Psychology & Neuroscience © 2017 American Psychological Association

2017, Vol. 10, No. 2, 225–242 1983-3288/17/$12.00 http://dx.doi.org/10.1037/pne0000081

Effects of Environmental Enrichment in a Mouse Model of

Posttraumatic Stress Disorder

Marília Bazan Blanco Azair Canto-de-Souza

and Rimenez Rodrigues de Souza Universidade Federal de São Carlos and
Universidade Federal de São Carlos Neuroscience and Behavioral Institute, Ribeirão
Preto, Brazil
This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.

Posttraumatic stress disorder (PTSD) may appear after an individual experiences a

This document is copyrighted by the American Psychological Association or one of its allied publishers.

stressful situation. It is characterized by the avoidance of situations that are related

to the trauma and persistent hyperarousal, which may become associated with
anxiety and cognitive disability. The present study evaluated the effects of envi-
ronmental enrichment on anxietylike behavior and memory in a mouse model of
PTSD that employed different reexposures to situational reminders (SRs; 1 or 10
min). Male Swiss mice were exposed or not exposed to environmental enrichment
and then subjected to the model of PTSD in a light-dark box. After exposure to the
aversive stimulation, the animals were evaluated in a light-dark box, a modified
light-dark box, the elevated plus maze (EPM), and the novel object recognition test.
Environmental enrichment attenuated the effects of footshock, did not alter anxi-
etylike behavior or memory in animals that were exposed to 1-min SRs, increased
anxietylike behavior and memory in animals that were exposed to 10-min SRs, and
reversed behavioral sensitization in the EPM. The results suggest that environmen-
tal enrichment does not mitigate the effects of reexposure to 1-min SRs on
traumatic memory and favors the maintenance of traumatic memories, even when
animals are reexposed to SRs for 10 min. However, environmental enrichment
reversed the effects of the aversive stimulation on behavioral sensitization. The effects of
environmental enrichment in the present model of PTSD are complex and depend on the
duration of SRs and the specific behaviors that are assessed.

Keywords: PTSD, situational reminders, environmental enrichment, aversive memory

PTSD can occur after an individual experi-
ences a stressful situation, such as a natural
disaster, physical violence, or an accident. It is
typically characterized by reliving the trauma
through nightmares and thoughts, the avoidance
of situations that remind the individual of the
trauma, and persistent hyperarousal (Pitman,
1989; Yehuda, 2000). It may also be associated
with the development of depression, excessive
This article was published Online First April 27, 2017.
Marília Bazan Blanco and Rimenez Rodrigues de Souza,
Graduate Program in Psychology, Universidade Federal de
São Carlos; Azair Canto-de-Souza, Department of Psychol-
ogy, Universidade Federal de São Carlos, and Neuroscience
and Behavioral Institute, Ribeirão Preto, Brazil.
Correspondence concerning this article should be addressed
to Marília Bazan Blanco, who is now at Universidade Estadual
do Norte do Paraná, Rodovia PR 160 - s/n, Km 0, CEP:
86300-000, Cornélio Procópio, Paraná, Brazil. E-mail:
mariliabazan@uenp.edu.br
225
anxiety, and cognitive disability (Bremner et al.,
1993). At the neurological level, a reduction of
hippocampal volume (Gross & Hen, 2004), ex-
aggerated amygdala responsivity, and impair-
ments in negative inputs from the ventromedial
prefrontal cortex to the amygdala (Likhtik, Pel-
letier, Paz, & Paré, 2005; Phelps, Delgado,
Nearing, & LeDoux, 2004) are at the core of
PTSD. This is generally followed by a failure of
the organism to return to normal levels of stress
hormones after the stressful event, with dys-
regulation of the hypothalamic-pituitary-
adrenal (HPA) axis and other stress systems
(Rauch, Shin, & Phelps, 2006).
According to the cognitive-behavioral model,
PTSD develops through classic Pavlovian fear
conditioning, in which the traumatic experience
results in an association between an uncondi-
tioned stimulus (US) that evokes intense fear
and perceived contextual cues (conditioned
 226 BLANCO, DE SOUZA, AND CANTO-DE-SOUZA
stimuli [CSs]) of the situation that later elicit
conditioned emotional responses. The mainte-
nance of PTSD can rely on a form of operant
conditioning. The avoidance of reminders of the
trauma (CSs) usually generate temporary relief,
supporting negative reinforcement and likely
preventing extinction of the conditioned re-
sponses (Brewin & Holmes, 2003; Knapp &
Beck, 2008). This hypothesis was confirmed by
studies in humans that showed that PTSD might
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involve excessive fear memory acquisition and
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consolidation, exaggerated conditioned fear re-
sponses to trauma-associated cues, and impair-
ments in fear extinction (Bremner et al., 2005;
Fani et al., 2012; Rauch et al., 2006). This
suggests that the establishment of PTSD might
involve the dysregulation of memory systems.
Numerous studies have suggested that long-
term exposure to reminders of fear memory
leads to the extinction of conditioned emotional
responses (Cammarota et al., 2005; de Quer-
vain, Aerni, Schelling, & Roozendaal, 2009; de
Quervain et al., 2011; Orsini & Maren, 2012).
Stress that is induced by fear retrieval can trig-
ger further excessive consolidation of the trau-
matic memory (Louvart et al., 2006; Pynoos,
Ritzmann, Steinberg, Goenjian, & Prisecaru,
1996), generalization, behavioral sensitization
(Siegmund & Wotjak, 2006, 2007), higher-
order fear conditioning (Paschall & Davis,
2002; Sears, Baker, & Frey, 1979; Souza, Dal
Bó, de Kloet, Oitzl, & Carobrez, 2014), and
impaired extinction (Fani et al., 2012; Sieg-
mund, Langnaese, & Wotjak, 2005; Stiedl et al.,
1999). Using an elegant experimental approach
in rats, Pynoos and colleagues (1996) demon-
strated that repeated exposure to SRs of intense
footshock fear conditioning led to observable
behavioral disturbances during each reminder
session and enhanced the startle reflex follow-
ing exposure to the reminders, suggesting the
emergence of an abnormal emotional state (Py-
noos et al., 1996). Similarly, Louvart and col-
leagues (Louvart, Maccari, Ducrocq, Thomas,
& Darnaudery, 2005, Louvart et al., 2006)
showed that exposing rats to SRs of a traumatic
event produced a long-term increase in anxiety-
like behavior, an increase in aversion to the
conditioned context, alterations in social inter-
action, blunted corticosterone release, an in-
crease in HPA feedback in response to a stres-
sor, an increase in mineralocorticoid receptor
mRNA in the hippocampus, and an increase in
corticotropin-releasing factor mRNA in the
paraventricular nucleus of the hypothalamus.
Collectively, these findings suggest that reexpo-
sure to trauma-associated reminders might con-
tribute to disturbances that are seen in PTSD as
a kind of dominolike effect.
Human studies have shown that exposure to
combinations of learning challenges, social net-
works, and physical exercise promotes resil-
ience to and recovery from stress (Hutchinson et
al., 2012). Environmental enrichment consists
of exposing young or adult animals for weeks or
months to environments that are rich in sensory
stimulation, physical exercise, and social rela-
tions (Diamond, 2001; Diamond, Rosenzweig,
Bennett, Lindner, & Lyon, 1972; Fernández-
Teruel et al., 2002; Leggio et al., 2005). Animal
studies that employ environmental enrichment
have demonstrated that it has buffering effects
against stressors and beneficial effects in animal
models of mental illness (Hendriksen, Olivier,
& Oosting, 2014; Hendriksen, Prins, Olivier, &
Oosting, 2010; Hutchinson et al., 2012; Pang &
Hannan, 2013). Moreover, environmental en-
richment has been shown to lead to improve-
ments in learning and memory (Arnaiz et al.,
2004; Bruel-Jungerman, Laroche, & Rampon,
2005; Fernández-Teruel et al., 2002; Soffié,
Hahn, Terao, & Eclancher, 1999) and facilitate
problem-solving strategies (Diamond, 2001;
Fernández-Teruel et al., 2002). Environmental
enrichment has also been shown to prevent ag-
ing-related cognitive impairments (Blanco,
Gon, & Estanislau, 2012; Harati et al., 2011)
and have antidepressantlike (Brenes, Padilla, &
Fornaguera, 2009; Brenes, Rodriguez, & Forna-
guera, 2008) and anxiolyticlike (Benaroya-
Milshtein et al., 2004); (Duman, Schlesinger,
Russell, & Duman, 2008) effects. To date, how-
ever, no reports have described the specific ef-
fects of environmental enrichment on behav-
ioral changes associated with chronic exposure
to traumatic reminders. The present study eval-
uated the long-term behavioral effects of envi-
ronmental enrichment in a mouse model of
PTSD.
Materials and Method
Animals
One hundred one male Swiss mice, 21 days
of age at the beginning of the study, were used.
 EFFECTS OF ENVIRONMENTAL ENRICHMENT ON PTSD
The animals were obtained from the vivarium of
the Universidade Federal de São Carlos
(UFSCar) and housed in the colony room in the
laboratory during the experimental procedures.
The animals were housed either with 10 animals
per homecage (41 cm ⫻ 34 cm ⫻ 16 cm) or
8 –10 animals per enriched chamber. They were
maintained under controlled temperature (24°C
⫾ 1°C) and humidity (55% ⫾ 5%) and a 12
h/12 h light/dark cycle (lights on at 7:00 a.m.).
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The animals had free access to food and water,
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except during the test sessions. The experiments
were performed during the light phase of the
light/dark cycle. The experiments were per-
formed in compliance with the recommenda-
tions of the National Council for the Control of
Animal Experimentation, and all of the proce-
dures were approved by the Ethics Committee
on Animal Use of UFSCar (Protocol No. 010/
2012).
Apparatus
Environmental enrichment. The enriched
environment consisted of a metal chamber (38
cm ⫻ 42.5 cm ⫻ 26 cm) that was divided into
three floors that were connected by stairs
(American Pets, Guarulhos, Brazil). Inside the
chambers were colored tubes that gave access to
different stimuli: an exercise wheel, a colorful
house, a drinking fountain, and two food dis-
pensers that were placed in different locations
of the chamber. Once per week, two different
objects were placed inside the chamber: colored
balls of different sizes, fitted playing pieces,
plastic toys, bells, a pipe, balls, and a brush with
soft bristles (Blanco et al., 2012; Simpson &
Kelly, 2011; Sztainberg & Chen, 2010).
Light-dark box test. The light-dark box
(Insight, Ribeirão Preto, Brazil) was con-
structed of dark acrylic (60 cm ⫻ 47 cm ⫻ 29
cm). The light-dark box test was performed as
previously described (Bourin & Hascoët, 2003).
The light compartment (28 cm ⫻ 47 cm ⫻ 29
cm) and dark compartment (20 cm ⫻ 47 cm ⫻
29 cm) were connected by a guillotine door (7
cm ⫻ 7 cm). The floor consisted of 35 metal
bars that were connected to a shock generator
(Insight, Ribeirão Preto, Brazil). The ceiling of
the light compartment was illuminated by a
light-emitting diode lamp (275 lux) that could
be turned on and off manually.
227
Modified light-dark box. The modified
light-dark box was constructed of wood (50
cm ⫻ 40 cm ⫻ 60 cm). The light compartment
(50 cm ⫻ 40 cm ⫻ 30 cm) and dark compart-
ment (50 cm ⫻ 40 cm ⫻ 30 cm) were connected
by a guillotine door (10 cm ⫻ 10 cm). The light
compartment had walls with vertical black
stripes and was illuminated by a 300 lux light
(Bourin & Hascoët, 2003).
EPM. The EPM was constructed of wood
(floor) and transparent glass (clear walls). The
maze consisted of four arms: two open arms (30
cm ⫻ 5 cm ⫻ 0.25 cm) and two closed arms (30
cm ⫻ 15 cm ⫻ 5 cm) that extended from a
common central platform (5 cm ⫻ 5 cm). The
maze was elevated 38.5 cm above the floor
(Lister, 1987). All of the tests were conducted
under moderate illumination (77 lux, measured
on the central platform).
Novel object recognition test. The novel
object recognition test apparatus consisted of a
clear acrylic round arena (28 cm diameter, 30
cm height (Insight, Ribeirão Preto, Brazil). The
objects consisted of two identical metal objects
(4 cm height, 3 cm diameter; Objects A and B)
and an opaque glass bottle (8.5 cm height, 3.5
cm diameter; Object C; Akkerman et al., 2012).
Experimental Design
Environmental enrichment. Nonenriched
animals (control) were housed in standard
homecages (8 –10 animals per cage). Enriched
animals were housed in enriched homecages
(8 –10 animals per cage) as previously de-
scribed. The animals were kept in their respec-
tive chambers for 32 days before the aversive
procedure began. The same housing conditions
were maintained during the entire experimental
protocol.
Aversive procedure.
Aversive conditioning. At the beginning of
this procedure, the animals were 53 days old. On
the day of conditioning, the animals were individ-
ually placed in the center of the dark side of the
light-dark box where they were confined for 2
min. After this period, the guillotine door was
opened, allowing the animals to explore the light
compartment of the apparatus. Upon the animals’
entry to the light compartment, the light was
turned on. As soon as the animal returned to the
dark compartment, the guillotine door was closed,
and a 10-s footshock was delivered (0.5 mA,
 228 BLANCO, DE SOUZA, AND CANTO-DE-SOUZA

scrambled). The animals were then removed from
the apparatus and returned to their respective
homecage. The animals in the control no-shock
(NoShock) group underwent the same procedure
but did not receive electrical stimulation.
SRs. The animals were exposed to SRs be-
ginning 7 days after aversive conditioning, with
7-day intervals between SRs, as proposed by
Louvart et al. (2005). On Days 39, 46, and 53,
the animals were confined to the light compart-
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box, with their head facing the open door be-
tween compartments. The session lasted 5 min,
and the light remained on in the light compart-
ment. The behavioral measures included the
latency to enter the dark compartment, the total
time spent in the dark compartment, the total
number of entries into the dark compartment,
rearing, and immobility in the light compart-
ment.
Experiment 1B. Generalization of aversive

ment of the light-dark box for 1 or 10 min, with memory in the modified light-dark box.
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the lights on, and immobility time was re- Two days after the aversive memory test in the
corded. The SRs were delivered in the light light-dark box, the animals were individually
compartment to prevent desensitization, which placed in the center of the light compartment of
could happen if the animals are exposed to the the modified light-dark box with the lights on
dark compartment without footshock. This pro- and the door open between compartments. The
cedure also was applied to replicate the disorder animals were placed in the light compartment
that is observed in humans, in which most peo- facing the open door. The latency to enter the
ple are not exposed to the traumatic situation
dark compartment, the total time spent in the
itself but rather to situations that remind them of
dark compartment, and the total number of en-
the trauma (Pynoos et al., 1996). Two groups
tries into the dark compartment were recorded
were not exposed to SRs (NoSR) and remained
in their respective homecages. Table 1 shows a for 5 min.
summary of the experimental groups.
Seven days after the last SR, the animals Experiment 2
were subjected to one of the following tests: Experiment 2A. Anxietylike behavior in
aversive memory test in the light-dark box, gen- the EPM. To evaluate emotional sensitiza-
eralization of aversive memory in the modified tion, a different group of animals was subjected
light-dark box, behavioral sensitization in the to the EPM 7 days after the last reexposure to
EPM, and the novel object recognition test. SRs for 5 min (Figure 1). The percentage of
Experiment 1 open arm entries, percentage of time spent in
the open arms, total number of entries into the
Experiment 1A. Aversive memory test in closed arms, and risk assessment behaviors (i.e.,
the light-dark box. Seven days after the last stretch attend posture and head dipping) were
SR, the animals were individually placed in the recorded.
center of the light compartment of the light-dark Experiment 2B. Novel object recognition
test. Twenty-four hours after the EPM test,
the animals were subjected to the novel object
Table 1 recognition test. The novel object recognition
Experimental Groups Formed During
test is a nonaversive memory test that is based
the Procedure
on the animal’s tendency to explore novel ob-
Environmental Situational jects more than familiar objects.
Group enrichment Shock reminders In the habituation session, the animals were
Control NoShock No No No individually placed in the center of the arena,
Enriched NoShock Yes No No without any object, and allowed to freely ex-
Control NoSR No Yes No plore the apparatus for 10 min. Twenty-four
Enriched NoSR Yes Yes No
Control SR1= No Yes 1 min
hours after the habituation session, the animals
Enriched SR1= Yes Yes 1 min were individually placed in the arena, facing
Control SR10= No Yes 10 min Objects A and B (arranged in parallel), where
Enriched SR10= Yes Yes 10 min they remained for 10 min. The test session was
Note. NoShock ⫽ no-shock; NoSR ⫽ no situational re- performed 24 hr after the training session when
minder; SR ⫽ situational reminder. the animals were allowed to explore the familiar
 EFFECTS OF ENVIRONMENTAL ENRICHMENT ON PTSD
Figure 1. General outline of the experimental design, including environmental enrichment,
the aversive procedure, and the experimental protocol.
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Object A and the novel Object C for 10 min
(Braida et al., 2013; Kazlauckas et al., 2011).
We recorded the percentage of time in con-
tact with Object A (% TA) and Object B (% TB)
on the day of the training, the percentage of
time in contact with Object A (% TA) and
Object C (% TC) on the test day, and the dis-
crimination index (d1 ⫽ TC/TA ⫹ TC), as
proposed by Akkerman et al. (2012).
Figure 1 shows a general outline of the ex-
perimental design.
Statistical Analysis
Immobility during reexposure to the SRs was
analyzed by repeated-measures analysis of vari-
ance (ANOVA). The behavioral measures in the
light-dark box, modified light-dark box, EPM,
and novel object recognition test were analyzed
by a two-way ANOVA (Housing ⫻ Aversive
Procedure). The percentage of time in the novel
object recognition test was analyzed by Stu-
dent’s t test for independent samples. Signifi-
cant main effects or interactions in the ANOVA
were followed by Duncan’s post hoc test. Val-
ues of p ⱕ .05 were considered statistically
significant.
Results
Effects of Aversive Stimulation and
Environmental Enrichment on Immobility
During Reexposure to SRs
The repeated-measures ANOVA of the per-
centage of immobility time during reexposure
to the 1-min SRs revealed a significant effect of
housing (F1,38 ⫽ 4.23, p ⬍ .05), indicating that
the enriched SR1= group spent more time im-
mobile than the control SR1= group, with no
change in immobility time during the sessions
229
(F2,76 ⫽ 1.17, p ⬎ .05). The repeated-measures
ANOVA of the percentage of immobility time
during reexposure to the 10-min SRs revealed a
significant effect of housing (F1,37 ⫽ 15.06, p ⬍
.05). Duncan’s post hoc test showed that the
enriched SR10= group spent more time immo-
bile than the SR1, SR2, and SR3 groups, with a
reduction of total immobility time in the SR2
and SR1 groups compared with the SR3 group
(F2,74 ⫽ 5.74, p ⬍ .05).
Experiment 1. Effects of the Aversive
Procedure and Environmental Enrichment
on Aversive Memory
Experiment 1A. Aversive memory test in
the light-dark box. Figure 2A shows the ef-
fects of the aversive procedure and environmen-
tal enrichment on the latency to enter the dark
compartment of the light-dark box. The two-
way ANOVA revealed a significant effect of the
aversive procedure (F3,68 ⫽ 66.54, p ⬍ .05) and
a significant Housing ⫻ Aversive Procedure
interaction (F3,68 ⫽ 27.28, p ⬍ .05) but no
effect of housing (F1,68 ⫽ .03, p ⬎ .05). Dun-
can’s post hoc test showed that the control
NoSR and control SR1= groups had a longer
latency to enter the dark compartment com-
pared with the control NoShock group. The
control SR10= group presented a shorter latency
to enter the dark compartment compared with
the control NoSR and control SR1= groups. The
enriched NoSR, enriched SR1=, and enriched
SR10= groups presented a longer latency to en-
ter the dark compartment compared with the
enriched NoShock group. The enriched NoSR
group presented a shorter latency to enter the
dark compartment compared with the control
NoSR group. The enriched SR10= group pre-
sented a longer latency to enter the dark com-
 230 BLANCO, DE SOUZA, AND CANTO-DE-SOUZA
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Figure 2. Effects of the aversive procedure and environmental enrichment on (A) the
latency to enter the dark compartment, (B) time spent in the dark compartment, and (C) total
entries into the dark compartment of the light-dark box. The data are expressed as mean ⫾
standard error of the mean. ⴱ p ⱕ .05, compared with respective NoShock group; # p ⱕ .05,
compared with control group; & p ⱕ .05, compared with NoSR group; $ p ⱕ .05, compared
with SR1= group. SR ⫽ situational reminder.

partment compared with the eontrol SR10=
group.
Figure 2B shows the effects of the aversive
procedure and environmental enrichment on the
total time spent in the dark compartment of the
light-dark box. The two-way ANOVA revealed
significant effects of housing (F1,68 ⫽ 4.96, p ⬍
.05) and the aversive procedure (F3,68 ⫽ 42.10,
p ⬍ .05) and significant Housing ⫻ Aversive
Procedure interaction (F3,68 ⫽ 19.89, p ⬍ .05).
Duncan’s post hoc test showed that the eontrol
NoSR, control SR1=, and control SR10= groups
 EFFECTS OF ENVIRONMENTAL ENRICHMENT ON PTSD
spent more time in the dark compartment com-
pared with the control NoShock group. The
control SR10= group spent more time in the dark
compartment compared with the control NoSR
and control SR1= groups.
The enriched SR1= and enriched SR10=
groups spent less time in the dark compartment
compared with the enriched NoSR group. The
enriched NoShock group spent less time in the
dark compartment compared with the control
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NoShock group. The enriched NoSR group
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spent more time in the dark compartment com-
pared with the control NoSR group. The en-
riched SR10= group spent less time in the dark
compartment compared with the control
SR10=group.
Figure 2C shows the effects of the aversive
procedure and environmental enrichment on the
total number of entries into the dark compart-
ment of the light-dark box. The two-way
ANOVA revealed a significant effect of the
aversive procedure (F3,68 ⫽ 45.11, p ⬍ .05) and
a significant Housing ⫻ Aversive Procedure
interaction (F3,68 ⫽ 16.06, p ⬍ .05) but no
effect of housing (F1,68 ⫽ .01, p ⬎ .05). Dun-
can’s post hoc test showed that the control
NoSR and control SR1= groups made fewer
total entries into the dark compartment com-
pared with the control NoShock group. The
control SR10= group made more total entries
into the dark compartment compared with the
control NoSR and control SR1= groups. The
enriched NoSR, enriched SR1=, and enriched
SR10= groups made fewer total entries into the
dark compartment compared with the enriched
NoShock group. The enriched SR1= and en-
riched SR10= groups made fewer total entries
into the dark compartment compared with the
enriched NoSR group.
The ANOVA also revealed significant effects
of housing (F1,68 ⫽ 33.12, p ⬍ .05) and the
aversive procedure (F3,68 ⫽ 6.85, p ⬍ .05) on
rearing and a significant Housing ⫻ Aversive
Procedure interaction (F3,68 ⫽ 2.96, p ⬍ .05).
The ANOVA revealed a significant effect of the
aversive procedure on total immobility time
(F3,68 ⫽ 12.65, p ⬍ .05) and a significant Hous-
ing ⫻ Aversive Procedure interaction (F3,68 ⫽
10.05, p ⬍ .05). Duncan’s post hoc test showed
that the control NoSR and control SR1= groups
presented a decrease in rearings compared with
the Ccontrol NoShock group. The enriched
SR10= group presented a decrease in rearings
231
compared with the enriched NoShock and en-
riched NoSR groups. The enriched NoShock,
enriched NoSR, and enriched SR1= groups pre-
sented an increase in rearings compared with
their respective control groups.
The control NoSR and control SR1= groups
presented an increase in immobility time com-
pared with the control NoShock group. The
enriched SR1= and enriched SR10=groups pre-
sented an increase in immobility time compared
with the enriched NoShock and enriched NoSR
groups. The enriched NoSR group presented a
decrease in immobility time compared with the
control NoSR group. The enriched SR10= group
presented an increase in immobility time com-
pared with the control SR10= group.
Experiment 1B. Generalization of aversive
memory in the modified light-dark box.
Figure 3A shows the effects of the aversive
procedure and environmental enrichment on the
latency to enter the dark compartment of the
modified light-dark box. The two-way ANOVA
revealed a significant effect of the aversive pro-
cedure (F3,68 ⫽ 10.25, p ⬍ .05) but no effect of
housing (F1,68 ⫽ 0 00, p ⬎ .05) and no Hous-
ing ⫻ Aversive Procedure interaction (F3,68 ⫽
2.09, p ⬎ .05). Duncan’s post hoc test showed
that the control SR1= and enriched SR1= groups
presented a longer latency to enter the dark
compartment compared with the control No-
Shock, control NoSR, enriched NoShock, and
enriched NoSR groups. The control SR10=
group presented a shorter latency to enter the
dark compartment compared with the control
SR1= group.
Figure 3B shows the effects of the aversive
procedure and environmental enrichment on the
total time spent in the dark compartment of the
modified light-dark box. The two-way ANOVA
revealed significant effects of housing (F1,68 ⫽
6.05, p ⬍ .05) and the aversive procedure
(F3,68 ⫽ 12.24, p ⬍ .05) and a significant Hous-
ing ⫻ Aversive Procedure interaction (F3,68 ⫽
6.26, p ⬍ .05). Duncan’s post hoc test showed
that the control SR1= group spent less time in
the dark compartment of the modified light-dark
box compared with the control NoShock group.
The control SR10= group spent more time in the
dark compartment compared with the control
NoShock, control NoSR, and control SR1=
groups.
The enriched NoSR and enriched SR1=
groups presented a decrease in the time spent in
 232 BLANCO, DE SOUZA, AND CANTO-DE-SOUZA
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Figure 3. Effects of the aversive procedure and environmental enrichment on (A) the
latency to enter the dark compartment, (B) time spent in the dark compartment, and (C) total
entries into the dark compartment of the modified light-dark box. The data are expressed as
mean ⫾ standard error of the mean. ⴱ p ⱕ .05, compared with respective NoShock group;
#
p ⱕ .05, compared with control group; & p ⱕ .05, compared with NoSR group; $ p ⱕ .05,
compared with SR1= group. SR ⫽ situational reminder.

the dark compartment compared with the en-
riched NoShock group. The enriched SR10=
group presented a decrease in the time spent in
the dark compartment compared with the con-
trol SR10= group.
Figure 3C shows the effects of the aversive
procedure and environmental enrichment on the
total number of entries into the dark compart-
ment of the modified light-dark box. The two-
way ANOVA revealed significant effects of
housing (F1,68 ⫽ 25.78, p ⬍ .05) and the aver-
sive procedure (F3,68 ⫽ 12.06, p ⬍ .05) and a
significant Housing ⫻ Aversive Procedure in-
teraction (F3,68 ⫽ 3.70, p ⬍ .05). Duncan’s post
 EFFECTS OF ENVIRONMENTAL ENRICHMENT ON PTSD
hoc test showed that the control SR1= group
made fewer total entries into the dark compart-
ment compared with the control NoShock, con-
trol NoSR, and control SR10= groups. The en-
riched NoSR, enriched SR1=, and enriched
SR10= groups made fewer total entries into the
dark compartment compared with the enriched
NoShock group. The enriched SR1= and en-
riched SR10= groups made fewer total entries
into the dark compartment compared with the
This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.
enriched NoSR group. The enriched NoShock
This document is copyrighted by the American Psychological Association or one of its allied publishers.
and enriched NoSR groups made more total
entries into the dark compartment compared
with the control NoShock and control NoSR
groups.
Experiment 2. Effects of the Aversive
Procedure and Environmental Enrichment
on Anxiety and Memory
Experiment 2A. Anxiety in the EPM.
Figure 4A shows the effects of the aversive
procedure and environmental enrichment on the
percentage of entries into the open arms of the
EPM. The two-way ANOVA revealed a signif-
icant effect of housing (F1,77 ⫽ 18.33, p ⬍ .05)
and a significant Housing ⫻ Aversive Proce-
dure interaction (F3,77 ⫽ 3.43, p ⬎ .05) but no
effect of the aversive procedure (F3,77 ⫽ 1.66,
p ⬎ .05). Duncan’s post hoc test showed that
the control SR1= group presented a decrease in
the percentage of entries into the open arms
compared with the control NoShock and control
NoSR groups. The enriched SR1= group pre-
sented an increase in the percentage of entries
into the open arms compared with the control
SR1= group.
Figure 4B shows the effects of the aversive
procedure and environmental enrichment on
the percentage of time spent in the open arms
of the EPM. The two-way ANOVA revealed a
significant effect of housing (F1,77 ⫽ 7.20,
p ⬍ .05) and a significant Housing ⫻ Aver-
sive Procedure interaction (F3,77 ⫽ 3.06, p ⬍
.05) but no effect of the aversive procedure
(F3,77 ⫽ .91, p ⬍ .05). Duncan’s post hoc test
showed that the control NoSR and control
SR1= groups presented a decrease in the per-
centage of time spent in the open arms com-
pared with the control NoShock group. The
enriched NoSR and enriched SR1= groups
presented an increase in the percentage of
time spent in the open arms compared with
233
the control NoSR and control SR1= groups,
respectively.
Figure 4C shows the effects of the aversive
procedure and environmental enrichment on the
total frequency of stretch attend posture in the
EPM. The two-way ANOVA revealed a signif-
icant effect of housing (F1,77 ⫽ 21.79, p ⬍ .05)
but no effect of the aversive procedure (F3,77 ⫽
.35, p ⬎ .05) and no Housing ⫻ Aversive
Procedure interaction (F3,77 ⫽ .72, p ⬎ .05).
Duncan’s post hoc test showed that the enriched
NoShock, enriched NoSR, and enriched SR1=
groups presented a decrease in the total fre-
quency of stretch attend posture compared with
the control NoShock, control NoSR, and control
SR1= groups, respectively.
We also evaluated the effects of the aversive
procedure and environmental enrichment on the
total number of entries into the closed arms of
the EPM and frequency of head dippings and
unprotected head dippings in the open arms of
the EPM. The two-way ANOVA revealed a
significant effect of housing (F1,77 ⫽ 69.73, p ⬍
.05) on the total number of entries into the
closed arms but no effect of the aversive pro-
cedure (F3,77 ⫽ 1.05, p ⬎ .05) and no Hous-
ing ⫻ Aversive Procedure interaction (F3,77 ⫽
1.49, p ⬎ .05). The two-way ANOVA re-
vealed significant effects of housing (F1,77 ⫽
10.17, p ⬍ .05) and the aversive procedure
(F3,77 ⫽ 4.56, p ⬍ .05) on head dippings and
a significant Housing ⫻ Aversive Procedure
interaction (F3,77 ⫽ 6.01, p ⬍ .05). Duncan’s
multiple-comparison post hoc test showed
that the control SR10= group presented an
increase in the frequency of head dippings
compared with the control NoSR and control
SR1= groups. The enriched SR1= group pre-
sented an increase in the frequency of head
dippings compared with the enriched No-
Shock, enriched NoSR, enriched SR10=, and
control SR1= groups. The ANOVA revealed a
significant effect of housing (F1,77 ⫽ 4.07, p ⬍
.05) on the frequency of unprotected head dip-
pings and a significant Housing ⫻ Aversive
Procedure interaction (F3,77 ⫽ 3.39, p ⬍ .05)
but no effect of the aversive procedure (F3,77 ⫽
.69, p ⬎ .05). Duncan’s multiple-comparison
post hoc test showed that the control SR1=
group presented a decrease in the frequency of
unprotected head dippings compared with the
control NoShock group. The enriched SR1=
group presented an increase in the frequency of
 234 BLANCO, DE SOUZA, AND CANTO-DE-SOUZA
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This document is copyrighted by the American Psychological Association or one of its allied publishers.

Figure 4. Effects of the aversive procedure and environmental enrichment on (A) the
percentage of entries into open arms, (B) the percentage of time spent on the open arms,
and (C) total stretch attend posture (SAP) in the EPM. The data are expressed as mean ⫾
standard error of the mean. ⴱ p ⱕ .05, compared with respective NoShock group; # p ⱕ
.05, compared with control group; $ p ⱕ .05, compared with SR1= group. SR ⫽ situational
reminder.

unprotected head dippings compared with the
control SR1=group.
Experiment 2B. Novel object recognition
test. We then evaluated the effects of the aver-
sive procedure and environmental enrichment on
the percentage of time in contact with Objects A
and C in the novel object recognition test (Figure
5B). Student’s t test showed that all groups pre-
sented a higher percentage of time in contact with
Object C compared with Object A: control No-
Shock (t9 ⫽ ⫺4.89, p ⬍ .05), control NoSR (t9 ⫽
⫺8.69, p ⬍ .05), control SR1= (t7 ⫽ ⫺10.61,
 EFFECTS OF ENVIRONMENTAL ENRICHMENT ON PTSD 235
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This document is copyrighted by the American Psychological Association or one of its allied publishers.

Figure 5. Effects of the aversive procedure and environmental enrichment on (A) the
percentage of time exploring objects A and B in the training session and (B) percentage of
time exploring objects A and C in the test session in the novel object recognition test. The data
are expressed as mean ⫾ standard error of the mean. ⴱ p ⱕ .05, compared with percentage of
time exploring object A in the respective group. SR ⫽ situational reminder.

p ⬍ .05), control SR10= (t9 ⫽ ⫺11.99, p ⬍ .05), Discussion

enriched NoShock (t9 ⫽ ⫺11.99, p ⬍ .05),
enriched NoSR (t9 ⫽ ⫺9.52, p ⬍ .05), enriched
SR1= (t9 ⫽ ⫺8.33, p ⬍ .05), and enriched
SR10= (t8 ⫽ ⫺8.86, p ⬍ .05).
We also evaluated the effects of the aversive
procedure and environmental enrichment on the
discrimination index (d1) in the novel object
recognition test. The two-way ANOVA re-
vealed no effect of housing (F1,77 ⫽ 3.30, p ⬎
.05) or the aversive procedure (F3,77 ⫽ 1.49,
p ⬍ .05) and no Housing ⫻ Aversive Procedure
interaction (F3,77 ⫽ .49, p ⬎ .05).
The present study investigated the effects of
an aversive procedure, with or without SRs, on
behavior in mice and the influence of environ-
mental enrichment. Experiment 1 showed that
exposure to 1-min SRs potentiated the effects of
aversive stimulation and favored its generaliza-
tion. Exposure to 10-min SRs resulted in the
possible extinction of aversive memory. In this
procedure, environmental enrichment appeared
to reverse the effects of aversive stimulation.
However, exposure to the SRs for 1 or 10 min
 236 BLANCO, DE SOUZA, AND CANTO-DE-SOUZA
enhanced the aversive memory. Experiment 2
showed that reexposure to the SRs for 1 min
increased anxietylike behavior and induced be-
havioral sensitization, and environmental en-
richment reversed these effects, increased ex-
ploration activity, and reduced risk assessment.
In the novel object recognition test, neither the
aversive procedure nor environmental enrich-
ment altered visual recognition memory.
With regard to the aversive procedure, Kohda
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et al. (2007); Siegmund and Wotjak (2007), and
This document is copyrighted by the American Psychological Association or one of its allied publishers.
Stam (2007) reported that animals that are sub-
jected to shock present an increase in condi-
tioned fear responses when they are exposed to
the environment where they received the aver-
sive stimulation or related environments. These
animals also exhibit exacerbated emotional re-
sponses to new environments or stressful
events. Some authors suggested that brief reex-
posure to SRs potentiates the effects of aversive
stimulation and favors the generalization of re-
breve reexposição potencializa o efeito
sponses to other contexts, indicating behavioral
sensitization (Geerse, van Gurp, Wiegant, &
Stam, 2006; Louvart et al., 2005; Pynoos et al.,
1996; Zhang et al., 2015). Such reexposure can
also return these memories to a labile state
during reconsolidation (Lee, Milton, & Everitt,
2006; Nader, 2003) and consequently increase
anxiety-related responses and avoidance of the
aversive environment, which is consistent with
the present study.
Reexposure of the animals to the SRs for 10
min consisted of prolonged exposure to aver-
sive contextual cues (CSs) without presentation
of the aversive stimulus (US), thus allowing a
new association and the formation of a new
memory that overlapped with the initial aver-
sive memory. This extinction process decreased
anxiety-related responses and avoidance of the
aversive environment, which was previously re-
ported by Cammarota et al. (2005) and Besnard,
Caboche, and Laroche (2012).
With regard to recognition memory, neither
the aversive procedure nor environmental en-
richment altered this type of visual memory. A
possible explanation for these results may in-
volve the intensity of the aversive stimulus and
the time interval between it and the memory
test. Other studies suggested that more intense
stimuli result in more intense and enduring
changes (Anderson, Arun, & Jensen, 2010;
Geerse et al., 2006; Hendriksen et al., 2010;
Siegmund & Wotjak, 2007; Zhang et al., 2015).
Most studies conduct memory assessments days
following the aversive stimulus (Kohda et al.,
2007; Luine, 2002).
With regard to environmental enrichment, the
assessment of immobility during reexposure to
SRs suggested that enriched animals spent more
time immobile than nonenriched animals, re-
sponding more intensely in context with re-
minders of the trauma. These results are consis-
tent with Woodcock and Richardson (2000) and
Vivinetto, Suarez, and Rivarola (2013), who
reported an increase in emotional memory in
environmentally enriched rats.
In Experiment 1, environmental enrichment
appeared to reverse the effects of aversive stim-
ulation. These results corroborate the findings
of Larsson, Winblad, and Mohammed (2002).
Although behavior can be altered in enriched
enr ambiental
animals that are exposed to a strong stressor, the=atividade fisica
effects are attenuated compared with nonen-
riched animals. Contact with many different
objects and situations and weekly variations in
sensory stimulation cause a moderate level of
stress in enriched animals, but the exploration
of these novel objects and situations does not
result in aversive consequences (Harris, D’Eath,
& Healy, 2009). Therefore, the reinforcement of
these exploratory behaviors favor better perfor-
mance on future stress situations (Larsson et al.,
2002).
Although, in the absence of SRs, environ-
mental enrichment attenuated the effects of
footshock but was unable to reverse the effects
of reexposure to SRs for 1 min. Moreover, with
reexposure to SRs for 10 min, environmental
enrichment strengthened the aversive memory,
with an increase in anxietylike behavior in both
the light-dark box and modified light-dark box.
Previous studies reported the facilitatory ef-
fects of environmental enrichment on learning
and memory processes (Bruel-Jungerman et al.,
2005; Larsson et al., 2002; Leggio et al., 2005),
including an enhancement of emotional mem-
ory (Vivinetto et al., 2013; Woodcock & Rich-
ardson, 2000). These results are related to the
better discrimination of contextual cues and
processing speed and structural and biochemi-
cal changes in the nervous system. Such bio-
chemical changes include an increase in hip-
pocampal neurogenesis, an increase in dendritic
branching, an increase in synapse formation, an
increase in long-term potentiation, increases in
norepinephrine and serotonin levels in the hip-
 EFFECTS OF ENVIRONMENTAL ENRICHMENT ON PTSD
pocampus and prefrontal cortex (Brenes et al.,
2008; Hirase & Shinohara, 2014; Hughes &
Collins, 2010; van Praag, Kempermann, &
Gage, 2000), increases in N-methyl-D-aspartate
and ␣-amino-3-hydroxy-5-methyl-4-isoxazole-
propionic acid glutamate receptors in the hip-
pocampus (Andin, Hallbeck, Mohammed, &
Marcusson, 2007), a decrease in glutamate
transporters (Andin et al., 2007), and an in-
crease in brain-derived neurotrophic factor lev-
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els in the hippocampus and cerebral cortex
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(Ickes et al., 2000; Zhu et al., 2006).
With regard to stress, previous studies sug-
gested that enriched animals are less affected by
stress (Benaroya-Milshtein et al., 2004), are
more resilient to severe stressors, and recover
more quickly from the effects of aversive con-
ditioning (Hendriksen et al., 2010; Hutchinson
et al., 2012; Larsson et al., 2002).
Moncek, Duncko, Johansson, and Jezova
(2004) and Bakos et al. (2009) reported that
enriched animals had higher basal levels of
corticosterone and adrenocorticotropic hormone
(ACTH) compared with animals that were
reared in standard environments. However,
Roy, Belzung, Delarue, and Chapillon (2001)
and Moncek et al. (2004) found that animals
that were reared in enriched environments had
lower levels of corticosterone and ACTH after
stressful situations than control animals.
Hutchinson et al. (2012) also found that en-
riched mice did not exhibit changes in the levels
of corticosterone after chronic stress. Thus, en-
vironmental enrichment appears to result in
lower reactivity of the HPA axis under stressful
conditions, with a consequent reduction of de-
fensive responses to stress. Additionally, ac-
cording to Sampedro-Piquero, Begega, and
Arias (2014), environmental enrichment in-
creases glucocorticoid receptors in the hip-
pocampus, which may be related to a better
ability to adapt to stressors and more active
coping strategies in anxiogenic situations.
Mitra and Sapolsky (2012) suggested that
enriched animals are less exposed to imminent
danger, thereby increasing their chances of sur-
vival. The maintenance of traumatic memory
may reduce the risk of reexposure to dangerous
situations, thus favoring survival of the species.
Environmental enrichment reversed the reduc-
tions of the percentage of time spent on the open
arms, the percentage of entries into the open arms,
and unprotected head dippings in the EPM. Envi-
237
ronmental enrichment also reduced risk assess-
ment and increased exploratory activity, thus fa-
voring the expression of more appropriate
emotional responses in different situations. Gen-
erally, environmental enrichment has an anxiolyti-
clike effect in the EPM (Benaroya-Milshtein et al.,
2004; Hellemans, Benge, & Olmstead, 2004;
Peña, Prunell, Rotllant, Armario, & Escorihuela,
2009; Simpson & Kelly, 2011). However, some
studies reported conflicting results (Hoffmann,
Schutte, Koch, & Schwabe, 2009; McQuaid, Au-
det, Jacobson-Pick, & Anisman, 2013; Mitra &
Sapolsky, 2012). Increases in exploratory activity
have also been shown to result from environmen-
tal enrichment in studies that used different exper-
imental designs (Hoffmann et al., 2009; Lima,
Silva, Padovan, Almeida, & Fukuda, 2014; Peña
et al., 2009; Roy et al., 2001; Sampedro-Piquero et
al., 2014).
With regard to the effects of environmental
enrichment on recognition memory, numerous
studies have reported that intellectual and phys-
ical stimulation associated with environmental
enrichment improved cognitive ability (Simp-
son & Kelly, 2011). However, these studies
evaluated the effects of environmental enrich-
ment in animals that exhibited cognitive impair-
ment that was caused by different experimental
manipulations, such as cerebral hypoxia
(Pereira et al., 2007; Pereira, Strapasson, Nabin-
ger, Achaval, & Netto, 2008), social isolation
(Zimmermann, Stauffacher, Langhans, & Wür-
bel, 2001), chronic stress (Hutchinson et al.,
2012), genetic alterations (Pamplona, Pandolfo,
Savoldi, Prediger, & Takahashi, 2009), and ag-
ing (Blanco et al., 2012; Escorihuela, Tobeña, &
Fernández-Teruel, 1995; Frick & Fernandez,
2003; Speisman et al., 2013). In the present
study, animals that were exposed to the aversive
procedure presented no cognitive impairment.
Therefore, we were unable to assess possible
effects of environmental enrichment on non-
emotional memory. In enriched animals, SRs
for 10 min enhanced aversive memory in the
light-dark box test and modified light-dark box
test but not visual recognition memory. These
results suggest that strengthening the traumatic
memory by environmental enrichment, regard-
less of whether it enhanced nonemotional visual
memory, may be related to an increase in pro-
tective behaviors that are important for survival
of the species (Mitra and Sapolsky (2012).
 238 BLANCO, DE SOUZA, AND CANTO-DE-SOUZA
Conclusion
The present study evaluated the effects of
environmental enrichment on behavior in a
mouse model of PTSD. The results showed that
aversive stimulation (.5 mA scrambled shock
for 10 s) increased anxietylike behaviors in the
light-dark box, modified light-dark box, and
EPM but did not alter memory in the novel
object recognition test. Reexposure to SRs for 1
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min increased anxietylike behavior and ap-
This document is copyrighted by the American Psychological Association or one of its allied publishers.
peared to favor the reconsolidation of traumatic
memory. Reexposure to SRs for 10 min reduced
anxietylike behavior and appeared to facilitate
the extinction of traumatic memory. Reexpo-
sure to the aversive procedure with SRs for 1
min favored generalization of the traumatic
memory and behavioral sensitization but did not
impair recognition memory.
The present results showed that environmen-
tal enrichment alleviated the effects of aversive
stimulation and reversed the effects of the aver-
sive procedure with reexposure to SRs for 1 min
in the EPM. However, environmental enrich-
ment did not reverse the effects of the aversive
stimulation in the light-dark box or modified
light-dark box; instead, it favored the mainte-
nance of the traumatic memory, even when the
animals were reexposed to SRs for 10 min.
Environmental enrichment appeared to hinder
extinction or facilitate discrimination of the CS
and did not alter visual recognition memory in
the novel object recognition test.
The effects of environmental enrichment in
the present model were quite complex. In the
absence of reexposure to SRs, environmental
enrichment decreased anxietylike behavior, mit-
igated the effects of aversive stimulation, and
facilitated exploratory activity. Reexposure to
SRs for 1 or 10 min intensified the traumatic
memory and increased anxietylike behavior in
the traumatic environment but reduced these
behaviors under anxiogenic conditions that
were unrelated to the initial trauma, thus revers-
ing behavioral sensitization in this model.
The present model of PTSD showed validity
in inducing behaviors that are similar to those of
PTSD patients. Environmental enrichment may
be somewhat associated with aspects of the
course of clinical PTSD. Further studies will
help elucidate the pathology of PTSD, including
the influence of the environment and history of
the subjects, and develop more effective thera-
pies and interventions.
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Received December 1, 2016
Revision received March 20, 2017
Accepted March 23, 2017 䡲