International Journal of Developmental Neuroscience 67 (2018) 19–32

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International Journal of Developmental Neuroscience

journal homepage: www.elsevier.com/locate/ijdevneu

Beneficial effects of environmental enrichment on behavior, stress reactivity T

and synaptophysin/BDNF expression in hippocampus following early life
stress
Εvgenia Dandia, Aikaterini Kalamaria, Olga Touloumic, Rosa Lagoudakic,
⁎ ⁎
Evangelia Nousiopoulouc, Constantina Simeonidoub, Evangelia Spandoub, , Despina A. Tataa,
a
Laboratory of Cognitive Neuroscience, School of Psychology, Aristotle University of Thessaloniki, Thessaloniki 541 24, Greece
b
Laboratory of Experimental Physiology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki 541 24, Greece
c
Laboratory of Neuroimmunology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki 541 24, Greece

A R T I C LE I N FO A B S T R A C T

Keywords: Exposure to environmental enrichment can beneficially influence the behavior and enhance synaptic plasticity.
Early stress The aim of the present study was to investigate the mediated effects of environmental enrichment on postnatal
Enriched environment stress-associated impact with regard to behavior, stress reactivity as well as synaptic plasticity changes in the
Visuospatial learning dorsal hippocampus. Wistar rat pups were submitted to a 3 h maternal separation (MS) protocol during postnatal
Spatial memory
days 1–21, while another group was left undisturbed. On postnatal day 23, a subgroup from each rearing
Recognition memory
Corticosterone
condition (maternal separation, no-maternal separation) was housed in enriched environmental conditions until
postnatal day 65 (6 weeks duration). At approximately three months of age, adult rats underwent behavioral
testing to evaluate anxiety (Elevated Plus Maze), locomotion (Open Field Test), spatial learning and memory
(Morris Water Maze) as well as non-spatial recognition memory (Novel Object Recognition Test). After com-
pletion of behavioral testing, blood samples were taken for evaluation of stress-induced plasma corticosterone
using an enzyme-linked immunosorbent assay (ELISA), while immunofluorescence was applied to evaluate
hippocampal BDNF and synaptophysin expression in dorsal hippocampus. We found that environmental en-
richment protected against the effects of maternal separation as indicated by the lower anxiety levels and the
reversal of spatial memory deficits compared to animals housed in standard conditions. These changes were
associated with increased BDNF and synaptophysin expression in the hippocampus. Regarding the neuroendo-
crine response to stress, while exposure to an acute stressor potentiated corticosterone increases in maternally-
separated rats, environmental enrichment of these rats prevented this effect. The current study aimed at in-
vestigating the compensatory role of enriched environment against the negative outcomes of adverse experi-
ences early in life concurrently on emotional and cognitive behaviors, HPA function and neuroplasticity markers.

1. Introduction
It is well established that environmental factors during the postnatal
period have long term effects on behavior, neuroendocrine function and
neuronal plasticity (Bock and Braun, 2011; Stiles, 2011). Epidemiolo-
gical and clinical studies support the idea that stress early in life may
lead to psychopathology in adulthood (Anda et al., 2009; Felitti et al.,
1998; Heim and Binder, 2012; Heim and Nemeroff, 2001; Palagini
et al., 2015). A well-established animal model of early stress that is
widely used over the last decades is the maternal separation (MS)
paradigm. During the first postnatal days, survival of rat pups depends
exclusively on maternal care. In addition, the role of tactile stimulation,
nourishing and passive contact is considered crucial for the regulation
of Hypothalamic-Pituitary-Adrenal (HPA) axis (Levine, 2002, 2001).
Prolonged MS disrupts the normal interaction between mother and
pups and affects brain and behavior. Reduction in neurogenesis (Korosi
et al., 2012; Lajud et al., 2012) and glucocorticoid receptors’ density
(Aisa et al., 2008; Enthoven et al., 2008a,b) has been reported as a
result of MS. Early life stress also reduces synaptic formation and en-
hances neuroendocrine stress response (Bock and Braun, 2011). At

Abbreviations: BDNF, brain-derived neurotrophic factor; CORT, corticosterone; EPM, elevated plus maze; EE, environmental enrichment; MS, maternal separation; NMS, no maternal
separation; OFT, open field test; PND, postnatal day; SYN, synaptophysin
⁎
Corresponding authors.
E-mail addresses: spandou@med.auth.gr (E. Spandou), dtata@psy.auth.gr (D.A. Tata).

https://doi.org/10.1016/j.ijdevneu.2018.03.003
Received 30 November 2017; Received in revised form 7 March 2018; Accepted 8 March 2018
Available online 12 March 2018
0736-5748/ © 2018 ISDN. Published by Elsevier Ltd. All rights reserved.
Ε. Dandi et al.
behavioral level, maternally separated rats show increased anxiety and
depressive-like behaviors in adulthood (Aisa et al., 2007; Veenema
et al., 2007; Vetulani, 2013; Wang et al., 2015a; Wigger and Neumann,
1999). Several studies have also reported impairments in various cog-
nitive functions (e.g., learning and memory), following early life stress
(Chocyk et al., 2014; Conrad, 2010; Cui et al., 2006; Mcewen, 1997;
Tata et al., 2015; Xiong et al., 2015). Based on existing evidence, these
effects have been associated with alterations in the expression of sy-
naptic plasticity markers, such as the brain-derived neurotrophic factor
(BDNF) and synaptophysin (SYN). BDNF, a main neurotrophin in
mammals’ central nervous system, is essential for cell survival and
regulation of dendritic and synaptic plasticity, mainly in the hippo-
campus (Fumagalli et al., 2007; Lu et al., 2005), while synaptophysin,
an indirect indicator of the synapse number, is a synaptic vesicle pro-
tein involved in neurotransmission (Valtorta et al., 2004). It has been
shown that adult rats exposed to MS condition exhibited reduced ex-
pression of BDNF and SYN in various brain regions (Andersen and
Teicher, 2004; Choy et al., 2008; Lippmann et al., 2007; Roth et al.,
2009).
In contrast to the effects of early stress, specific housing conditions,
such as environmental enrichment (EE), can exert a neuroprotective
role (Baroncelli et al., 2010; Gelfo et al., 2011; Jha et al., 2011). En-
vironmental enrichment (EΕ) promotes neurogenesis (Monteiro et al.,
2014) and synaptogenesis (Rampon et al., 2000), enhances hippo-
campal LTP (Cortese et al., 2018) and increases dendritic arborization
as well as spine density, thus facilitating neuronal communication
(Leggio et al., 2005). In addition, it improves learning and memory
(Frick and Fernandez, 2003; Harburger et al., 2007; Leggio et al., 2005;
Rampon and Tsien, 2000) and protects from cognitive deficits caused
by brain damage or exposure to stress (Hralová et al., 2013; Nozari
et al., 2014; Schreiber et al., 2014; Wright and Conrad, 2008) or aging
(Cortese et al., 2018). Furthermore, enriched housing promotes social
interaction, locomotor activity and exploratory behavior (Brenes Sáenz
et al., 2006) and regulates emotional behavior. Rodents housed in EE
show decreased anxiety and depressive-like behaviors in various be-
havioral tasks (Chapillon et al., 1999; Friske and Gammie, 2005;
Ishihama et al., 2010; Nicolas et al., 2015). These effects seem to be
associated with a number of neuronal and neuroendocrine changes that
may, in turn, result in greater resistance to stress later in life (Connors
et al., 2014; Crofton et al., 2015; Fox et al., 2006).
These findings support the hypothesis that EE could compensate for
the detrimental effects of stress. In fact, existing evidence indicates that
EE attenuates the chronic stress impact on behavior and hippocampal
integrity, thus protecting against the stress-associated cognitive deficits,
emotional dysregulation, dendritic atrophy and reduced neurogenesis
(Hutchinson et al., 2012; Veena et al., 2009; Wright and Conrad, 2008).
Similar to adult stress, restorative effects of EE have been reported in
rodents submitted to prenatal (Laviola et al., 2008; Pascual et al., 2015;
Yang et al., 2007) or juvenile stress (Ilin and Richter-Levin, 2009). To
the best of our knowledge, there is limited information regarding the
compensatory action of EE following early stress (do Prado et al., 2016;
Francis et al., 2002; Koe et al., 2016; Vivinetto et al., 2013) and so far,
no study has looked at the interaction of maternal separation and
subsequent exposure to EE conditions concurrently at cognitive and
emotional behavior, HPA function and markers of synaptic plasticity
(BDNF, SYN) during adulthood. The critical importance of the first
postnatal weeks to normal development (Rice and Barone, 2000), fur-
ther supports the significance of exploring the hypothesis that EE ex-
perience may overcome behavioral and neurobiological deficits in-
duced by early postnatal stress.
The purpose of the current study was to explore the hypothesis that
EE during adolescence may protect against the negative outcomes of
maternal separation on spatial and non-spatial learning and memory,
anxiety, as well as neuroendocrine stress response during adulthood.
Furthermore, given the essential role of BDNF and SYN in cognitive
functions and emotional behavior, we aimed at exploring the
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International Journal of Developmental Neuroscience 67 (2018) 19–32
expression of these two proteins in the hippocampus, a structure par-
ticularly vulnerable to stress.
2. Materials and method
2.1. Animals
Female Wistar rats on the second gestational week were in-
dividually housed until delivery. The day of birth was designated as
postnatal day 0 (PND 0). Totally, 47 neonates were included in the
experiment and all animals participated in behavioral testing, as well as
in corticosterone and BDNF/synaptophysin measurements. In order to
ensure normal growth and development, body weight measurements of
neonates were taken on the 3rd, 8th, 14th and 21st postnatal days. All
animals were maintained on a 12:12 light/dark cycle (08:00 light/
20:00 dark) and standard temperature (22 ± 2 °C), with food and
water available ad libitum. Handling of the pups and behavioral testing
were performed by the same personnel since familiarity with the ex-
perimenter increases consistency in animal testing (van Driel and
Talling, 2005). Εxperimental procedures were conducted in accordance
to the Institutional Animal Ethics EL 54 BIO 20.
2.2. Rearing conditions
On PND 1 litters were assigned randomly to no-maternal separation
(NMS) (NS = 23) or maternal separation (MS) (N = 24) conditions. The
pups of the NMS condition remained undisturbed in their cages with
their dams until weaning (PND 23). The MS condition involved a daily
3 h separation of the pups from their dams during the three postnatal
weeks (PND1-21) (Huot et al., 2001). The maternal separation proce-
dure took place between 0900 and 1400 h and was performed as pre-
viously described (Tata et al., 2015). Briefly, dams were first removed
from their cages, followed by the pups, which were placed, as a litter, in
plastic containers. Upon completion of the 3 h separation period, pups
were returned to their home cage, followed by their dam. Litter size
ranged from 6 to 10 rats.
2.3. Post-weaning housing conditions
On PND 23 rats from each of the two rearing conditions were ran-
domly assigned to either standard housing (SH) (N = 22) or enriched
environment (EE) conditions (N = 25). In the SH condition, rats in
same-gender pairs were housed in standard laboratory cages. In the EE
condition (PND23-65), same-gender groups of 5–7 rats were housed in
large cages (60 cm × 45 cm × 76 cm) containing various non chewable
toys, climbing platforms, tunnels and running wheels (Griva et al.,
2017). To promote exploratory and novelty-seeking behavior, every
3–4 days toys as well as food and water containers were moved to a new
location in the cage. Once a week the cages were cleaned and platforms,
tunnels and running wheels were rearranged, and toys were replaced by
new ones. On PND 65, animals of the EE condition were transferred in
pairs to standard laboratory cages, where they were kept until beha-
vioral testing began.
The final four groups that emerged after experimental manipula-
tions were the following: a) non-maternally separated rats housed in
standard housing conditions (NMS/SH; N = 10), b) non-maternally
separated rats housed in enriched environment condition (NMS/EE;
N = 13) c) maternally-separated rats housed in standard housing con-
ditions (MS/SH; N = 12), d) maternally-separated rats housed in en-
riched environment (MS/EE; N = 12). The animals of each experi-
mental group were obtained from 2 to 3 litters.
2.4. Behavioral testing
Behavioral testing took place at approximately 2.5 months of age in
order to examine emotional and exploratory behavior as well as
Ε. Dandi et al.
learning and memory. Anxiety-like behavior was assessed by the
Elevated Plus Maze (EPM) (PND 67), locomotion and exploratory be-
havior was estimated by the Open Field Test (OFT) (PND 69), while
non-spatial and spatial (visual) learning and memory were assessed by
the Novel Object Recognition (NORT) (PND 70) and the Morris Water
Maze (MWM) (PND72-76) tests, respectively. Behavioral testing oc-
curred during the light cycle between 09:00–15:00. Rat behavior was
recorded by means of a ceiling-mounted camera placed 160 cm above
the experimental arena. Animals’ swimming behavior in the MWM was
analyzed by a data acquisition system (Ethovision v.2.3, Noldus
Information Technology), while in case of NORT and EPM testing re-
corded behavior was manually scored by two researchers.
Experimenters were blind to experimental conditions both during
behavioral testing and data analysis. All experimental apparatuses were
cleaned thoroughly with a 25% ethanol solution after each trial to
eliminate odor cues.
2.4.1. Elevated plus maze (EPM)
EPM is a behavioral test that is used to measure anxiety (Handley
and Mithani, 1984) based on rodents’ innate preference of dark and
enclosed spaces and their unconditioned fear of height/open areas. The
apparatus was cross-shaped, with two open and two closed arms
(50 cm × 10 cm); the height of the walls was 40 cm and the maze was
positioned 50 cm from the floor. The animals were placed in the open
central square (10 cm × 10 cm) formed at the four arms intersection,
facing the open arm opposite to experimenter, and were left un-
disturbed for 5 min to explore the maze. Increased time and number of
entries in the open arms (Walf and Frye, 2007) as well as smaller oc-
currence of risk assessment behaviors (i.e., stretch attend posture,
closed arms returns) (Cruz et al., 1994; Doremus et al., 2006; Rodgers
and Dalvi, 1997) indicate lower levels of anxiety. In the present study
assessment of anxiety was based on a) the ratio of open arms entries
(open arms entries/total entries), b) the ratio of the open arms time
(open arms time/total arms time), c) the number of stretch attend
posture/SAP (the animal’s two hind legs remain in an arm while it
elongates its head and shoulders out of the arm and then returns to its
initial position) and d) the number of returns to closed arms (the ani-
mal’s half front body exits a closed arm followed by return to the closed
arm). In addition, the total number of arm entries was estimated as an
index of general activity.
2.4.2. Open field test (OFT)
Open field test is used to measure locomotion and exploration (Lau
et al., 2008). The apparatus used in the current study consisted of a
wooden square arena (100 cm × 100 cm) surrounded by 40 cm high
wall to prevent escape. The arena’s floor was divided into 16 equally
sized sectors (25 cm × 25 cm). The four squares in the center formed
the inner zone of the arena, while the twelve outer squares formed the
outer zone (peripheral squares). Rats were placed facing the wall and
they were allowed to freely explore the arena for 6 min.
Two behavioral parameters were analyzed in order to estimate
general activity and exploration, the number a) of square visits (am-
bulation) (Bubser et al., 1992; Suchecki et al., 2000) and b) rearings
(Gamberini et al., 2015; Kulesskaya and Voikar, 2014), respectively. A
square visit was recorded if at least half of animal’s torso was in a
sector, while rearing when animals kept hind paws on the floor with the
front limbs off it.
2.4.3. Novel object recognition test (NORT)
This task was administered in order to examine the non-spatial
episodic memory (Ennaceur and Meliani, 1992). It took place in a
wooden open field arena (see above, 2.4.2.), and it was preceded by two
habituation sessions (6 min duration each).
The testing consisted of two trials separated by a 70 min delay. In
trial 1 (sample phase; 4 min duration), rats were allowed to freely ex-
plore two identical objects that were placed at the two adjacent back
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International Journal of Developmental Neuroscience 67 (2018) 19–32
corners of the arena at a distance of 25 cm from the wall. The objects
used were made of metal or glass and were heavy enough so that they
could not be displaced by the rats. In addition, we ensured that the
objects were of similar attractiveness for the rats. Object exploration
was operationally defined as directly attending to the object with the
head no more than 2 cm from the object (Ennaceur and Delacour,
1988). In trial 2 (choice phase; 3 min duration) one of the two objects
was replaced by a new one of similar height (Bevins and Besheer, 2006;
Ennaceur and Delacour, 1988). Under normal conditions, rats tend to
explore more the novel object, a preference that is indicative of non-
spatial memory. The length of the specific inter-trial interval (ITI) was
based on finding that under control conditions rats can still recognize
the novel object and subsequently the danger of floor effects due to
longer ITIs is eliminated (Ennaceur et al., 1997). In the current study,
we estimated the discrimination ratio, defined as the exploration time
of the novel object in trial 2 divided by the total time exploring both
objects in the same trial. Exploration time of each object during the
sample phase was also calculated in order to ensure that any preference
shown for the new object would be due to its novelty and not to dif-
ference in the exploration during the sample phase.
2.4.4. Morris water maze (MWM)
Morris Water Maze is used to estimate spatial learning and memory
(Morris, 1984). In the present study the maze consisted of a plastic
circular tank (1.6 m/diameter × 50 cm/height) filled with water in
standard temperature (25 ± 1 °C) and was virtually divided into four
quadrants: north-west (NW), north-east (NE), south-west (SW) and
south-east (SE). An escape platform (10 cm × 10 cm) made of trans-
parent Plexiglas was placed in the middle of one of the quadrants (SW),
half-way between the center and the wall, and 1.5 cm below the water
surface. Pictures on the walls and the furniture of the room were used as
extra-maze cues. Animals entered the maze facing the wall of the pool
and were allowed to swim for 60 s in order to locate the submerged
platform. In case an animal failed to locate the platform within the
specified period, it was gently guided to the platform by the experi-
menter; once the animal had reached the platform it was allowed to
remain there for 15 s. The interval between trials of each day was 1 min
(including the 15 s period spent on the platform). The animals’ swim-
ming behavior was recorded by a video camera connected to a data
acquisition system (Ethovision v.2.3, Noldus Information Technology).
2.4.4.1. Spatial acquisition phase (spatial learning). In this phase animals
had to learn to navigate to the platform based on external cues. Spatial
acquisition phase lasted 4 days and animals were given 4 trials/day
(totally 16 trials). The platform was permanently located in the SW
quadrant of the maze and start position was different for each trial. A
semi-random approach of start positions for each trial was chosen
(Vorhees and Williams, 2006). Time to reach the platform (escape
latency; s) was measured to estimate visuospatial learning. Normally,
animals spend less time to locate the platform from day to day as a
result of learning (Morris, 1984). Swim speed (velocity; cm/s) was also
estimated in order to ensure that any differences in time to locate the
platform was attributed to learning acquisition and not to differences in
swimming speed.
2.4.4.2. Probe trial. In order to assess spatial memory, a probe trial was
administered 24 h after the last acquisition day. During this trial (60 s),
the escape platform was removed and each animal entered the maze
from a novel position (180° from the platform position during the
acquisition phase). The time spent to the target quadrant (SW) as well
as the frequency of entries in the area where the platform used to be
located during the acquisition phase were used as indices of spatial
memory (D’Hooge and De Deyn, 2001; Shinohara and Hata, 2014).
Ε. Dandi et al.
2.5. Corticosterone assessment
One week after completion of behavioral testing blood was collected
from the rats’ carotid artery in order to determine corticosterone levels
before and after stress induction. Stress was induced by placing the rat
cage on a rotating disk (78 revolutions per minute) for a 15 min period.
According to existing evidence, cage rotation for durations of 10 or
20 min can produce significant increase in circulating corticosterone
concentrations (Gein and Sharavieva, 2016; Riley, 1981). In order to
avoid circadian variability, blood sampling was carried out between
0900 and 1100 for all control and experimental groups. Samples were
centrifuged (12.000 r.p.m. for 10 min at 4 °C) and plasma was extracted
and maintained at −80 °C until assay. Corticosterone concentrations
were assessed using an enzyme-linked immunosorbent assay (ELISA) kit
(MB Biomedicals, 07DE9922).
2.6. Tissue preparation and immunofluorescence
All animals aged approximately 3.5 months were euthanized fol-
lowing anaesthetization. Brains were removed immediately and post-
fixed (4% paraformaldehyde in 0.1 M phosphate-buffer saline, 3 × 24 h
at 4 °C). Coronal blocks were gradually hydrated and embedded in
paraffin. Serial coronal sections of 5 μm thick were taken at the level of
dorsal hippocampus (−3.24 mm to −3.36 mm posterior to bregma)
(Paxinos and Watson, 2007). Expression of BDNF and SYN on these
sections was evaluated using immunofluorescence. Following depar-
affinization and hydration, steamer was used for antigen retrieval
(pH = 6, 1 h). Next, sections were incubated to blocking buffer (10%
Fetal Bovine Serum, 2% Normal Goat Serum, 30 min) and treated with a
primary antibody against BDNF (1:400, rabbit polyclonal, Santa Cruz),
or synaptophysin (1:100, mouse monoclonal, CloneSY38, DAKO)
overnight (4 °C). Depending on the primary antibody, sections were
exposed to goat anti-rabbit IgC (Biotium 488) or goat Anti-MouseIgG
(Biotium 555) for BDNF or SYN, respectively. Nuclei were counter-
stained with DAPI and mounted with the corresponding Biotium
medium (Biotium 23004).
Tissue images were captured from sections with a fluorescent
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International Journal of Developmental Neuroscience 67 (2018) 19–32
Fig. 1. (A) Entries and (B) time spent in the open
arms expressed as a ratio of the total entries and time
spent in both open and closed arms of the EPM.
Maternal separation significantly decreased number
of entries and time spent in the open arms in SH
animals (NMS/SH vs MS/SH, entries:**p < 0.001,
time:*p < 0.01), but exposure to EE reversed this
outcome (MS/SH vs. MS/EE, #p < 0.05). (C)
Number of returns to closed arms of the EPM. MS
animals reared in standard housing performed more
closed arms returns, compared to NMS treated ani-
mals (NMS/SH vs. MS/SH: *p < 0.01). Exposure of
maternally-separated animals to EE decreased the
number of returns compared to MS animals of the SH
condition (MS/SH vs. MS/EE: #p < 0.001). (D)
Among animals raised in SH condition, MS rats ex-
hibited increased number of Stretch-Attend Postures
(SAPs) in relation to non-maternally separated ani-
mals (NMS/SH vs. MS/SH, *p < 0.01), but EE re-
duced occurrence of this behavior (MS/SH vs. MS/
EE: #p < 0.001).
microscope Zeiss Axioplan-2 using a CCD camera (Nikon DS–5 M). Two
sections per animal were chosen with care to allow comparison of si-
milar regions across experimental conditions. Immunoreactivity for
BDNF and SYN was estimated within the CA3, CA2 and CA1 stratum
radiatum and the dentate gyrus (DG) molecular layer with 40x objec-
tive lens. Approximately sixteen microscopic fields per section/animal
were analyzed. Expression of BDNF and SYN was measured using the
ImageJ software (version 1.45b, NIH) and is presented as mean
Integrated Density (arbitrary density units) of all four hippocampal
subregions.
2.7. Statistical analyses
All statistical analyses were performed using the statistical program
SPSS Statistics (v. 22). Effects of the two experimental conditions
(rearing: NMS, MS and housing: SH, EE) on behavior (elevated plus
maze, open field test, novel object recognition, MWM retention phase),
plasma corticosterone levels and immunofluorescence markers (BDNF,
SYN) were tested using 2 × 2 ANOVAs with rearing and housing as the
between-subjects factors. A three factor repeated-measures ANOVA
(between-subjects factors: rearing, housing; within-subjects factor: day)
was applied to analyze the latency to locate the platform (MWM; ac-
quisition phase). Body weight measurement during the first three
postnatal weeks (3rd, 8th, 14th, 21st postnatal days) were analyzed by
a two factor repeated-measures ANOVA (between-subjects factors:
rearing; within-subjects factor: postnatal day). Pairwise comparisons
with Bonferroni correction were used in order to explore simple effects
(i.e., effect of each factor within each level of the other factor). Data are
presented as mean values ( ± SEM). Statistical significance was set at
a < 0.05 for all measures.
3. Results
3.1. Body weight
A repeated-measures analysis showed a significant increase in body
weight over the three postnatal weeks [F(3, 126) = 1593.8, p < 0.001,
Ε. Dandi et al.
Fig. 2. (A) Number of rearings in the outer zone of the open field during the 6-min period.
Environmental enrichment significantly increased the number of rearings (#p < 0.05, EE
main effect). (B) Total entries (ambulation). There were no significant effects for the total
number of square visits caused by either the MS or EE manipulations (p > 0.05) and the
two factors did not interact with each other (p > 0.05) (Fig. 2B).
partial η2 = 0.974; 3rd day: 9.19 (0.255), 8th day: 17.43 (0.481), 14th
day: 30.65 (0.783), 21st day: 47.1 (0.974)]. Maternal separation did not
affect the body weight [F(1, 42) = 1.602, partial η2 = 0.037] neither
interacted with postnatal day [F(3, 126) = 2.541, p > 0.05, partial
η2 = 0.057].
3.2. Elevated plus maze
Ratio of open arm entries: Analysis revealed a significant main effect
of EE [F(1, 43) = 11.68, p < 0.01, partial η2 = 0.214; SH:
0.262(0.021), EE: 0.361(0.02)], but not of MS condition [F(1,
43) = 0.23, p > 0.05, partial η2 = 0.005, NMS: 0.305 (0.021.), MS:
0.319 (0.02)] (Fig. 1A). Pairwise comparisons to explore the significant
MS × EE interaction [F(1, 43) = 47.97, p < 0.001, partial η2 = 0.527]
showed that while maternal separation in standard-housed animals
decreased the number of entries [F (1, 43) = 19.47, p < 0.001, partial
η2 = 0.312, NMS/SH: 0.355 (0.031) vs. MS/SH: 0.169(0.028)], this
effect was completely reversed by EE housing [F(1, 43) = 55.13,
p < 0.001, MS/SH: 0.169 (0.028) vs. MS/EE: 0.468 (0.028)].
Ratio of time spent in open arms: Both MS and EE conditions af-
fected time spent in open arms [MS: F(1, 43) = 6.90, p < 0.05, partial
η2 = 0.138, NMS: 0.226 (0.022.), MS: 0.306 (0.021); EE: F(1,
43) = 15.87, p < 0.001, partial η2 = 0.270, SH: 0.205 (0.022), EE:
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International Journal of Developmental Neuroscience 67 (2018) 19–32
0.327 (0.021)] (Fig. 1B).The two manipulations significantly interacted
with each other [MS × EE: F(1, 43) = 52.85, p < 0.001, partial
η2 = 0.551]. Maternal separation in standard-housed condition de-
creased the time spent in open arms [F(1, 43) = 19.47, p < 0.01,
partial η2 = 0.19, NMS/SH: 0.276 (0.033) vs. MS/SH: 0.134 (0.030)].
Housing of MS rats in EE condition significantly reversed the decreased
time seen in MS animals that lived in SH conditions [F(1, 43) = 65.264,
p < 0.001; MS/SH: 0.134 (0.030) vs. MS/EE: 0.478 (0.030)].
Closed arm returns: Housing conditions affected the specific beha-
vior, with animals exposed to EE performing less returns compared to
SH animals [F(1, 43) = 23.48, p < 0.001, partial η2 = 0.353]
(Fig. 1C). Results revealed a tendency of the effect of rearing on closed
arm returns, which did not reach statistical significance [F(1,
43) = 3.73, p = 0.06, partial η2 = 0.08]. The two factors interacted
with each other [F(1, 43) = 8.23, p < 0.01, partial η2 = 0.161]. Spe-
cifically, MS increased closed arms returns for SH animals [F(1,
43) = 10.793, p < 0.01; NMS/SH: 2.5 (0.525) vs. MS/SH: 4.833
(0.479)], but exposure of MS rats to EE condition totally reversed this
effect [F(1, 43) = 16.898, p < 0.001; MS/SH: 4.833 (0.479) vs. MS/
EE: 1.083 (0.479)].
Stretch Attend Posture (SAP): There were no significant effects for
the number of SAPs caused by either the rearing or housing manip-
ulations [MS: F(1, 43) = 0.276, p > 0.05, partial η2 = 0.006; EE: F(1,
43) = 2.28, p > 0.05, partial η2 = 0.05]. However, the effect of EE
differed as a function of rearing, as indicated by the significant inter-
action [F(1, 43) = 17.78, p < 0.001, partial η2 = 0.292]. MS increased
the SAPs in the standard-housed condition [F(1, 43) = 10.532,
p < 0.01, partial η2 = 0.197; NMS/SH: 2.8 (0.785) vs. MS/SH: 6.25
(0.717)]. While EE condition did not affect occurrence of this behavior
in the NMS rats [F(1, 43) = 3.556, p > 0.05, partial η2 = 0.076], it
caused a significant decrease in the MS groups [F(1, 43) = 16.898,
p < 0.001, partial η2 = 0.282; MS/SH: 6.25 (0.717), MS/EE: 2.083
(0.717)] (Fig. 1D).
Total arm entries: Neither MS nor EE conditions affected the total
arm entries [MS: F(1, 43) = 0.110, p > .05, partial η2 = 0.003, NMS:
16.45 (0.641), MS: 16.75 (0.622); EE: F (1, 43) = 0.092, p > .05,
partial η2 = 0.002, SH: 16.47 (0.653), EE: 16.74 (0.61)].
3.3. Open field
Rearings (vertical activity): Analysis of rearings performed in the
outer zone revealed a significant effect of EE [F(1, 43) = 4.943,
p < 0.05, partial η2 = 0.103]. EE treated animals performed more
rearings than SH treated animals [EE: 12.18 (1.22), SH: 8.22 (1.3)]. MS
condition did not affect vertical activity [F(1, 43) = 2.264, p > 0.005,
partial η2 = 0.050]. The effect of EE was not a function of rearing, as
indicated by the non-significant interaction [F(1, 43) = 0.064,
p > 0.05, partial η2 = 0.001] (Fig. 2A).
Ambulation: There were no significant effects for the total number
of square visits caused by either the MS [F(1, 43) = 1.373, p > 0.05,
partial η2 = 0.031] or EE manipulations [F(1, 43) = 2.103, p > 0.05,
partial η2 = 0.047], and the two factors did not interact with each other
[F(1, 43) = 0.101, p > 0.05, partial η2 = 0.002] (Fig. 2B).
3.4. Novel object recognition task (NORT)
MS or EE did not differentiate the groups in exploration time of the
object A1 [MS: F(1, 43) = 3.102, p > 0.05, partial η2 = 0.067; EE: F(1,
43) = 0.188, p > 0.05, partial η2 = 0.004] or object A2 [MS: F(1,
43) = 2.431, p > 0.05, partial η2 = 0.054; EE: F(1, 43) = 0.520,
p > 0.05, partial η2 = 0.012] during the sample phase (trial 1).
Analysis of the exploration time from the choice phase (trial 2) in-
dicated no significant effect of MS [F(1, 40) = 0.494, p > 0.05, partial
η2 = 0.012], EE [F(1, 40) = 1.635, p > 0.05, partial η2 = 0.039] or a
significant interaction between the two factors [F(1, 40) = 1.48,
p > 0.05, partial η2 = 0.036] on discrimination ratio (Fig. 3). This
Ε. Dandi et al.
Fig. 3. Mean values of the discrimination ratio of all groups in the novel object re-
cognition. Preference for the novel object did not differ among groups as a function of
maternal separation or environmental enrichment (p > 0.05).
finding indicates that all animals reacted similarly to novelty regardless
of experimental conditions.
3.5. Morris water maze
Spatial learning: Spatial learning was significantly affected by EE,
with EE treated animals being faster to locate the platform compared to
the SH condition [F(1, 43) = 62.47, p < 0.001, partial η2 = 0.592]
(Fig. 4). The rearing condition exerted no influence on latency [MS: F(1,
43) = 0.848, p > 0.05, partial η2 = 0.019] neither interacted with EE
[MS × EE: F(1, 43) = 1.419, p > 0.05, partial η2 = 0.032]. The per-
formance of rats was improved over the 4-day period [day: F(3,
129) = 174.309, p < 0.001, partial η2 = 0.802]. Bonferroni’s pairwise
comparisons revealed a gradual decrease in latency from the first to the
third day (p < 0.001), but no significant difference was found between
the third and the fourth day (p > 0.05). The “day” factor interacted
with housing [day × EE: F(3, 129) = 6.42, p < 0.001, partial
η2 = 0.13].
In order to ensure that differences in latency to locate the platform
were not due to differences in swim speed, a two-way ANOVA
(MS × EE) was conducted on mean velocity/day. No statistically sig-
nificant difference was found as a result of MS [day 1: F(1, 43) = 3.72,
p > 0.05, partial η2 = 0.08, day 2: F(1, 43) = 4.01, p > 0.05, partial
η2 = 0.085), day 3: F(1, 43) = 3.17, p > 0.05, partial η2 = 0.07, day 4:
F(1, 43) = 0.08, p > 0.05, partial η2 = 0.002] or EE [day 1: F(1,
43) = 1.86, p > 0.05, partial η2 = 0.041, day 2: F(1, 43) = 0.44,
Fig. 4. Latency to find the platform of all groups in the spatial learning protocol of the
Morris water maze. All animals improved their performance over the 4-day period
(#
p < 0.05). EE treated animals exhibited lower latencies compared to SH treated ani-
mals (*p < 0.001).
24
International Journal of Developmental Neuroscience 67 (2018) 19–32
p > 0.05, partial η2 = 0.011, day 3: F(1, 43) = 0.82, p > 0.05, partial
η2 = 0.019, day 4: F(1, 43) = 2.76, p > 0.05, partial η2 = 0.06].
Probe trial: A two-way ANOVA revealed a significant effect for MS
on time spent in the target quadrant (SW) [F(1, 43) = 6.74, p < 0.05,
partial η2 = 0.135] as well as on frequency of entries into the platform
area [F(1, 43) = 9.113 p < 0.01, partial η2 = 0.175] (Fig. 5). MS an-
imals spend less time and did fewer entries than the non-maternally
separated animals. The EE condition did not affect any of the two
measures [time: F(1, 43) = 0.43, p > 0.05, partial η2 = 0.01; fre-
quency: F(1, 43) = 0.866, p > 0.05, partial η2 = 0.020]. Interestingly,
housing in EE seems to protect against stress-induced impairments, as
indicated by the significant interaction between MS and ΕΕ for the
duration [F(1, 43) = 7.57, p < 0.01, partial η2 = 0.150] and fre-
quency of entries [F(1, 43) = 4.474, p < 0.05, partial η2 = 0.094].
Specifically, MS rats raised in standard housing conditions exhibited
poorer performance compared to NMS rats as indicated by duration
time [F(1, 43) = 13.395, p < 0.01; NMS/SH: 23.544 (1.183) vs. MS/
SH: 17.68 (1.08)] and frequency [F(1, 43) = 12.349, p < 0.01; NMS/
SH: 3.30 (1.418) vs. MS/SH: 1.33 (1.154). Housing of maternally se-
parated rats in EE reversed this effect [duration: F(1, 43) = 5.93,
p < 0.05; MS/SH: 17.68 (1.08) vs. MS/EE: 21.42 (1.080); frequency: F
(1, 43) = 4.78, p < 0.05, MS/SH: 1.33 (1.154) vs. MS/EE: 2.50
(1.243)]. As far as the time spent in the remaining (non-target) quad-
rants (NE, NW, SE), there was no difference as a function of MS or EE in
time spent in the a) NE quadrant [MS: F(1, 43) = 1.24, p > 0.05,
partial η2 = 0.028; EE: F (1, 43) = 0.012, p > 0.05, partial η2 = 0.00;
NMS/SH = 11.15 (1.21), NMS/EE = 12.05 (1.18), MS/SH = 13.53
(1.36), MS/EE = 12.37 (1.02)], b) NW quadrant [MS: F(1, 43) = 0.057,
p > 0.05, partial η2 = 0.001; EE: F(1, 43) = 0.417, p > 0.05, partial
η2 = 0.01; NMS/SH = 11.58 (0.65), NMS/EE = 13.41 (1.32), MS/
SH = 14.57 (1.62), MS/EE = 11.04 (1.26)] and c) SE quadrant [MS: F
(1, 43) = 0.819, p > 0.05, partial η2 = 0.019; EE: F (1.43) = 0.006,
p > 0.05, partial η2 = 0.00; NMS/SH: 12.34 (1.07), NMS/EE: 12.28
(1.51), MS/SH: 13.41 (1.32), MS/EE: 13.68 (1.38)].
3.6. Plasma corticosterone analysis
Basal levels: There were no significant effects for the basal corti-
costerone levels by either MS [F (1, 38) = 1.04, p > 0.05, partial
η2 = 0.027] or EE manipulations [F(1, 38) = 0.028, p > 0.005, partial
η2 = 0.001], and the two factors did not interact with each other [F(1,
38) = 0.000, p > 0.05, partial η2 = 0.000; NMS/SH: 25.58 (6.695),
NMS/EE: 24.59 (5.316), MS/SH: 19.73 (5.484), MS/EE: 18.11 (4.854)].
Stress-induced levels: Analysis revealed a significant effect of
housing condition on corticosterone elevations in response to acute
stress [F(1, 41) = 6.168, p < 0.017, partial η2 = 0.131], with the SH
rats expressing significantly higher levels than the animals of the EE
manipulation (Fig. 6). The effect of EE was a function of MS as indicated
by their significant interaction [MS × EE: F(1, 41) = 7.920, p < 0.01,
partial η2 = 0.162]. Pairwise comparisons revealed that maternal se-
paration caused significant elevations in animals of the SH condition
compared to NMS rats [F(1, 41) = 4.887, p < 0.05; NMS/SH: 261
(20.102) vs. MS/SH: 321.167 (18.35)]. On the contrary, exposure to EE
blocked this effect [F(1, 41) = 13.788, p < 0.01, partial η2 = 0.252;
MS/SH: 321.167 (18.35) vs. MS/EE: 220.100 (20.102)].
3.7. Immunoreactivity of BDNF and synaptophysin in the hippocampus
BDNF: Analysis of variance revealed a significant main effect of MS
on the expression of hippocampal BDNF [F (1, 19) = 9.134, p < 0.001,
partial η2 = 0.325], with MS animals expressing lower levels compared
to NMS (Fig. 7). In addition, immunoreactivity was significantly higher
in animals that were housed in enriched environment, as indicated by
the main effect of the EE condition [F(1, 19) = 27.068, p < 0.001,
partial η2 = 0.588]. In NMS condition, BDNF expression was sig-
nificantly higher in rats that were exposed to enriched conditions
Ε. Dandi et al.
Fig. 5. (A) Time spent in the target quadrant and (B) frequency of entries in the platform area during the probe trial of the Morris water maze. MS rats housed in standard conditions spent
less time and did fewer entries, compared to NMS/SH rats (*p < 0.01), while exposure to EE increased the time and the number of entries for MS rats (MS/SH vs MS/EE, #p < 0.05).
Fig. 6. MS animals housed in SH had higher plasma corticosterone levels after exposure
to acute stress, compared to NMS animals (NMS/SH vs MS/SH,*p < 0.05). Rearing of
maternally-separated animals in EE significantly reduced these corticosterone elevations
(MS/SH vs MS/EE, #p < 0.05).
compared to standard housing [F(1, 19) = 7.470, p < 0.05, NMS/SH:
21.64 (1.91) vs NMS/EE: 28.72 (1.74), partial η2 = 0.282]. Pairwise
comparisons revealed that maternal separation significantly reduced
the BDNF levels in SH animals [F(1, 19) = 9.545, p < 0.001, partial
η2 = 0.334, NMS/SH: 21.64 (1.91) vs. MS/SH: 13.9 (1.62)]. Environ-
mental enrichment of maternally separated rats counteracted this MS-
associated decrease [F(1, 19) = 21.696, p < 0.001, partial η2 = 0.533,
MS/SH: 13.9 (1.62) vs MS/EE: 25.57 (1.91)].
Synaptophysin (SYN): Both MS and EE conditions significantly af-
fected the SYN immunoreactivity in the hippocampus [MS: F(1,
19) = 14.172, p < 0.01, partial η2 = 0.427; EE: F(1, 19) = 25.848,
p < 0.001, partial η2 = 0.57], with MS decreasing and EE increasing
its levels (Fig. 8). Additionally, environmental enrichment significantly
increased SYN expression in the non-maternally separated rats [F(1,
19) = 12.769, p < 0.01, NMS/SH: 27.59 (2.21) vs NMS/EE: 38.31
(2.02), partial η2 = 0.402]. In SH rats, maternal separation significantly
decreased SYN expression [F(1, 19) = 7.125, p < 0.05, partial
η2 = 0.273; NMS/SH: 27.59 (2.85), MS/SH: 19.84 (1.74)]. Housing of
maternally-separated rats in enriched conditions reversed this effect [F
(1, 19) = 13.093, p < 0.01, partial η2 = 0.408; MS/SH: 19.84 (1.74)
vs. MS/EE: 30.34 (2.80)].
4. Discussion
It is well known that environmental conditions can exert a strong
influence on brain development, behavior and neuroplasticity. In par-
ticular, early adverse experiences may lead to psychopathology and
cognitive impairments that can persist until adulthood, while environ-
mental enrichment (EE) appears to have a beneficial role on different
aspects of brain function and behavior. Τhe aim of the present study
was to explore whether Environmental Enrichment (EE) during ado-
lescence can counterbalance the negative impact of early stress in the
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International Journal of Developmental Neuroscience 67 (2018) 19–32
form of maternal separation (MS). In particular, we investigated the
effects of these environmental manipulations as well as their interaction
on cognition, emotionality, stress reactivity and expression of BDNF
and synaptophysin in the hippocampus.
4.1. Effect of maternal separation and environmental enrichment on anxiety
and stress reactivity
Emotionality was evaluated by the EPM task, a reliable behavioral
test of anxiety. Analysis of four behavioral indices (open-arms time and
entries, closed-arms returns and SAPs) showed that daily MS during the
first three postnatal weeks increased anxiety-like behavior during
adulthood. Specifically, our data suggest that MS potentiated the
standardly housed (SH) rodent’s unconditioned fear towards open
spaces and increased closed arm returns as well as SAPs, a behavioral
profile indicative of increased anxiety. The reduced open-arms pre-
ference seems not to be associated with a general decrease in activity,
as indicated by the comparable ambulation scores and arm entries in
our Open Field and EPM task, respectively, among the MS and NMS
groups. Specifically, there was no significant difference between NMS
and MS groups with respect to the horizontal (ambulation) or vertical
(rearing) activity, a finding also reported previously (Markostamou
et al., 2016; Shalev and Kafkafi, 2002; Vivinetto et al., 2013).
Interestingly, post-weaning housing of maternally separated rats in
EE conditions acted beneficially, decreasing significantly the risk as-
sessment behaviors (i.e., stretch attend posture, closed arms returns)
while increased open arms entries and time. These behavioral effects of
MS and EE conditions appear to be mediated by alterations in the HPA
axis reactivity. Indeed, maternally separated rats that were housed in
standard post-weaning conditions (MS/SH group) expressed sig-
nificantly higher levels of corticosterone in response to a stress chal-
lenge as adults. On the contrary, housing in EE condition (MS/EE
group) counterbalanced the MS effects, thus attenuating adrenocortical
responses to levels similar to those of the NMS group. It should be also
reported that basal corticosterone concentrations did not differ sig-
nificantly as a function of rearing or housing.
Our findings are in accordance with previous studies reporting the
detrimental effects of early adversity on emotionality and stress re-
activity. In fact, it has been shown that MS over the first 2–3 postnatal
weeks significantly increases anxiety and depression-like behaviors and
enhances HPA responses to stressors during adulthood (Aisa et al.,
2007; Kalinichev et al., 2002; Koe et al., 2016; Liu et al., 2000;
Veenema et al., 2007). In our experiment, the potentiated corticos-
terone increase of MS/SH rats in response to an acute stressor (i.e., cage
rotation) was not associated with elevated basal corticosterone in MS/
SH compared to NMS/SH groups. This finding is in accordance with
studies reporting no differences in basal concentrations in animals
previously exposed to early-life stress (Knuth and Etgen, 2007; Koe
et al., 2016; Rüedi-Bettschen et al., 2005; Slotten et al., 2006; Wigger
and Neumann, 1999). Given the significant role of the hippocampus,
Ε. Dandi et al. International Journal of Developmental Neuroscience 67 (2018) 19–32

Fig. 7. (A) BDNF expression in the dorsal hippocampus expressed as integrated density. MS animals raised in SH had lower BDNF levels compared to NMS animals of the same rearing
condition (NMS/SH vs MS/SH, *p < 0.001). Environmental enrichment significantly increased protein expression in standard housed rats (NMS/SH vs NMS/EE, #p < 0.05) and
counteracted the decreased levels in maternally-separated rats (MS/SH vs MS/EE, ##p < 0.001). (B) Representative photomicrographs of BDNF immunofluorescence staining in the
hippocampal CA1 region. Total magnification 400x, scale bar = 100 μm.

particularly its glucocorticoid receptor (GR) system, in the HPA nega-
tive feedback mechanism, it may be suggested that hippocampal al-
terations may be associated with the potentiated stress response. In fact,
decreases in the GR density and mRNA expression have been reported
following early stress (Aisa et al., 2008; Enthoven et al., 2008b). This
downregulation appears to dysregulate the negative feedback me-
chanism, thus leading to further elevations of plasma corticosterone
and increased anxiety (Sampedro-Piquero et al., 2014). Existing data
suggest that glucocorticoids elevations play an important role in the
effects of MS. Specifically, both recognition memory deficits and de-
pressive-like behavior are completely reversed after administration of a
GR antagonist (Aisa et al., 2008, 2007), while treatment with the cor-
ticosterone synthesis inhibitor, metyrapone, reduces the increased
vulnerability to kindling epileptogenesis seen in maternally separated
rats (Koe et al., 2016).
Contrary to early stress, EE appears to have beneficial behavioral
and neurobiological effects. A body of evidence suggests that EE
housing attenuates anxiety and HPA-mediated endocrine responses

26
Ε. Dandi et al. International Journal of Developmental Neuroscience 67 (2018) 19–32

Fig. 8. (A) Synaptophysin (SYN) expression in the dorsal hippocampus expressed as integrated density. Environmental enrichment significantly increased protein expression in standard
housed rats (NMS/SH vs NMS/EE, #p < 0.01). Maternal separation significantly decreased SYN expression in rats of the SH condition (NMS/SH vs MS/SH, *p < 0.05). Enriched
environment reversed the decreased SYN levels in maternally separated rats (MS/SH vs MS/EE, #p < 0.01). Representative photomicrographs of SYN immunofluorescence staining in
the hippocampal CA1 region. Total magnification 400x, scale bar = 100 μm.

evoked by stressors in adulthood (Chapillon et al., 1999; Fox et al.,
2006), while it increases GR expression in hippocampal areas (Vivinetto
et al., 2013). The regulatory impact of EE is also supported by studies of
reduced anxiety in animal models of anxiety, depression or mental
disorders (e.g., schizophrenia and ADHD) (Brenes Sáenz et al., 2006;
Ishihama et al., 2010; Nicolas et al., 2015) as well as in gestational
inflammation-induced HPA axis hyperactivity in young rats (Connors
et al., 2014). The majority of existing data highlighting the
compensatory role of EE mainly refer to chronic stress paradigms ad-
ministered during adulthood. Specifically, EE housing prior to or fol-
lowing periods of adult chronic stress reduces emotionality and HPA
activity to an acute stressor (Wright and Conrad, 2008). Recently, it has
been shown that a short episode of EE during adulthood reduces an-
xiety-like behavior in MS rats (Koe et al., 2016). The increases in GR
expression seen in EE-housed rats that were subsequently exposed to
both chronic and acute stress as adults appear to contribute to a more

27
Ε. Dandi et al.
adaptive response to chronic stress (Zanca et al., 2015). To the best of
our knowledge, so far only one study has investigated the role of EE on
enhanced HPA reactivity in maternally-separated rats, reporting a total
reversal of corticosterone to normal levels under conditions of EE
(Francis et al., 2002).
4.2. Effect of maternal separation and environmental enrichment on
cognitive function
Behavioral testing in the Morris Water Maze (MWM) revealed a
beneficial effect of EE on visuospatial learning during adulthood. The
groups that were housed in post-weaning EE conditions exhibited
shorter latencies, compared to the SH rats, to locate the platform, an
indication of improved spatial acquisition. Our findings are in line with
previous studies demonstrating the beneficial effects of environmental
enrichment on cognitive function. In fact, EE has been associated with
improvement in spatial learning, memory, and orientation in adult and
aged rodents (Frick and Fernandez, 2003; Harburger et al., 2007;
Hullinger et al., 2015; Leggio et al., 2005) as well as protection against
age-associated impairment in LTP induction (Stein et al., 2016).
MS did not impair spatial acquisition, a finding also reported by
other studies following postnatal stress (Aisa et al., 2009; Akatsu et al.,
2015; Grace et al., 2009). However, it did impair spatial memory, as
indicated by our probe trial data. These long term negative con-
sequences in memory (spatial or fear-associated) are in line with pre-
vious reports demonstrating spatial and fear-associated memory im-
pairments following maternal separation (Chocyk et al., 2014; Sousa
et al., 2014; Xiong et al., 2015). This finding may imply that spatial
memory is more vulnerable than visuospatial learning to the effect of
early adversity, as it has been also suggested regarding the effects of
chronic stress (Conrad, 2010). In fact, it has been reported that the
spatial learning deficits seen in juvenile rats following postnatal social
isolation are restored in adulthood, an over-time improvement possibly
associated with compensatory changes that take place (i.e., damaged
dendrites are repaired, new ones are sprouting) within the hippo-
campus (Frisone et al., 2002).
Interestingly, post weaning EE offered total protection against the
spatial memory deficits associated with MS. While MS rats spent less
time in the target quadrant and made fewer frequencies of entries into
the platform area, compared to NMS group, EE completely reversed this
effect. As far as the impact of EE on spatial memory of MS rats, previous
findings also support its compensatory role against cognitive deficits
following chronic adult (Hutchinson et al., 2012; Veena et al., 2009;
Wright and Conrad, 2008), juvenile (Ilin and Richter-Levin, 2009) or
early stress induced by MS or limited nesting/bedding material (Cui
et al., 2006; do Prado et al., 2016; Vivinetto et al., 2013). However, the
present study is the first to demonstrate the ameliorating role of EE
against spatial memory impairments in rodents that experienced early
stress in the form of maternal separation.
Behavioral analysis of the NORT data revealed that neither the type
of rearing nor housing manipulations affected non-spatial recognition
memory. While a number of studies showed impairments in recognition
memory following early adversity (Aisa et al., 2008; Daniels et al.,
2009; Hulshof et al., 2011), other investigators report no effect of
postnatal stress on this type of memory (Grace et al., 2009; Mourlon
et al., 2010; Vivinetto et al., 2013). It is possible that differences in
duration and type of postnatal stress as well as time of testing may
account for this discrepancy. It should be mentioned that the absence of
deficits in the non-spatial recognition memory reported here is in line
with previous research conducted in our lab (Tata et al., 2015). The fact
that MS rats were impaired in the probe trial of the MWM, but not in
the NOR task, implies that our MS paradigm selectively affects spatial
memory. In fact, Leret and colleagues have also shown impairments in
spatial memory but not object recognition both in adolescent and adult
rats that had been exposed to MS (Leret et al., 2010). Regarding the role
of housing, so far there is limited information concerning the effect of
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International Journal of Developmental Neuroscience 67 (2018) 19–32
post-weaning EE on NORT performance. Although EE housing of 3-
month old adult rats for 3 weeks (PND90-111) improves novel object
recognition (Bechara and Kelly, 2013), EE exposure for a longer period
of time during an earlier developmental stage (PND21-60) does not
seem to affect it (Vivinetto et al., 2013). These findings probably sug-
gest that the effectiveness of EE on recognition memory may be a
function of time of exposure and duration of exposure.
4.3. Effects of maternal separation and environmental enrichment on BDNF
and synaptophysin expression in the hippocampus
Increasing evidence underlines the essential role of hippocampal
BDNF and synaptophysin (SYN) in cognition as well as anxiety and
depression-related behaviors (Heldt et al., 2007; Liu et al., 2005;
Mitchelmore and Gede, 2014). In the present study, MS rats expressed
behaviors indicative of impaired memory and increased anxiety, while
EE exerted a beneficial effect in the MS group. In order to estimate
whether these behavioral changes were mediated by alterations in
neuronal plasticity, we evaluated the expression of these two markers
(BDNF and SYN) in the dorsal hippocampus. BDNF plays an essential
role in synapse formation, neuronal survival and growth and it has been
suggested to be involved in transducing the effects of environmental
manipulation on brain function during early developmental stages
(Cirulli et al., 2003). In addition, BDNF expression in multiple brain
regions is sensitive to adverse life experiences (Han et al., 2011; Meng
et al., 2011).
According to our analysis, MS significantly decreased the expression
of BDNF in the dorsal hippocampus. This finding is in line with previous
studies showing a downregulation in BDNF mRNA or protein expression
following 2–3 weeks of MS (Aisa et al., 2009; Lippmann et al., 2007;
MacQueen et al., 2003). However, the investigation of the MS impact
on BDNF expression has yielded inconsistent results due to the in-
volvement of various factors. The duration of adverse postnatal ma-
nipulation appears to be a determining factor, since postnatal stress of
shorter duration did not affect BDNF levels (i.e., a single episode of 24 h
maternal deprivation, or 6-day MS) (Choy et al., 2008; Markostamou
et al., 2016; Roceri et al., 2002). Furthermore, the developmental
period of the animals as well as the brain region seem to influence MS
effects. In fact, it has been shown that MS induces different patterns (up
or down-regulation) of BDNF expression along with age in different
brain regions (Pardon et al., 2009; Récamier-Carballo et al., 2017;
Wang et al., 2015b). This differential impact of MS on BDNF expression
reflects the complex functional consequences of adverse early life ex-
periences in synaptic plasticity mechanisms.
In addition to the BDNF changes, we found that prolonged MS
significantly decreased SYN in the dorsal hippocampus. SYN, a synaptic
vesicle-associated protein, is a general synaptic marker and its presence
indicates the efficiency of synaptic transmission (Thiel, 1993; Valtorta
et al., 2004). It has been previously reported that MS can cause down-
regulation of SYN during adulthood, and this effect might be re-
sponsible for impaired hippocampal neurotransmission and behavioral
changes (Aisa et al., 2009; Andersen and Teicher, 2004).
The possibility that BDNF and SYN expression may have been af-
fected by the acute stress (i.e., rotation) applied at the end of the ex-
periments cannot be excluded. Previous studies have shown that acute
stress alters the expression of neurotrophic factors and synaptic reg-
ulatory proteins and these alterations may be responsible for some of
the morphological and behavioral changes observed after stress ex-
posure (Amin et al., 2015; Gao et al., 2006; Smith et al., 1995; Thome
et al., 2001). Specifically, acute stress induces a brain region-dependent
differential expression of BDNF (Lakshminarasimhan and Chattarji,
2012). Furthermore, the type of stressor, the duration of stress or the
age of the animals are factors that also affect the outcome (Badowska-
Szalewska et al., 2017; Fuchikami et al., 2009; Molteni et al., 2009).
However, the present study did not intend to explore the possible effect
of acute stress on markers of synaptic plasticity or its functional
Ε. Dandi et al.
consequences. Furthermore, it should be mentioned that all animals
were subjected to the acute stress condition, which implies that all they
would have been equally affected.
Contrary to the effects of MS, exposure to EE increased the levels of
both SYN and BDNF expression in the non-stressed group. Previous
studies have also supported the beneficial effects of EE as indicated by
the enhanced BDNF both at mRNA and protein levels (Cao et al., 2014;
Griva et al., 2017; Ickes et al., 2000; Mosaferi et al., 2015). In addition,
our analysis showed that EE upregulated SYN in control animals, an
increase which is in agreement with previous studies (Birch et al., 2013;
Lambert et al., 2005; Nithianantharajah et al., 2004) and indicates the
involvement of this synaptic protein in experience-dependent neuro-
plasticity.
In our study, the beneficial role of EE is mainly supported by its
compensatory effect against the MS-associated BDNF and SYN de-
creases. Specifically, the levels in the stressed rats exposed to EE were
significantly higher than those in the MS animals housed in standard
conditions, and did not differ from those in the non-stressed groups.
Existing evidence supports the compensatory effect of EE on brain da-
mage in models of chronic stress or aging. In particular, EE protects
from dendritic atrophy as well as reductions in neurogenesis and neu-
rotrophin expression following adult stress (Hutchinson et al., 2012;
Shilpa et al., 2017; Veena et al., 2009; Vega-Rivera et al., 2016). Pre-
vious studies have also shown that exposure to EE during a critical
developmental period (0–2 postnatal months), is more effective at in-
creasing BDNF levels (Jha et al., 2016). In this respect, our BDNF in-
creases are in line with existing evidence, indicating a beneficial role of
post-weaning EE (PND23-65) against MS-associated decreases in BDNF
expression. Given that EE completely restored spatial memory impair-
ments in MS group and improved spatial acquisition, it could be sug-
gested that EE-associated increases in SYN and BDNF may be a pre-
requisite for these behavioral effects. In fact, higher levels of BDNF and
SYN have been correlated with cognitive improvement (Chen et al.,
2013; Frick and Fernandez, 2003), while according to others may not
be a prerequisite (Bennett et al., 2006; Griva et al., 2017; Pereira et al.,
2009).
5. Conclusion
In the current study we explored the interaction of a 3-week MS and
subsequent exposure to enriched conditions on various behaviors,
neuroendocrine stress response and two plasticity markers, BDNF and
synaptophysin. To date, existing studies suggest the negative impact of
MS or, on the contrary, the beneficial role of EE on emotional and
cognitive behaviors, stress response and brain anatomy and neu-
rochemistry. Besides the main effects of the two environmental condi-
tions on learning and memory, corticosterone levels and BDNF and SYN
expression, here we report that EE can compensate against stress-as-
sociated spatial memory deficits, increased anxiety and downregulation
of BDNF and SYN. To the best of our knowledge, this is the first study to
show that MS and EE interact with each other, modulating adult’s be-
havior and neuroendocrine response to stress as well as neuroplasticity.
Given the important role of early experiences, the need to better un-
derstand the underlying mechanisms that mediate behavioral changes
is further emphasized.
Funding
This research has been financially supported by the General
Secretariat for Research and Technology (GSRT) and the Hellenic
Foundation for Research and Innovation (HFRI) (Scholarship Code:
95144 to E.D).
Acknowledgements
The authors are grateful to Alexandros Antonellos (Microanalysi
29
International Journal of Developmental Neuroscience 67 (2018) 19–32
Medical Athens S.A) for his excellent technical assistance in corticos-
terone measurements (ELISA).
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