Ethowatcher® software.
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Tail Suspension Test (TST)
The TST measures coping strategy to an acute inescapable stress (Commons et al., 2017). This
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test was developed by Steru and colleagues (1985) based on the premise that an animal subjected
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to a stressful and inescapable situation presents two types of alternating behaviors, the agitation
characteristic of the attempt to escape from the stressful situation, and immobility. This pattern of
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behavior can also be called searching-behavior, characterized by the alternation of intense motor
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activity and energy expenditure with immobility. Mice both acoustically and visually isolated
were suspended 30 cm above the floor by adhesive tape placed approximately 1 cm from the tip of
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the tail. Immobility time was manually recorded during a 6-min period by an experienced
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observer. The observer was in the room where experiments were performed and were blind to the
animal condition.
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Open Field Test (OFT)
The open field was used to evaluate the locomotor and exploratory activities induced by a
novel environment. The natural tendency of the animal in a new environment is to exploit it,
despite the stress and conflict caused by the new environment (Prut & Belzung, 2003). This test

assess unconditioned emotional reactions based on natural approach/avoidance conflict (Ramos,
2008). The anxiety-related behavior in the OFT is triggered by two factors: individual testing and
agoraphobia. Higher levels of anxiety should mainly lead to decreases in the ratio ‘number of
squares visited in center/number of squares visited on periphery’. The apparatus, made of wood
and covered with impermeable Formica, had 50 cm wide × 50 cm deep × 40 cm high. Each
mouse was placed in the center of the open field and allowed to freely explore the apparatus for 5
min. The following behavioral parameters were evaluated: % of crosses in the center, total number
of crosses and time in the center.
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Biochemical analysis
The glucose tolerance test was assessed by i.p. administration of D-(+)-glucose (Sigma Aldrich,
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St Louis, MO) at a concentration of 2 g/kg body weight. Blood glucose was measured from the
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tail tip at baseline and 15, 30, 60, and 120 minutes after glucose overload using a glucometer
(Accu-Chek Performa®). For the area under the curve (AUC) calculation, it was used the mean
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baseline value (time 0) for each group (baseline for the area). Total cholesterol, triglycerides, and
total proteins were measured in plasma using the enzymatic kit according to the manufacturer’s
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instructions (Gold Analisa, Belo Horizonte, MG, Brazil). The results were expressed as mg/dl.
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Statistical analysis
The results are presented as mean + standard error of the mean (SEM). Statistical analyses
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were carried out using a two-way or three-way analysis of variance (ANOVA). Following
significant ANOVAs, multiple post hoc comparisons were performed using Newman Keul’s
test. P values less than 0.05 (P < 0.05) were considered as indicative of significance. All tests
were performed using the STATISTICA ® software package (StatSoft Inc, Tulsa, OK, USA).

Results
Open Field Test (OFT)
The two-way ANOVA indicated a significant main effect for treatment [F(1, 36) = 5.18, p <
0.05] and for housing conditions by treatment interaction [F(1, 36) = 6.93, p < 0.05] on center time
(Fig 2a). Subsequent post hoc comparisons revealed a significant decreased in the time spent in
the center of the apparatus in the SE Dexa group in comparison with the SE Saline group and with
the EE Dexa group (p < 0.05). On the other hand, the two-way ANOVA indicated no significant
effects on the percentage of center crossings (Fig 2b) and on the total crossings (Fig 2c).
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Tail Suspension Test (TST)
The two-way ANOVA indicated no significant effects on the latency to immobility (Fig 3a).
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On the other hand, the two-way ANOVA indicated a significant main effect for treatment [F(1,
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36) = 8.58, p < 0.05] and for housing conditions by treatment interaction [F(1, 36) = 9.65, p <
0.005] on the immobility time in the TST. Subsequent post-hoc analysis revealed a significant
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increase in the immobility time in the SE Dexa group when compared with the SE Saline group
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and with the EE Dexa group (p < 0.05) (Fig 3b).

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Metabolic analysis
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The two-way ANOVA revealed a significant main effect for treatment [F(1, 36) = 27.98, p <
0.0005] and for housing conditions by treatment interaction [F(1, 36) = 4.06, p < 0.05] on body
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mass variation (Fig 4a). Subsequent post-hoc analysis revealed a significant loss of body mass (%)
in the SE Dexa group when compared with the SE saline group and with the EE Dexa group (p <
0.05).
The two-way ANOVA analysis indicated a significant main effect for treatment [F(1, 36) =
153.86, p < 0.0005] on plasma cholesterol concentration (Fig 4b). Subsequent post-hoc analysis

revealed a significant elevation on plasma cholesterol concentrations in both the SE Dexa group
and the EE Dexa group when compared with their respective saline control groups (p < 0.05). On
the other hand, the two-way ANOVA indicated a significant main effect for treatment [F(1, 36) =
5.12, p < 0.05] and for housing conditions by treatment interaction [F(1, 36) = 6.26, p < 0.05] on
plasma triglyceride concentration (Fig 4c). Subsequent post-hoc analysis revealed a significant
decrease in triglycerides concentrations in the SE Dexa group when compared with the SE Saline
group and with the EE Dexa group (p < 0.05). Moreover, the two-way ANOVA revealed a
significant main effect for treatment [F(1, 36) = 5,85, p < 0.05] and for housing conditions by
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treatment interaction [F(1, 36) = 5,61, p < 0.05] on plasma total protein concentration. Subsequent
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post-hoc analysis revealed a significant increase in total protein concentration in the SE Dexa
group when compared with the SE Saline group and with the EE Dexa group (p < 0.05) (Fig 4d).
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The two-way ANOVA analysis indicated a significant main effect for housing conditions [F(1,
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36) = 10.72, p < 0.005] on basal glucose levels (Fig 4e). Subsequent post-hoc analysis revealed a
significant decrease on basal glucose levels specifically in the EE Dexa group when compared
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with its respective saline control group (p < 0.05). The three-way ANOVA indicated significant
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main effects for housing conditions [F(1, 180)= 11.68, p < 0.001], treatment [F(1, 180)= 4.81, p <
0.05], time [F(1, 180)= 11.68, p < 0.0001], and housing conditions by treatment interaction [F(1,
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180)= 11.35, p < 0.0005] (Fig 4f). Subsequent post-hoc comparisons indicated no significant
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differences among groups at each time point. On the other hand, the two-way ANOVA revealed a
significant main effect for treatment [F(1, 36) = 5.81, p < 0.05] on the area-under-the-glucose-
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curve (AUC) (Fig 3g) that was based on blood glucose values from Figure 4g.

Discussion
The present study aimed to investigate the effects of EE on dexamethasone-induced behavioral
reactivity and metabolic disturbances in mice. Our findings that chronic dexamethasone treatment
induces anxiety-like and passive stress-coping behaviors in mice replicate the findings of the study
conducted by Skupio et al. (2015), upon which our experimental protocol was based. Regarding
the metabolic effects of dexamethasone, mice presented weight loss, decreased plasma
triglycerides levels, increased total plasma proteins, and cholesterol levels and glucose intolerance.
Of importance, we demonstrated that EE blunted the dexamethasone-induced behavioral reactivity
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in the TST and OFT, respectively. EE also mitigated dexamethasone-induced weight loss,
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hypotriglyceridemia, and hyperproteinemia.
Herein, chronic dexamethasone treatment-induced behavioral despair and anxiety-like behavior
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in mice, as observed in the TST and OFT, respectively. These findings agree with data observed in
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the literature. There is compelling evidence that dexamethasone administration, in an acute or
chronic regimen, at doses varying from 16 µg/kg to 4 mg/kg, induces depressive-like and anxiety-
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like behavior in mice (Wróbel et al., 2014; Skupio et al., 2015; De Souza et al., 2019b). Moreover,
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Pazini et al. (2017) have shown that swiss mice treated with corticosterone, at a dose of 20 mg/kg,
for 21 days, showed an increase in the immobility time in the TST. The authors associated such
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depressive-like effect with decreased hippocampal cell proliferation and neuronal differentiation
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and increased glial fibrillary acid protein (GFAP) immunostaining (suggestive of astrogliosis) in
dentate gyrus (DG) of the hippocampus (Pazini et al., 2017). Moreover, Zhang and colleagues
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have shown that C57Bl/6 mice treated with corticosterone, at a dose of 40 mg/kg, for 35 days
showed significantly less time spent in the center zone of the OFT (Zhang et al., 2016). The
behavioral despair and anxiety-like behavior seen here probably reflect the spectrum of mood
disorders observed in patients with hypercortisolemia, as in Cushing’s syndrome (Newell-Price et
al., 2006) and the common comorbidity of depression and anxiety observed in patients (Wu &

Fang, 2014). In the present study, it was found that treatment with EE mitigated both behavioral
despair and anxiety-like behavior induced by dexamethasone in the TST and OFT, respectively. In
this regard, previous studies reported that EE decreases corticosterone concentrations in rodents
(Belz et al., 2003). In an elegant study, Sztainberg and colleagues (2010) demonstrated that EE
significantly reduced CRHr-1 mRNA expression in limbic regions, suggesting a molecular
mechanism that might explain the EE effects on decreasing corticosterone concentrations: the
downregulation of this receptor decreases the release of ACTH by adenohypophysis and,
consequently, decreases the release of corticosterone by the adrenal cortex. Furthermore, Hare and
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colleagues (2014) showed that four weeks of voluntary wheel running resulted in a shorter time to
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peak corticosterone concentration and a more rapid decay of plasma corticosterone levels
following restrain stress in mice. The authors conclude that exercise renders animals particularly
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resilient, capable of producing a robust response to stress while limiting the damage that might
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result from overexposure to glucocorticoids during periods of stress (Hare et al., 2014). Indeed,
numerous clinical and experimental studies demonstrated that many environmental factors, such
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as EE, may affect both the physiological functions of the central nervous system and its ability to
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counteract pathological changes (Van Praag, Kempermann & Gage, 2000). Of note, the changes
provided following EE exposure are commonly described as experience-dependent, indicating that

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they result from active interaction between the animal and the affordances available in the
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environment (Nithianantharajah & Hannan, 2006).
Herein it was observed that chronic dexamethasone treatment induced a significant metabolic
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dysfunction, characterized by glucose intolerance, decreased body weight and plasma triglyceride
levels, as well as increased plasma total protein and cholesterol levels. The increase in plasma total
protein concentrations is related to the catabolic properties of glucocorticoids, i.e., increasing
protein degradation and decreasing protein synthesis in skeletal muscle (Morgan et al., 2016),
which may also support the weight loss observed in dexamethasone-treated rodents (Tonolo et al.,

1988; Noh et al., 2014). This reduction in body weight during dexamethasone treatment is also
related to a negative water balance (Thunhorst et al., 2007) and higher levels of anorexigenic
insulin levels (Protzek et al., 2014). The dexamethasone treatment also induced a significant
decrease in plasma triglyceride concentration. However, in this case, our results diverge from prior
findings (Barbosa et al., 2016). For instance, dexamethasone treatment, at a dose of 0.5 mg/kg for
15 days, resulted in a significant increase in triglyceride levels in rats (Barbosa et al., 2016).
Although most data have shown that glucocorticoids induce lipolysis and consequently, plasma
free fatty acid increases (Divertie et al., 1991), the effects of glucocorticoids on lipid mobilization
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are still controversial. For instance, some studies report an inhibitory effect of cortisol on lipolytic
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activity (Ottosson et al., 2000). Of note Chen and colleagues (2018) found a significant decrease
of triglyceride in plasma and in the liver of goats after intramuscular administration of
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dexamethasone, at a concentration of 0.2 mg/kg for 21 days. Finally, the significant increase in
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plasma cholesterol concentration here observed is in accordance with previous studies. For
instance, Kumar and colleagues (2011) showed that dexamethasone administration, at a dose of 10
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mg/kg for 8 days, increased the plasma cholesterol levels in rats. Most importantly herein, we
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demonstrated that EE mitigated the dexamethasone effects on body weight, plasma triglycerides,
and total protein levels. EE has a general tendency to increase activity and activity energy
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expenditure in rodents, which has downstream effects on other aspects of energy balance (Novak
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et al., 2012). In this regard, it is noteworthy that our EE protocol included a running wheel, and
since physical exercise is associated with increased protein synthesis (Tipton & Wolfe, 2001), one
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may associate with the maintenance of body mass observed in EE dexamethasone-treated mice.
After ~ 3 weeks in running well, skeletal muscles (gastrocnemius, tibialis anterior, triceps) of male
mice show enhanced citrate synthase activity, a validated biomarker for mitochondrial density in
skeletal muscle, resulting in better endurance capacity (Manzanares et al., 2018). Moreover, Barel

and colleagues showed that run on a treadmill attenuated the dexamethasone-induces muscular
glycogen loss, muscular atrophy and body weight loss in rats (Barel et al., 2010).
Glucocorticoids, per se, are considered diabetogenic agents, as they may promote an increase in
hepatic glucose production and a decrease in peripheral uptake (e.g., in muscle and adipose
tissues) (Pasieka and Rafacho, 2016). Because of such properties, up to 60% of patients with
Cushing’s syndrome, characterized by hypercortisolemia, have glucose intolerance (Newell-Price
et al., 2006). The glucose intolerance caused by dexamethasone treatment in our study is in
agreement with previous studies (Pandey et al., 2014; Lee et al., 2018). The reduction of insulin-
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stimulated protein kinase B phosphorylation in skeletal muscle seems to be consensual at this
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chronic dexamethasone exposure (Pandey et al., 2014, Lee et al., 2018), which can imply in
reduced glucose disposal. Despite to be not effective in preventing the glucose intolerance caused
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by glucocorticoids, EE significantly improved the basal glycemic values and kept blood glucose
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levels bellow of that observed in dexamethasone-treated mice in SE during all period of the test.
In this regard, McMurphy and colleagues (2018) observed that EE exposure induces a robust
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reduction of brown and white adipose tissue, in addition to decreasing hepatic steatosis, hepatic
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glucose production and increasing glucose uptake by the liver and adipose tissue, contributing to
glycemic control in mice. Specifically, the authors demonstrated that EE induced a significant
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reduction in the expression of two important enzymes involved in the gluconeogenesis pathway,
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phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6-phosphatase (G6PC) (McMurtphy et
al., 2018). The apparent lack of effect of EE upon beta cell function may be due to the genomic
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impact of dexamethasone on insulin secretory machinery of beta cells (Lambillotte et al., 1997).
Although we do not discard new approaches of EE aiming to be more effective, we consider EE as
a valuable tool for improvement of neurometabolic outcomes as previously demonstrated in a
model of glucose intolerance associated to hypercholesterolemic diet where EE mitigated the
glucose intolerance (De Souza et al., 2019a).

An inevitable limitation of our study is that multiple sensory, social, and physical activity
factors are typically combined in the EE treatment, making it difficult to determine which of the
various enrichment factors or the interaction contributes to the observed effects on behavioral and
metabolic outcomes. In this regard, it is not possible to distinguish between the effects of EE vs
exercise alone, since a running wheel was provided. Of note, McMurphy and colleagues (2018)
evaluated the extent to which physical activity is the responsible for the EE-induced healthy aging.
In that study, the gene expression profile of the major organs involved in systemic glucose
homeostasis (liver, fat and muscle) revealed distinct patterns between EE and running wheel
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animals, where animals exposed to running wheel showed minor changes in these parameters
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(McMurphy et al., 2018). Thus, it is noteworthy that no social interaction, novelty, or any other
single variable can account for all of the effects of EE (van Praag et al., 2000).
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Conclusion
Collectively, our results reproduce the depressive- and anxiety-like behavior caused by
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supraphysiologic doses of dexamethasone in Swiss male mice, as well as metabolic disturbances

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(glucose intolerance, hyperproteinemia, hypercholesterolemia, and body weight loss). Most
importantly, EE mitigates the depressive- and anxiety-like behaviors. Moreover, our findings
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suggest that EE is able to promote healthier metabolic function in mice, even in situations of
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chronic administration of synthetic glucocorticoids. It is noteworthy, however, that although our
results reinforce the positive infuences of EE on stress response, the interpretation of our data
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should be seen as prophylactic rather than therapeutic since EE started before the dexamethasone
treatment. Taken together, our results support the hypothesis that EE enhances resilience to
stressors, such as the chronic administration of synthetic glucocorticoids used herein, a strategy
that may be translated to the clinical perspective. Both, behavioral and metabolic improvements,
are of particularly importance, since the prevalence of metabolic disturbance is 58% higher in

psychiatric patients than in the general population, which suggests that metabolic disturbance is a
general comorbidity seen in different psychiatric patient groups, including patients with major
depressive disorder (Vancampfort et al., 2015) and anxiety disorder (Tang et al., 2017).
Acknowledgments
Research supported by grants from the Brazilian funding agencies: CAPES, CNPq (Universal
424799/2018-9) and FAPESC.
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Declaration of interest
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The authors have no financial or personal conflicts of interest related to this work.
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Figure Legends
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Figure 1. Experimental design.
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Figure 2. EE mitigates the dexamethasone-induced anxiety-like behavior in the open field test.
(A) Center time (s), (B) Percentage of center crossings, (C) Total crossings. Data are expressed as
mean + SEM (n = 10 animals per group). *p < 0.05 vs. Saline/SE group and &p < 0.05 vs.
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Dexa/SE group (two-way analysis of variance followed by Newman–Keuls post-hoc test). Dexa
(dexamethasone); SE (standard environment); EE (environmental enrichment).
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Figure 3. EE mitigates the dexamethasone-induced behavioral despair in the tail suspension test.
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(A) Latency to immobility (s) and (B) Immobility time (s). Data are expressed as mean + SEM
(n = 10 animals per group). *p < 0.05 vs. Saline/SE group and &p < 0.05 vs. Dexa/SE group (two-
way analysis of variance followed by Newman–Keuls post-hoc test). Dexa (dexamethasone); SE
(standard environment); EE (environmental enrichment).

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Figure 4. EE effects on dexamethasone-induced metabolic alterarions. (A) Body mass variation
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(%), (B) Plasma cholesterol levels, (C) Plasma triglycerides levels, (D) Plasma total proteins
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levels, (E) Basal glucose levels, (F) Glucose tolerance test, and (F) Area under curve of GTT. Data
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are expressed as mean + SEM (n = 10 animals per group). *p < 0.05 vs. Saline/SE group and &p <
0.05 vs. Dexa/SE group (two-way analysis of variance followed by Newman–Keuls post-hoc test).
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£p < 0.05 treatment factor (two-way analysis of variance followed). #p < 0.05 environment factor
(two-way analysis of variance). Dexa (dexamethasone); SE (standard environment); EE

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(environmental enrichment).
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Stress

The International Journal on the Biology of Stress

ISSN: 1025-3890 (Print) 1607-8888 (Online) Journal homepage: https://www.tandfonline.com/loi/ists20

Enriched environment ameliorates

Eslen Delanogare, Raul Marin de Souza, Giovana Karoline Rosa, Fernando

To link to this article: https://doi.org/10.1080/10253890.2020.1735344

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reactivity and metabolic parameters in mice.

Running title: EE prevents dexamethasone behavior outcomes.

Guanabara3, Alex Rafacho1,4, Eduardo Luiz Gasnhar Moreira1,2,4*.

Eduardo Luiz Gasnhar Moreira, PhD (eduardo.luiz@ufsc.br); Departamento de Ciências

Fisiológicas, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brasil.

Phone: +55 48 3721 4617.

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adrenal cortex. Chronic hypercortisolism typically characterizes stress-related illnesses, such as

environmental enrichment (EE) mitigates the deleterious effects of stress on neurobiological

systems, we hypothesized that EE can confer resiliency against prolonged glucocorticoid

prevents the dexamethasone-induced anxiety-like and passive stress-coping behaviors, as observed

Keywords: dexamethasone; environmental enrichment; behavior dysfunctions, metabolic

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The stress-responsive hypothalamic-pituitary-adrenal (HPA) axis is causally implicated in the

in Cushing’s disease or after prolonged glucocorticoid administration, can result in depression or

anhedonia, behavioral despair, weight loss, and anxiety-like behavior.

(Nithianantharajah & Hannan, 2006). EE leads to improved cerebral health as defined by

Yuri Santos Novais - novaisyuri5@gmail.com - CPF: 866.508.285-99

alterations induced by chronic dexamethasone administration in mice. Thus, in order to evaluate

facility of Universidade Federal de Santa Catarina (UFSC, Florianópolis, Brazil), weighing

the local Ethics Committee on the Use of Animals (protocol 5497310718).

treatments occurred between 7:00 and 9:00 a.m.

Yuri Santos Novais - novaisyuri5@gmail.com - CPF: 866.508.285-99

Yuri Santos Novais - novaisyuri5@gmail.com - CPF: 866.508.285-99

was administered (Figure 1).

videos were analyzed using the Ethowatcher® software.

Open Field Test (OFT)

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of crosses and time in the center.

Yuri Santos Novais - novaisyuri5@gmail.com - CPF: 866.508.285-99

Open Field Test (OFT)

and with the EE Dexa group (p < 0.05) (Fig 3b).

Yuri Santos Novais - novaisyuri5@gmail.com - CPF: 866.508.285-99

Yuri Santos Novais - novaisyuri5@gmail.com - CPF: 866.508.285-99

The present study aimed to investigate the effects of EE on dexamethasone-induced behavioral

Of importance, we demonstrated that EE blunted the dexamethasone-induced behavioral reactivity

Herein, chronic dexamethasone treatment-induced behavioral despair and anxiety-like behavior

disorders observed in patients with hypercortisolemia, as in Cushing’s syndrome (Newell-Price et

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significantly reduced CRHr-1 mRNA expression in limbic regions, suggesting a molecular

downregulation of this receptor decreases the release of ACTH by adenohypophysis and,

provided following EE exposure are commonly described as experience-dependent, indicating that

environment (Nithianantharajah & Hannan, 2006).

protein concentrations is related to the catabolic properties of glucocorticoids, i.e., increasing

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