Psychoneuroendocrinology 68 (2016) 202–209

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Psychoneuroendocrinology
journal homepage: www.elsevier.com/locate/psyneuen

A diet high in fat and sugar reverses anxiety-like behaviour induced

by limited nesting in male rats: Impacts on hippocampal markers
Jayanthi Maniam, Christopher P. Antoniadis, Vivian Le, Margaret J. Morris ∗
Department of Pharmacology, School of Medical Sciences, UNSW Australia, Sydney 2052, New South Wales, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Stress exposure during early development is known to produce long-term mental health deficits. Stress
Received 2 December 2015 promotes poor lifestyle choices such as poor diet. Early life adversity and diets high in fat and sugar
Received in revised form 1 February 2016 (HFHS) are known to affect anxiety and memory. However additive effects of HFHS and stress during
Accepted 9 March 2016
early development are less explored. Here, we examined whether early life stress (ELS) simulated by
limited nesting (LN) induces anxiety-like behaviour and cognitive deficits that are modulated by HFHS
Keywords:
diet. We examined key hippocampal markers involved in anxiety and cognition, testing the hypothesis
Early life stress
that post-weaning HFHS following ELS would ameliorate anxiety-like behaviour but worsen memory
Anxiety-like behaviour
Akt3
and associated hippocampal changes. Sprague-Dawley rats were exposed to LN, postnatal days 2–9, and
Cognition at weaning, male siblings were given unlimited access to chow or HFHS resulting in (Con-Chow, Con-
Glucocorticoid receptor HFHS, LN-Chow, LN-HFHS, n = 11–15/group). Anxiety-like behaviour was assessed by Elevated Plus Maze
Glycogen synthase kinase (EPM) at 10 weeks and spatial and object recognition tested at 11 weeks of age. Rats were culled at 13
High-fat and sugar diet weeks. Hippocampal mRNA expression was measured using TaqMan® Array Micro Fluidic cards (Life
Hippocampus Technologies). As expected HFHS diet increased body weight; LN and control rats had similar weights
Limited nesting at 13 weeks, energy intake was also similar across groups. LN-Chow rats showed increased anxiety-like
behaviour relative to control rats, but this was reversed by HFHS diet. Spatial and object recognition mem-
ory were unaltered by LN exposure or consumption of HFHS diet. Hippocampal glucocorticoid receptor
(GR) protein was not affected by LN exposure in chow rats, but was increased by 45% in HFHS rats rel-
ative to controls. Hippocampal genes involved in plasticity and mood regulation, GSK␣ and GSK␤ were
affected, with reductions in GSK␤ under both diet conditions, and reduced GSK␣ only in LN-HFHS versus
Con-HFHS. Interestingly, HFHS diet and LN exposure independently reduced expression of Akt3 mRNA,
a key gene involved post-natal brain development. In summary, while an energy rich diet ameliorated
anxiety-like behaviour induced by LN exposure, it significantly altered key genes that are essential for
hippocampal development.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction utilised animal models, predominantly in rats, to simulate ELS in

Early life stress (ELS) is prevalent in today’s society, being mani-
fested in a number of different ways such as child abuse, childhood
trauma and neglect. ELS has been shown to have significant adverse
effects lasting into adulthood, altering the brain and behavioural
aspects such as mood and anxiety, as well as increasing the risk
of psychiatric disorders (Bremne and Vermetten, 2001; Heim and
Nemeroff, 1999; Sanchez et al., 2001). These effects on emotions
have been demonstrated to be a result of changes to the hippocam-
pus and amygdala (Aust et al., 2014). Further research efforts have
∗ Corresponding author.
E-mail address: m.morris@unsw.edu.au (M.J. Morris).
order to investigate its effects. A variety of methods have been
employed to induce ELS, including maternal separation and depri-
vation (Maniam et al., 2014). A more recently developed model,
limited nesting (LN), involving dams being supplied with inade-
quate nesting materials, has been proven to be robust in mimicking
the behavioural patterns of maternal neglect in humans (Ivy et al.,
2008; Prusator and Greenwood-Van Meerveld, 2015).
The modern diet is rich in foods high in both fats and sugars,
and people commonly crave these palatable ‘comfort foods’ when
under stress. With diet known to be an important factor for its
effects on the brain and behaviour, the combined effects of ELS
along with a poor quality diet high in fat and sugar needs to be
considered as a possible outcome for those who undergo ELS. The
detrimental effects of a high fat diet on metabolism are numerous

http://dx.doi.org/10.1016/j.psyneuen.2016.03.007
0306-4530/© 2016 Elsevier Ltd. All rights reserved.
 J. Maniam et al. / Psychoneuroendocrinology 68 (2016) 202–209
and well-known (Nakamura and Terauchi, 2013), but its effects,
and the mechanisms underpinning these effects, on the brain and
mental health are less clearly understood.
A high fat diet has been linked to adverse effects on cognition,
learning and memory, although there are a number of proposed
potential underlying mechanisms (Cordner and Tamashiro, 2015;
Freeman et al., 2014). Diets high in fats and sugars have been linked
to stress and chronic stressful conditions have previously been
shown to increase the intake of palatable foods (Teegarden and
Bale, 2008). This bidirectional association has been demonstrated
in response to both acute and chronic stress in rodents (Maniam and
Morris, 2012). However, limited work has examined the effects of
ELS on post-weaning high fat diet intake. While conflicting data has
been obtained regarding overall energy intake following ELS, it has
been demonstrated that adult female rats had an increased prefer-
ence for palatable “comfort foods” high in sucrose and fats when
exposed to ELS by limiting access to nesting materials (Machado
et al., 2013). Additional negative effects of an unhealthy diet on the
brain and mental health that have also been established include
modifications to the inflammatory response, leading to a worse
outcome following stroke (Maysami et al., 2015). Of particular
interest to our study, it has also been shown to induce neurochem-
ical changes in the hippocampus (Krishna et al., 2015). A high fat
diet has been demonstrated to ameliorate anxiety-like behaviour
in rats that had previously been exposed to ELS (induced by mater-
nal separation) (Maniam and Morris, 2010a, b), but its effects on
hippocampal gene expression are unclear.
The aims of this paper were to firstly investigate whether HFHS
consumption may moderate anxiety-like behaviour and cognitive
impairment induced by ELS in the form of LN, and secondly to
explore whether the diet has any additional effects on hippocam-
pal markers involved in pathways known to be affected by ELS.
Therefore, we hypothesized that ELS (via limited nesting) would
induce anxiety-like behaviour and cognitive deficits in offspring,
and given that foods rich in sugar and fat are reported to reduce
stress levels in humans (Adam and Epel, 2007) and rodents (la
Fleur et al., 2005), that voluntary intake of HFHS would ame-
liorate ELS induced anxiety-like behaviour and cognitive deficit.
Further, we hypothesized that chronic consumption of HFHS would
worsen hippocampal markers that regulate pathways (neuroplas-
ticity, neurogenesis, stress response, inflammatory, mitochondrial
biogenesis) that are known to regulate a range of behaviours con-
sistently associated with ELS.
2. Materials and methods
2.1. Animals
All animal procedures were approved by the Animal Care and
Ethics Committee of UNSW Australia. Male and female Sprague-
Dawley rats (Animal Resource Centre, Perth, WA, Australia) were
maintained in a temperature controlled (23 ◦ C) colony room on a
12 h light/dark cycle (lights on at 0700) with ad libitum access to
standard laboratory chow and water. Mating was carried out in
house to generate litters. After mating females were housed singly
and littered normally.
Litters comprising 9–15 pups were included and standardized
to 12 pups per litter on postnatal day (PND) 1 using pups from
dams littering on the same day to minimize alterations in mater-
nal behaviour and pup nutrition. Litters were housed with the dam
in polypropylene cages (20 cm × 32 cm × 19 cm) on wood shavings
with a metal lid. From post-natal days 2–9 cages of the ELS group
had a metal grid floor inserted that allowed waste to be excluded,
and given only half a sheet of paper towel as nesting material.
Both groups were left undisturbed between PND 2–9, then ELS rats
203
were returned to normal bedding at PND 10. The control group had
normal bedding throughout.
2.2. Post-weaning diet
At PND 21, the pups were weaned and housed 3–4 rats per cage.
Male offspring were assigned to either standard laboratory chow
(11 kJ/g, energy 12% fat, 21% protein, 65% carbohydrate, Gordon’s
Specialty Stockfeeds, NSW, Australia) or standard laboratory chow
and palatable HFHS, SF03-020 (20 kJ/g, energy 43% fat, 17% pro-
tein, 40% sucrose supplied by Specialty Feeds, Glen Forrest, WA,
Australia). Chow, water, and HFHS were available ad libitum. This
generated four groups: Con-Chow; Con-HFHS; LN-Chow; and LN-
HFHS, comprising, n = 10–15/group. Rats were weighed at weaning
and at regular intervals throughout the study. Food intake over 24 h
was recorded by weighing food and bottles before presentation to
the rats and again after 24 h. Energy intake per cage was measured
and average intake was calculated assuming equal intake for each
rat.
2.3. Elevated plus maze (EPM)
The rats undertook the EPM as previously described (Maniam
and Morris, 2010a, b) during the light phase at 10 weeks of age.
Opaque perspex was used to construct a plus-shaped maze con-
sisting of 2 opposite open arms (50 cm × 10 cm) and 2 closed arms
(50 cm × 10 cm with 40 cm walls). Once the rat was placed in the
arena the experimenter left the room. Behaviour was analysed
using footage obtained from directly above the arena using a video-
camera. Macropod’s OD log software was used to score the data,
both by the experimenter and a second observer unaware of the
allocation of the groups. The time and entries of the rats in each
of the open and closed arms was recorded over a 10-min period.
Data are presented as percentage time in open arms, percentage
entries into open arms and number of entries into open arms. We
also recorded number of rearing events.
2.4. Novel objects and place recognition task
This task was conducted as previously described (Beilharz et al.,
2014). Rats were given 10 min on 2 consecutive days to acclimatize
themselves to the empty arena (a black acrylic open field arena
(60 cm × 60 cm × 60 cm)), and behavioural assessment started on
the following day. The rats from each diet group were split into 2
equal groups, and on the first day one half undertook the object task
whilst the remainder were assessed on the place recognition task.
The groups then switched tasks on the following day of testing.
At the commencement of every test, 2 identical objects were
placed into two of the middle four sections. The novel objects were
commercial products that varied in size and texture (e.g., mugs,
tins). The familiarisation phase of the test involved positioning the
rat in the middle of the arena and giving it 5 min to explore. At
the conclusion of this phase, the rat was removed for 5 min (the
retention phase). For the test phase of the object task, the objects
were then set up in the same formation as during the familiarisa-
tion phase, except one of the objects was replaced with a novel one.
In the test phase of the place recognition task, the same objects that
had been used for the familiarisation phase were put back into the
arena, except one of them was placed in one of the corner quad-
rants instead of its original position. These test phases were 3 min
in duration. Each rat was only allowed exposure to the same object
for one trial, and testing took place at the same time of day for
each repeated trial. The order and locations of the objects were
counterbalanced across trials and between the rats.
Behaviour was analysed using footage obtained from directly
above the arena using a video-camera. Exploration was defined
204 J. Maniam et al. / Psychoneuroendocrinology 68 (2016) 202–209
as the rat extending its neck to position its head within 2 cm of
the object followed by moving its vibrissae around the object.
Macropod’s OD log software was used to score the data, both by
the experimenter and a second observer unaware of the alloca-
tion of the groups. The Exploration Ratio was used to represent
the data—this was calculated by dividing the time spent explor-
ing the novel object by the total time spent exploring both objects
(tnovel /(tnovel + told )).
2.5. Reverse-transcription PCR (RT-PCR)
RNA was extracted using Tri-reagent (Sigma-Aldrich, Sydney,
NSW, Australia) and treated with DNase I (Invitrogen, Mel-
bourne, VIC, Australia) to remove any contaminating genomic
DNA and stored at −80 ◦ C. RNA concentration was determined
using a Biospec-nano spectrophotometer (Shimadzu, Sydney, NSW,
Australia). RNA was reverse transcribed to cDNA using Omnis-
cript Reverse Transcription kit (Qiagen, Melbourne, VIC, Australia)
and stored at −20 ◦ C. We performed RT-PCR using TaqMan®
Array Micro Fluidic Cards (Life Technologies) which were pre-
customised with Taqman probes (Applied Biosystems, Melbourne,
VIC, Australia) for nuclear factor of kappa light polypeptide gene
enhancer in B-cells 1(Nfkb1-Rn01399583 m1), toll-like receptor 3
(Tlr3-Rn01488472 g1), toll-like receptor 4 (Tlr4-Rn00569848 m1),
tumor necrosis factor (Tnf-Rn99999017 m1), S100 calcium
binding protein B (S100b-Rn04219408 m1), nuclear respira-
tory factor 1(Nrf1-Rn01455958 m1), peroxisome proliferator-
activated receptor gamma, coactivator 1 alpha (Ppargc1a-
Rn00580241 m1), sirtuin 3 (Sirt3-Rn01501410 m1), superoxide
dismutase 1, soluble (Sod1-Rn00566938 m1), v-akt murine
thymoma viral oncogene homolog 1 (Akt1-Rn00583646 m1),
v-akt murine thymoma viral oncogene homolog 2 (Akt2-
Rn00567290 m1), v-akt murine thymoma viral oncogene homolog
3 (protein kinase B, gamma) (Akt3-Rn00442194 m1), brain-
derived neurotrophic factor (Bdnf-Rn01484924 m1), neuritin
1(Nrn1-Rn00584304 m1), cAMP responsive element binding
protein 1(Creb1-Rn00578829 g1), reelin (Reln-Rn00589609 m1),
caspase 3 (Casp3-Rn00563902 m1), glycogen synthase kinase
3 alpha (Gsk3a-Rn00569232 m1), glycogen synthase kinase 3
beta (Gsk3b-Rn00583429 m1), glycogen synthase kinase 3 alpha
(Gsk3a-Rn00569232 m1), gamma-aminobutyric acid (GABA-A)
receptor, subunit alpha 2 (Gabra2-Rn01413643 m1), homer
homolog 1 (Drosophila) (Homer1-Rn00581785 m1), nuclear
receptor subfamily 3, group C, member 1/glucocorticoid recep-
tor (Nr3c1-Rn00561369 m1), 5-hydroxytryptamine (serotonin)
receptor 1A (Htr1a-Rn00561409 s1), 5-hydroxytryptamine (sero-
tonin) receptor 2A (Htr2a-Rn00568473 m1), mitogen activated
protein kinase 1 (Mapk1-Rn00587719 m1), mitogen-activated
protein kinase 8 (Mapk8-Rn01453358 m1), mitogen activated
protein kinase 3 (Mapk3-Rn00820922 g1), mitogen activated pro-
tein kinase 14 (Mapk14-Rn00578842 m1), signal transducer and
activator of transcription 6 (Stat6-Rn01505881 m1), signal trans-
ducer and activator of transcription 3 (Stat3-Rn00562562 m1),
signal transducer and activator of transcription 5A (Stat5a-
Rn00567011 m1).
We used 1.5 ␮g of cDNA. 100 ml of cDNA/Mastermix (Advanced
Fast Mastermix, Life Technologies) mixture was pipetted into the
micro fluid cards, after centrifugation the cards were sealed and
RT-PCR was performed. The geometric mean of two housekeepers,
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activa-
tion protein (Ywhaz) and hypoxanthine phosphoribosyltransferase
1 (Hprt1) was used as reference. Analysis was performed using the
CT method and data expressed relative to a calibrator sample
of all groups.
2.6. Western blot
Whole tissue lysates from powdered hippocampus were pre-
pared using a Precellys 24 homogenizer (Sapphire Bioscience,
Sydney, NSW, Australia) in radioimmunoprecipitation assay buffer
(RIPA) buffer containing 65 mM Tris (pH 7.4), 150 mM NaCl, 5 mM
EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate (SDS), 10% glycerol, 1 ␮g/ml aprotinin, 1 ␮g/ml
leupeptin, 10 mM sodium fluoride, 1 mM sodium orthovanadate
(Na3 VO4 ) and 1 mM phenylmethanesulfonylfluoride or phenyl-
methylsulfonyl fluoride (PMSF) according to methods described
elsewhere (Hoy et al., 2007). The hippocampus homogenate was
then incubated for 2 h at 4 ◦ C and then centrifuged at 10 000g
for 15 min to remove insoluble material. Protein concentration
was determined by Bradford protein assay (Bio-Rad) using bovine
serum albumin (BSA) as standard. Proteins were resolved by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) electrophoresis on a 4–15% polyacrylamide gel (BioRad) and
transferred to a polyvinylidene difluoride (PVDF) membrane. Non-
specific binding sites were blocked in Tris-buffered saline with 4%
BSA and 0.1% Tween-20 for 1 h at room temperature. Membranes
were incubated on a roller at 4 ◦ C overnight with glucocorticoid
receptor antibody (Cell Signaling; 1:1000) followed by anti-rabbit
IgG horse radish peroxidase (HRP)-linked antibody (Cell Signal-
ing; 1:10 000); and anti-alpha-tubulin (Millipore; 1:1000) followed
by anti-mouse IgG HRP-linked antibody (Cell Signaling; 1:10 000).
After washing in Tris-buffered saline with 0.1% Tween-20, mem-
branes were visualised with enhanced chemiluminescence (ECL,
Millipore) with images captured using Image Quant LAS-4000
(GE Healthcare, Sydney, NSW, Australia). Protein expression lev-
els were quantified by densitometry using ImageJ 1.47v software
(NIH, Maryland, USA).
3. Results
3.1. The effects of LN exposure and HFHS diet on body weight and
food intake
Body weight was measured at termination (Con-Chow:
357.8 ± 7.83 g LN-Chow: 330.11 ± 8.62 g, Con-HFHS: 478.1 ± 17.3 g,
LN-HFHS: 425.0 ± 14.0 g). In both chow and HFHS fed groups, the
body weight of LN rats were not significantly different from con-
trol rats as evidenced by no significant interaction between LN
and HFHS (F(1,53) = 1.104, p = 0.298). However, as expected a main
effect of HFHS diet was observed (F(1,53) = 79.26, p < 0.0001). HFHS
fed rats were heavier than their chow counterparts.
In line with THE body weight data, LN exposure had no effect on
energy intake in the chow [Con-Chow (2536 ± 97 kJ/rat), LN-Chow
(2448 ± 68 kJ/rat)], or HFHS fed rats [Con-HFHS (3290 ± 107 kJ/rat),
LN-HFHS (3013 ± 88 kJ/rat)] as evidenced by no significant interac-
tion between LN and HFHS diet [LN × HFHS, F(1, 11) = 1.10, p > 0.05].
However, as expected, HFHS increased energy intake across groups
as evidenced by a main effect of HFHS (F(1, 11) = 53.49, p < 0.0001).
3.2. The effects of LN exposure and HFHS diet on anxiety-like
behaviour
Anxiety-like behaviour was assessed at 10 weeks of age using
EPM. Increased percentage of open arm entries or time spent in
open arm is indicative of reduced anxiety-like behaviour (Korte
and De Boer, 2003; Rodgers and Dalvi, 1997). Here, in those con-
suming chow, LN rats made approximately 20% less open arm
entries compared to control rats (post-hoc p < 0.05 LN versus con-
trol, Fig. 1A). A significant interaction was observed between LN
and diet; LN × Diet, F(1,29) = 4.21, p = 0.04. Interestingly, provision
 J. Maniam et al. / Psychoneuroendocrinology 68 (2016) 202–209
Fig. 1. Anxiety-like behaviour assessed by elevated plus maze at 10 weeks of age. (A) Percentage open arm entries, (B) percentage time in open, (C) number of open arm
entries, (D) number of rearing events. Results are expressed as mean ± S.E.M; n = 7–10, two-way ANOVA followed by LSD. Post-hoc analysis was performed when a significant
interaction between diet and LN was present.
†<0.05 versus control rats consuming the same diet (LN effect).
**p < 0.01 versus rats consuming chow (diet effect)
of a palatable HFHS diet to LN rats almost doubled the percentage
of open arm entries versus chow fed LN rats (p < 0.05, Fig. 1A).
While the percentage of time spent in open arms (Fig. 1B)
appears to show a similar trend as the percentage of open
arm entries, we observed no statistically significant interaction
between LN exposure and diet consumed (two-way ANOVA anal-
ysis, (F(1,29) = 1.34, p > 0.05), see Fig. 1B). In addition, there were
no significant differences in two-way ANOVA analysis of general
activity, as evidenced by the number of entries into open arms
(p > 0.05, see Fig. 1C) and rearing (p > 0.05, see Fig. 1D). While LN-
HFHS appears to have reduced rearing activity, two-way ANOVA
analysis showed no significant interaction between LN exposure
and diet (F (1, 29) = 0.1578, P = 0.67).
3.3. The effects of LN exposure and HFHS diet on spatial and
object recognition memory
Tests of spatial and object recognition memory were conducted
using novel object and place recognition tasks. We observed no
statistical differences across groups in terms of times of exploration,
on object (F(1,35) = 3.16, p > 0.05, Fig. 2A) and place (F(1,35) = 0.09,
p > 0.05, Fig. 2C) testing. Neither LN exposure nor HFHS diet had
any effect on performance of the object recognition task (LN × Diet
F(1,35) = 1.001, p = 0.32, Fig. 2), however, there was a main effect
of LN exposure (F(1,35) = 4.23, p = 0.047). The average exploration
ratio in LN rats consuming chow appears to be slightly higher than
control rats consuming the same diet.
In terms of the place recognition task, while there appears to
be a trend for HFHS effects in the control condition, the inter-
action between LN exposure and diet did not reach significance
(F(1,35) = 3.15, p = 0.08).
205
3.4. The effects of LN exposure and HFHS diet on hippocampal GR
protein levels
The hippocampal GR plays a key role in HPA axis activity reg-
ulation, and here we examined GR protein expression by western
blot. A significant interaction was observed between LN and diet;
LN × Diet, F(1,43) = 5.14, p = 0.03. LN exposure had no effect on hip-
pocampal GR protein expression in the chow fed groups. However,
in those consuming HFHS, LN exposure increased hippocampal GR
mRNA expression by about 45 percent relative to control rats (post
hoc p < 0.05, Fig. 3). Further, LN-HFHS rats had an approximate
50% increase in hippocampal GR versus their chow counterparts
(LN-HFHS versus LN-chow p < 0.01, Fig. 3).
3.5. The effects of LN exposure and HFHS diet on hippocampal
genes
A targeted array of hippocampal genes associated with neuro-
plasticity, neurogenesis, stress response, serotonin, inflammatory,
mitochondrial biogenesis including MAP kinases and STATs were
examined. All of these processes are critical in brain function and
behavioural regulation of anxiety, depression, stress response and
cognition. Several genes showed a main effect of LN exposure. An
interaction was observed between LN and diet for GSK isoforms
(LN × HSHS interaction, GSKB␣ F(1,28) = 5.14, p = 0.031 and GSK3␤
F(1,27) = 4.74, p = 0.039). In those consuming chow, LN exposure
significantly reduced hippocampal GSK␤ mRNA expression by
about 30% (p < 0.01, Table 1), but had no effect on GSKB␣ mRNA
expression. In those rats consuming HFHS, LN exposure markedly
reduced the expression of both GSKB␣ and GSK3␤ compared to
control rats (p < 0.01, Table 1). Akt3 was affected by both LN expo-
sure and HFHS (LN × HFHS interaction F(1,28) = 18.83, p = 0.0002).
LN exposure alone significantly reduced the expression of Akt3
mRNA by about 34% in the chow fed rats (p < 0.01, Table 1), and had
206 J. Maniam et al. / Psychoneuroendocrinology 68 (2016) 202–209
Fig. 2. Object and place recognition memory was assessed with novel object and place tasks at 11 weeks. Object task exploration time (A) and ratio (B) and place task
exploration time (C) and ratio (D) are expressed as mean ± SEM, with n = 9–10 rats per group. Data were analysed by two-way ANOVA followed by LSD.
Fig. 3. Glucocorticoid receptor protein measured at 13 weeks of age. Data are
expressed as mean ± S.E.M; two-way ANOVA followed by LSD, n = 11–12/group.
Post-hoc analysis performed when a significant interaction between diet and LN
present.
† p < 0.05 versus control rats consuming the same diet (LN effect).
** p < 0.01 versus rats consuming chow (diet effect)
no effect of LN in the HFHS fed rats. Interestingly, HFHS diet alone
produced similar effects as LN exposure on Akt3 mRNA expression
in control rats, where consumption of HFHS reduced the expres-
sion of Akt3 mRNA by about 20% (p < 0.01, Table 1). There was no
additive effect when LN and HFHS diet were combined.
4. Discussion
Our finding that post-weaning HFHS ameliorated the increased
anxiety-like behaviour induced by ELS in the form of LN is the first
demonstration of a post-weaning diet interaction with ELS in this
paradigm. Several of the findings of this study are consistent with
our previous observations using maternal separation as a model of
ELS (Maniam and Morris, 2010a, b). Here we observed markedly
upregulated GR protein levels in response to the combination of
LN and HFHS exposure, which may be associated with the rever-
sal of anxiety-like behaviour by the diet. Surprisingly, contrary to
the findings of others, in our hands, object and spatial recognition
memory were not affected by LN exposure or chronic consumption
of HFHS diet. Interestingly, of the array of hippocampal genes that
are involved in pathways that regulate anxiety, depression and cog-
nition measured, only GSKB␣, GSKB␤ and Akt3 were significantly
affected by LN exposure.
Given the percentages of entries and time spent in each arm con-
stitute an index of anxiety (Korte and De Boer, 2003; Rodgers and
Dalvi, 1997), here in our hands, we concluded that LN-Chow rats
exhibited increased anxiety-like behaviour when compared to Con-
Chow rats. Others using elevated plus maze and different batteries
of anxiety-like behaviour tests such as open-field, light-dark box
and novelty-induced hypophagia have consistently demonstrated
that LN exposure increases anxiety-like behaviour in pups. Thus
an LN induced anxiety-like behaviour phenotype was observed in
mice (Malter Cohen et al., 2013; Wang et al., 2012) and rats (Dalle
Molle et al., 2012; Machado et al., 2013). This is suggestive of LN
being a robust model of ELS across species. It is important to note
that, the main effect of diet was significant in both measures of time
spent in open arm and percentage open arm entries which suggests
that diet has a robust effect on anxiety-like behaviour across control
and LN groups.
Changes in maternal behaviour have been consistently shown
to impact offsprings’ neurodevelopment including hypothalamic-
pituitary-adrenal axis activity which governs stress response
activity (Caldji et al., 2000; Ziabreva et al., 2003, 2000). Work from
our lab and others has previously demonstrated that ELS in the form
of maternal separation significantly reduces the quality of maternal
care provided by the dams to their pups and this was associated
with behavioural outcomes such as increased stress responsive-
ness, anxiety- and depression-like behaviours (Macri et al., 2008;
 J. Maniam et al. / Psychoneuroendocrinology 68 (2016) 202–209 207

Table 1
The effects of LN exposure and post-weaning HFHS diet on the expression of hippocampal genes. Results are expressed as mean ± S.E.M; data were analysed by two-way
ANOVA followed by LSD, n = 7–9 rats/group. mRNA expression was measured using the microfluidic card platform (Life Technologies).

Significance

Con-Chow LN-Chow Con-HFHS LN-HFHS Interaction Diet LN

Inflammatory genes
nfkb1 0.94 ± 0.07 0.82 ± 0.09 1.00 ± 0.12 0.74 ± 0.05 ns ns 0.035
Tlr3 1.15 ± 0.30 1.09 ± 0.27 1.28 ± 0.37 0.92 ± 0.12 ns ns ns
Tlr4 1.09 ± 0.20 0.98 ± 0.08 0.97 ± 0.10 0.97 ± 0.17 ns ns ns
TNF 1.49 ± 0.44 2.22 ± 0.37 1.86 ± 0.30 1.57 ± 0.30 ns ns ns
s100b 1.02 ± 0.08 0.76 ± 0.07 1.06 ± 0.10 0.90 ± 0.04 ns ns 0.009

Mitochondrial biogenesis and energy metabolism

Nrf1 1.01 ± 0.05 0.76 ± 0.08 1.01 ± 0.10 0.62 ± 0.05 ns ns p < 0.0001
ppargc1a 1.02 ± 0.07 1.13 ± 0.17 1.15 ± 0.12 0.80 ± 0.06 ns ns ns
SIRT3 1.02 ± 0.07 0.99 ± 0.09 1.18 ± 0.12 0.99 ± 0.06 ns ns ns
SOD1 1.03 ± 0.09 0.77 ± 0.05 1.16 ± 0.07 0.72 ± 0.05 ns ns p < 0.0001
Akt1 1.00 ± 0.03 1.07 ± 0.12 0.98 ± 0.04 1.02 ± 0.04 ns ns ns
Akt2 1.01 ± 0.06 0.89 ± 0.06 1.17 ± 0.13 0.90 ± 0.03 ns ns p = 0.012
Akt3 1.01 ± 0.06 0.66 ± 0.04† 0.80 ± 0.03* 0.82 ± 0.04 0.0002 ns 0.001

Neurogenesis and plasticity

BDNF 1.02 ± 0.07 1.01 ± 0.06 1.33 ± 0.14 0.98 ± 0.07 ns ns ns
nrn1 0.90 ± 0.05 0.86 ± 0.09 1.01 ± 0.08 0.69 ± 0.05 ns ns 0.019
creb1 1.02 ± 0.08 1.06 ± 0.09 1.17 ± 0.11 0.96 ± 0.04 ns ns ns
reln 1.04 ± 0.11 0.80 ± 0.04 1.14 ± 0.12 0.65 ± 0.05 ns ns 0.0002
Casp3 1.03 ± 0.09 0.79 ± 0.08 0.83 ± 0.09 0.71 ± 0.07 ns ns 0.044
GSK3␤ 0.95 ± 0.07 0.87 ± 0.07† 1.19 ± 0.08 0.78 ± 0.09† 0.039 ns 0.004
GSK3␣ 1.02 ± 0.06 0.92 ± 0.06 1.22 ± 0.07 0.86 ± 0.04† 0.031 ns 0.001
Gabra2 1.02 ± 0.06 1.10 ± 0.09 1.16 ± 0.13 1.12 ± 0.06 ns ns ns

Stress response
GR 1.03 ± 0.09 0.94 ± 0.06 1.02 ± 0.06 0.80 ± 0.07 ns ns p = 0.043
Homer1 1.01 ± 0.06 0.97 ± 0.07 1.01 ± 0.06 0.79 ± 0.04 ns ns 0.028

Serotonin
Htr1a 1.01 ± 0.05 1.05 ± 0.06 1.14 ± 0.09 1.05 ± 0.06 ns ns ns
Htr2b 1.05 ± 0.11 0.90 ± 0.08 0.98 ± 0.07 0.70 ± 0.08 ns ns 0.021

MAP kinases
Mapk1 1.02 ± 0.08 1.04 ± 0.17 1.36 ± 0.30 0.96 ± 0.13 ns ns ns
Mapk3 1.02 ± 0.07 0.85 ± 0.10 1.01 ± 0.06 0.83 ± 0.05 ns ns 0.022
Mapk14 1.02 ± 0.07 0.73 ± 0.05 1.05 ± 0.04 0.69 ± 0.03 ns ns p < 0.0001
Mapk8 1.01 ± 0.06 0.79 ± 0.06 0.95 ± 0.09 0.71 ± 0.05 ns ns 0.001

Signal transducer and activator of transcription

STAT6 0.96 ± 0.03 0.79 ± 0.06 1.01 ± 0.06 0.80 ± 0.06 ns ns 0.002
STAT3 1.01 ± 0.04 0.969 ± 0.08 1.03 ± 0.07 0.79 ± 0.04 ns ns 0.032
STAT5a 1.03 ± 0.10 0.965 ± 0.08 1.19 ± 0.10 0.95 ± 0.10 ns ns ns
†
p < 0.01 versus control rats consuming the same diet (LN effect).
*
p < 0.01 versus rats consuming chow (diet effect).

Maniam and Morris, 2010a, b; Meaney et al., 1991, 1988). There-
fore, one possible reason for the changes in anxiety-like behaviour
induced by the LN model in this study could be alterations in mater-
nal behaviour. The lab that developed the LN material as a model
of ELS has systematically demonstrated that dams exposed to LN
regularly leave the pups in the nesting area and so the duration of
each nurturing behaviour (licking, grooming and feeding) is short
and variable (Ivy et al., 2008). This altered maternal behaviour
has been proposed to be linked with altered stress response as
evidenced by changes in the hypothalamo-pituitary-adrenal axis
activity (Avishai-Eliner et al., 2001; Brunson et al., 2005).
In terms of cognitive measurements, we observed no effects
of ELS in the form of LN on spatial and object recognition tasks.
However, other studies using similar ELS paradigms showed LN
exposure produced spatial memory deficits (Ivy et al., 2010;
Naninck et al., 2015). The order of behavioural tests conducted
may have had an influence on the cognitive measures in our study.
Even though the cognitive task was conducted one week after the
anxiety-like behaviour test, we cannot exclude the possibility that
this test may have impacted the cognitive measures. Future work
may need to consider increasing the gap between anxiety and cog-
nitive testing and counterbalancing the order of the anxiety and
cognitive tests. Although we observed differences in anxiety-like
behaviour across the LN and diet groups, the overall exploration
time was not different in the novel object tasks, so we do not think
this influenced the outcomes of the cognitive measures.
While we observed an increase in anxiety-like behaviour in LN
rats, GR protein was not affected. At the level of mRNA expres-
sion, we observed a main effect of LN exposure but no significant
interaction with HFHS diet. We previously showed that ELS induced
by maternal separation reduced the expression of hippocampal GR
mRNA (Maniam and Morris, 2010a, b) and a similar effect was
observed in female rats exposed to LN versus controls (Maniam
& Morris unpublished data). In the current study, we provide the
first evidence that chronic consumption of HFHS altered hippocam-
pal GR protein levels in those exposed to LN relative to their chow
counterparts. The marked increase in GR protein levels in LN rats
consuming HFHS diet versus LN rats consuming chow appears to
be in line with the reversal of anxiety-like behaviour. In support,
we previously showed that cafeteria style diet normalised the ELS
induced reduction in hippocampal GR mRNA expression (Maniam
and Morris, 2010a, b). There are several other studies that simu-
lated ELS with LN exposure (Cui et al., 2006; Naninck et al., 2015),
however, no data on hippocampal GR were reported. In summary,
across two different ELS paradigms, we showed that HFHS diet can
interact with ELS on hippocampal GR.
208 J. Maniam et al. / Psychoneuroendocrinology 68 (2016) 202–209
We targeted several genes that mediate important pathways
known to be affected by early life adversity such as plasticity &
neurogenesis, stress response, serotonin, inflammatory and down-
stream mediators such as MAP kinases and STATs. However, only 3
genes were significantly affected by LN and diet interaction. Firstly,
we observed a marked decrease in hippocampal GSK3a and GSK3b
expression of LN rats in those consuming HFHS diet relative to
control rats. Serine/threonine protein kinase synthase is known
to mediate many brain functions including mood and neuroge-
nesis. Antipsychotics and antidepressants such as fluoxetine are
shown to directly or indirectly block the action of GSK3␣/␤, and
selective inhibition of GSK3 produces antidepressant behaviour in
the forced swim test (Ackermann et al., 2010). Transgenic mice
expressing elevated GSK3 activity display memory deficits, reduced
brain size and alterations in mood behaviour and social interac-
tions (Hernandez et al., 2002; Polter et al., 2010; Prickaerts et al.,
2006; Spittaels et al., 2002). Several other studies have outlined
the role of GSK3 in mediating the effects of antidepressant drugs
(Duman and Aghajanian, 2014; Li et al., 2014). Antidepressant drugs
have also been shown to increase neurogenesis through recruit-
ment of new neurons (Surget et al., 2011). Here, a reduction of
expression in hippocampal GSK3␣/␤ mRNA was observed in chow
and HFHS fed rats exposed to ELS, but their memory task perfor-
mance was not affected by LN exposure or by the combination with
HFHS diet. However, the reduced expression of GSK3␣/␤ mRNA
by the HFHS diet in LN rats is supportive of the amelioration of
anxiety-like behaviour in LN-HFHS rats. It is important to note that
altering mRNA expression may not reflect the pathological condi-
tion in which GSK3 activity is dysregulated in the application of
mood stabilisers and antidepressants (Polter et al., 2010, 2012).
However, this is the first evidence of GSK3 mRNA alterations fol-
lowing ELS, particularly when combined with post-weaning HFHS
diet, and provides impetus for future work to further examine the
role of GSK3 activity under conditions of early life insults and later
diet manipulation.
Another gene that was affected by LN exposure and chronic con-
sumption of HFHS is Akt3. Hippocampal expression of Akt3 mRNA
was reduced by about 40% by LN exposure in chow fed rats only
and the HFHS diet only reduced its expression in control rats; hence
providing evidence that an unhealthy diet produces similar effects
to LN exposure on the expression of Akt3 mRNA. There appears
to be limited work examining the impact of HFHS on Akt related
pathways in the brain. Akt plays an important role in brain devel-
opment, synaptic plasticity and neurodegeneration (Easton et al.,
2005; Tschopp et al., 2005; Wang et al., 2003). Among all the iso-
forms, Akt3 is predominantly expressed in the brain and represents
50% of the total Akt in the cerebral cortex and hippocampus (Easton
et al., 2005). Moreover, Akt3 is involved in postnatal brain devel-
opment (Tschopp et al., 2005). Of concern is our finding that the
chronic consumption of HFHS led to a similar degree of reduc-
tion in Akt3 mRNA as LN exposure. If a similar process occurs in
the human, the consumption of HFHS may increase the risk for
psychopathologies.
In summary, LN exposure produced long-lasting effects in adult
behaviour as evidenced by increased anxiety-like behaviour in
those consuming chow and LN was associated with a marked reduc-
tion in genes associated with plasticity and mood regulation which
are also a main target of antidepressant drugs. A diet rich in fat and
sugar ameliorated the increased anxiety-like behaviour induced by
LN exposure suggesting it may have “therapeutic value” (Machado
et al., 2013) but this was at the expense of adverse effects on the
expression of hippocampal genes related to mood regulation and
neurogenesis; GSK␣ and GSK3␤. Moreover, we observed the HFHS
diet produced equivalent adverse effects on hippocampal Akt3 gene
that may have implications for brain development. Hence, we pro-
vide evidence for the first time that LN exposure and HFHS diet
independently affect expression of hippocampal genes that are vital
for mood and brain development. There is evidence in human stud-
ies that people prefer diets that are rich in sugar and fat as a stress
coping strategy (Adam and Epel, 2007). However, given that such
diet produces deleterious effects at a molecular level, it is impor-
tant for the public to be educated on the adverse effects of diets rich
in fat and sugar. Moreover, we simulated childhood trauma via LN
exposure which is thought to mimic conditions of human mater-
nal neglect (Ivy et al., 2008) and so if similar processes are at play
in the human, other interventions to offset the adverse effects on
brain and behaviour are crucial.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Role of the funding source
Funding for this study was provided by Diabetes Australia
Research Trust (DART). The DART had no further role in the study
design, in the collection, analysis and interpretation of data; in
writing of the report; and in the decision to submit the paper for
publication.
Author contributions
Designed the experiments: JM and MJM. Performed the exper-
iments: JM, CPA, and VL. Analysed the data: JM, CPA and VL. All
authors contributed to the writing of the manuscript.
Acknowledgment
This work was supported by project funding from the Diabetes
Australia Research Trust awarded to MJM.
References
Ackermann, T.F., Kempe, D.S., Lang, F., Lang, U.E., 2010. Hyperactivity and
enhanced curiosity of mice expressing PKB/SGK-resistant glycogen synthase
kinase-3 (GSK-3). Cell. Physiol. Biochem. 25, 775–786.
Adam, T.C., Epel, E.S., 2007. Stress, eating, and the reward system. Physiol. Behav.
91, 449–458.
Aust, S., Stasch, J., Jentschke, S., Alkan Hartwig, E., Koelsch, S., Heuser, I., Bajbouj,
M., 2014. Differential effects of early life stress on hippocampus and amygdala
volume as a function of emotional abilities. Hippocampus 24, 1094–1101.
Avishai-Eliner, S., Gilles, E.E., Eghbal-Ahmadi, M., Bar-El, Y., Baram, T.Z., 2001.
Altered regulation of gene and protein expression of
hypothalamic-pituitary-adrenal axis components in an immature rat model of
chronic stress. J. Neuroendocrinol. 13, 799–807.
Beilharz, J.E., Maniam, J., Morris, M.J., 2014. Short exposure to a diet rich in both fat
and sugar or sugar alone impairs place, but not object recognition memory in
rats. Brain Behav. Immun. 37, 134–141.
Bremne, J.D., Vermetten, E., 2001. Stress and development: behavioral and
biological consequences. Dev. Psychopathol. 13, 473–489.
Brunson, K.L., Kramar, E., Lin, B., Chen, Y., Colgin, L.L., Yanagihara, T.K., Lynch, G.,
Baram, T.Z., 2005. Mechanisms of late-onset cognitive decline after early-life
stress. J. Neurosci. 25, 9328–9338.
Caldji, C., Diorio, J., Meaney, M.J., 2000. Variations in maternal care in infancy
regulate the development of stress reactivity. Biol. Psychiatry 48, 1164–1174.
Cordner, Z.A., Tamashiro, K.L., 2015. Effects of high-fat diet exposure on learning &
memory. Physiol. Behav. 152, 363–371.
Cui, M., Yang, Y., Yang, J., Zhang, J., Han, H., Ma, W., Li, H., Mao, R., Xu, L., Hao, W.,
Cao, J., 2006. Enriched environment experience overcomes the memory
deficits and depressive-like behavior induced by early life stress. Neurosci.
Lett. 404, 208–212.
Dalle Molle, R., Portella, A.K., Goldani, M.Z., Kapczinski, F.P., Leistner-Segal, S.,
Salum, G.A., Manfro, G.G., Silveira, P.P., 2012. Associations between parenting
behavior and anxiety in a rodent model and a clinical sample: relationship to
peripheral BDNF levels. Transl. Psychiatry 2, e195.
Duman, R.S., Aghajanian, G.K., 2014. Neurobiology of rapid acting antidepressants:
role of BDNF and GSK-3beta. Neuropsychopharmacology 39, 233.
 J. Maniam et al. / Psychoneuroendocrinology 68 (2016) 202–209
Easton, R.M., Cho, H., Roovers, K., Shineman, D.W., Mizrahi, M., Forman, M.S., Lee,
V.M., Szabolcs, M., de Jong, R., Oltersdorf, T., Ludwig, T., Efstratiadis, A.,
Birnbaum, M.J., 2005. Role for Akt3/protein kinase Bgamma in attainment of
normal brain size. Mol. Cell. Biol. 25, 1869–1878.
Freeman, L.R., Haley-Zitlin, V., Rosenberger, D.S., Granholm, A.C., 2014. Damaging
effects of a high-fat diet to the brain and cognition: a review of proposed
mechanisms. Nutr. Neurosci. 17, 241–251.
Heim, C., Nemeroff, C.B., 1999. The impact of early adverse experiences on brain
systems involved in the pathophysiology of anxiety and affective disorders.
Biol. Psychiatry 46, 1509–1522.
Hernandez, F., Borrell, J., Guaza, C., Avila, J., Lucas, J.J., 2002. Spatial learning deficit
in transgenic mice that conditionally over-express GSK-3beta in the brain but
do not form tau filaments. J. Neurochem. 83, 1529–1533.
Hoy, A.J., Bruce, C.R., Cederberg, A., Turner, N., James, D.E., Cooney, G.J., Kraegen,
E.W., 2007. Glucose infusion causes insulin resistance in skeletal muscle of rats
without changes in Akt and AS160 phosphorylation. Am. J. Physiol. Endocrinol.
Metab. 293, E1358–1364.
Ivy, A.S., Brunson, K.L., Sandman, C., Baram, T.Z., 2008. Dysfunctional nurturing
behavior in rat dams with limited access to nesting material: a clinically
relevant model for early-life stress. Neuroscience 154, 1132–1142.
Ivy, A.S., Rex, C.S., Chen, Y., Dube, C., Maras, P.M., Grigoriadis, D.E., Gall, C.M., Lynch,
G., Baram, T.Z., 2010. Hippocampal dysfunction and cognitive impairments
provoked by chronic early-life stress involve excessive activation of CRH
receptors. J. Neurosci. 30, 13005–13015.
Korte, S.M., De Boer, S.F., 2003. A robust animal model of state anxiety:
fear-potentiated behaviour in the elevated plus-maze. Eur. J. Pharmacol. 463,
163–175.
Krishna, S., Keralapurath, M.M., Lin, Z., Wagner, J.J., de La Serre, C.B., Harn, D.A.,
Filipov, N.M., 2015. Neurochemical and electrophysiological deficits in the
ventral hippocampus and selective behavioral alterations caused by high-fat
diet in female C57BL/6 mice. Neuroscience 297, 170–181.
Li, G., Liu, T., Kong, X., Wang, L., Jin, X., 2014. Hippocampal glycogen synthase
kinase 3beta is critical for the antidepressant effect of cyclin-dependent kinase
5 inhibitor in rats. J. Mol. Neurosci. 54, 92–99.
Machado, T.D., Dalle Molle, R., Laureano, D.P., Portella, A.K., Werlang, I.C., Benetti
Cda, S., Noschang, C., Silveira, P.P., 2013. Early life stress is associated with
anxiety, increased stress responsivity and preference for comfort foods in adult
female rats. Stress 16, 549–556.
Macri, S., Chiarotti, F., Wurbel, H., 2008. Maternal separation and maternal care act
independently on the development of HPA responses in male rats. Behav. Brain
Res. 191, 227–234.
Malter Cohen, M., Jing, D., Yang, R.R., Tottenham, N., Lee, F.S., Casey, B.J., 2013.
Early-life stress has persistent effects on amygdala function and development
in mice and humans. Proc. Natl. Acad. Sci. U. S. A. 110, 18274–18278.
Maniam, J., Morris, M.J., 2010a. Palatable cafeteria diet ameliorates anxiety and
depression-like symptoms following an adverse early environment.
Psychoneuroendocrinology 35, 717–728.
Maniam, J., Morris, M.J., 2010b. Voluntary exercise and palatable high-fat diet both
improve behavioural profile and stress responses in male rats exposed to early
life stress: role of hippocampus. Psychoneuroendocrinology 35, 1553–1564.
Maniam, J., Morris, M.J., 2012. The link between stress and feeding behaviour.
Neuropharmacology 63, 97–110.
Maniam, J., Antoniadis, C., Morris, M.J., 2014. Early-life stress, HPA axis adaptation,
and mechanisms contributing to later health outcomes. Front. Endocrinol. 5,
73.
Maysami, S., Haley, M.J., Gorenkova, N., Krishnan, S., McColl, B.W., Lawrence, C.B.,
2015. Prolonged diet-induced obesity in mice modifies the inflammatory
response and leads to worse outcome after stroke. J. Neuroinflammation 12,
140.
Meaney, M.J., Aitken, D.H., van Berkel, C., Bhatnagar, S., Sapolsky, R.M., 1988. Effect
of neonatal handling on age-related impairments associated with the
hippocampus. Science 239, 766–768.
209
Meaney, M.J., Aitken, D.H., Bhatnagar, S., Sapolsky, R.M., 1991. Postnatal handling
attenuates certain neuroendocrine, anatomical, and cognitive dysfunctions
associated with aging in female rats. Neurobiol. Aging 12, 31–38.
Nakamura, A., Terauchi, Y., 2013. Lessons from mouse models of high-fat
diet-induced NAFLD. Int. J. Mol. Sci. 14, 21240–21257.
Naninck, E.F., Hoeijmakers, L., Kakava-Georgiadou, N., Meesters, A., Lazic, S.E.,
Lucassen, P.J., Korosi, A., 2015. Chronic early life stress alters developmental
and adult neurogenesis and impairs cognitive function in mice. Hippocampus
25, 309–328.
Polter, A., Beurel, E., Yang, S., Garner, R., Song, L., Miller, C.A., Sweatt, J.D.,
McMahon, L., Bartolucci, A.A., Li, X., Jope, R.S., 2010. Deficiency in the inhibitory
serine-phosphorylation of glycogen synthase kinase-3 increases sensitivity to
mood disturbances. Neuropsychopharmacology 35, 1761–1774.
Polter, A.M., Yang, S., Jope, R.S., Li, X., 2012. Functional significance of glycogen
synthase kinase-3 regulation by serotonin. Cell. Signal. 24, 265–271.
Prickaerts, J., Moechars, D., Cryns, K., Lenaerts, I., van Craenendonck, H., Goris, I.,
Daneels, G., Bouwknecht, J.A., Steckler, T., 2006. Transgenic mice
overexpressing glycogen synthase kinase 3beta: a putative model of
hyperactivity and mania. J. Neurosci. 26, 9022–9029.
Prusator, D.K., Greenwood-Van Meerveld, B., 2015. Gender specific effects of
neonatal limited nesting on viscerosomatic sensitivity and anxiety-like
behavior in adult rats. Neurogastroenterol. Motil. 27, 72–81.
Rodgers, R.J., Dalvi, A., 1997. Anxiety, defence and the elevated plus-maze.
Neurosci. Biobehav. Rev. 21, 801–810.
Sanchez, M.M., Ladd, C.O., Plotsky, P.M., 2001. Early adverse experience as a
developmental risk factor for later psychopathology: evidence from rodent
and primate models. Dev. Psychopathol. 13, 419–449.
Spittaels, K., Van den Haute, C., Van Dorpe, J., Terwel, D., Vandezande, K., Lasrado,
R., Bruynseels, K., Irizarry, M., Verhoye, M., Van Lint, J., Vandenheede, J.R.,
Ashton, D., Mercken, M., Loos, R., Hyman, B., Van der Linden, A., Geerts, H., Van
Leuven, F., 2002. Neonatal neuronal overexpression of glycogen synthase
kinase-3 beta reduces brain size in transgenic mice. Neuroscience 113,
797–808.
Surget, A., Tanti, A., Leonardo, E.D., Laugeray, A., Rainer, Q., Touma, C., Palme, R.,
Griebel, G., Ibarguen-Vargas, Y., Hen, R., Belzung, C., 2011. Antidepressants
recruit new neurons to improve stress response regulation. Mol. Psychiatry 16,
1177–1188.
Teegarden, S.L., Bale, T.L., 2008. Effects of stress on dietary preference and intake
are dependent on access and stress sensitivity. Physiol. Behav. 93, 713–723.
Tschopp, O., Yang, Z.Z., Brodbeck, D., Dummler, B.A., Hemmings-Mieszczak, M.,
Watanabe, T., Michaelis, T., Frahm, J., Hemmings, B.A., 2005. Essential role of
protein kinase B gamma (PKB gamma/Akt3) in postnatal brain development
but not in glucose homeostasis. Development 132, 2943–2954.
Wang, Q., Liu, L., Pei, L., Ju, W., Ahmadian, G., Lu, J., Wang, Y., Liu, F., Wang, Y.T.,
2003. Control of synaptic strength, a novel function of Akt. Neuron 38,
915–928.
Wang, X.D., Labermaier, C., Holsboer, F., Wurst, W., Deussing, J.M., Muller, M.B.,
Schmidt, M.V., 2012. Early-life stress-induced anxiety-related behavior in
adult mice partially requires forebrain corticotropin-releasing hormone
receptor 1. Eur. J. Neurosci. 36, 2360–2367.
Ziabreva, I., Schnabel, R., Braun, K., 2000. Parental deprivation induces
N-methyl-d-aspartate-receptor upregulation in limbic brain areas of Octodon
degus: protective role of the maternal call. Neural Plast. 7, 233–244.
Ziabreva, I., Poeggel, G., Schnabel, R., Braun, K., 2003. Separation-induced receptor
changes in the hippocampus and amygdala of Octodon degus: influence of
maternal vocalizations. J. Neurosci. 23, 5329–5336.
la Fleur, S.E., Houshyar, H., Roy, M., Dallman, M.F., 2005. Choice of lard, but not
total lard calories, damps adrenocorticotropin responses to restraint.
Endocrinology 146, 2193–2199.