Behavioural Brain Research 379 (2020) 112399
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Behavioural Brain Research
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Research report
Early life stress and the programming of eating behavior and anxiety: Sexspecific relationships with serotonergic activity and hypothalamic
neuropeptides
T
Randriely Merscher Sobreira de Limaa,*, Lucas Victor dos Santos Bentob,
Marcelo di Marcello Valladão Lugonb, Valerio Garrone Baraunac,
Athelson Stefanon Bittencourtb,d, Carla Dalmaza,e,
Ana Paula Santana de Vasconcellos Bittencourtc
a
Programa de Pós-Graduação em Neurociências, Instituto de Ciências Básicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
Programa de Pós-Graduação em Bioquímica e Farmacologia, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Espírito Santo, Brazil
c
Departamento de Ciências Fisiológicas, Universidade Federal do Espírito Santo, Espírito Santo, Brazil
d
Departamento de Morfologia, Universidade Federal do Espírito Santo, Espírito Santo, Brazil
e
Departamento e PPG Bioquímica, Instituto de Ciências Básicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Maternal separation
Maternal deprivation
Food consumption
POMC
Serotonin
Early life experiences have strong influences on brain programming and can affect eating behavior control and
body weight later in life. However, there is no consensus about the relationship between neonatal stress and
feeding behavior. We evaluated whether maternal deprivation (MD) and maternal separation (MS) alter body
weight and appetite using standard rat chow consumption and palatable food. Also, we evaluated anxiety and
the expression of the leptin receptor, neuropeptides POMC, CART, NPY in the hypothalamus, as well as the
serotoninergic system in the amygdala and hypothalamus as possible modulators of these behaviors. We found a
decrease in standard rat chow consumption in MD. However, both neonatal stress protocols increased the
consumption of palatable food and led to anxiogenic behavior in male animals. MD led to decreased hypothalamic POMC levels in adult males. Serotonin in the hypothalamus was decreased by both stress models in males
and females. In the amygdala, MS decreased serotonin levels while MD increased its metabolite levels. We
observed that males are more vulnerable and females are more resilient to the effects of neonatal stress on
anxiety-like behavior, as well as on food consumption and on the central changes observed. These data together
add support to the concept that the early environment contributes to the development of eating disorders later in
life.
1. Introduction
environmental factors, acute or chronic exposure to stress evokes
physiological and behavioral responses that alter the metabolic and
behavioral state of animals [4,5]. The effects of stress exposure on food
intake are complex and can be bidirectional. Studies have shown that
effects are dependent on the level of stress: acute stress is associated
with reduced food intake while chronic social stress is associated with
increased food intake [6]. These early life stressors hold great importance as they may lead to effects that persist during the entirety of
an animal’s life [7–13]. Studies with animal models that have evaluated
the effects of neonatal stress on feeding behavior and weight of the
animals observed different results for different models of neonatal
stress. Maternal separation for 180 min per day for fourteen days may
Obesity is a disorder characterized by an excess of body fat and it
may lead to health complications such as cardiovascular disorders,
hypertension, and type-2 diabetes [1]. It is a multifactorial problem,
which has been prevalent for many years in developed countries, but it
is also becoming increasingly present in developing countries [2].
Given that obesity has become a worldwide public health problem, it is
important to try and understand the processes and mechanisms involved in eating behavior and in body weight gain.
Causes of the obesity epidemic include environmental factors such
as lifestyle, physical activity, diet, and pollution [3,1]. Among
⁎
Corresponding author.
E-mail addresses: randriely@gmail.com, randriely.lima@ufrgs.br (R.M.S. de Lima).
https://doi.org/10.1016/j.bbr.2019.112399
Received 28 June 2019; Received in revised form 28 November 2019; Accepted 28 November 2019
Available online 29 November 2019
0166-4328/ © 2019 Elsevier B.V. All rights reserved.
Behavioural Brain Research 379 (2020) 112399
R.M.S. de Lima, et al.
2. Materials and methods
from 6:00 AM to 6:00 PM). At postnatal day 0 (PND0), the litters were
standardized (with 4 males and 4 females each) and divided into three
distinct groups: control, maternal separation (MS), and maternal deprivation (MD). The control animals remained with their mothers until
weaning (PND 21), with only standard procedures for cage cleaning.
Maternal Separation was performed for 3 h per day for 14 consecutive
days from PND 2 through 14. Following this process, the mothers were
transferred from their home cage to a different box in another room.
The pups were also transferred to new boxes containing eight divisions,
in which each animal was placed in a separated compartment. The
procedures always took place at the same time, starting between 12 PM
and 1 PM. Maternal deprivation was performed on postnatal days 9 and
11, in which pups were separated from their mothers for 24 -h period
each time. To perform this procedure, the mother was removed from
the litter to another box and transferred to another room, following
which all the pups were removed from the nest and moved into a single
box. Then, the litter remained together for 24 h in a different room.
After that period, the pups along with the dams returned to standard
housing boxes. The procedures always took place at the same time,
starting between 9:00am and 11:00am (modified from Ellenbroek,
et al., 2004 [24]). We standardized our maternal deprivation model to
be more representative of early life stress observed in the clinic. In such
cases, babies remain separated from their mothers for long periods of
time, ranging from days to months. Considering the importance of
parental contact in the early years of life, neonatal stress models close
to those observed in the clinic help us understand the long-term effects
of exposure to early life adversity. After the neonatal stress period, the
animals remained with the mother until weaning (PND 21).
After weaning, body weight was measured once a week. At PND 60,
standard chow in addition to palatable food consumption was measured
and the behavioral tests (elevated plus maze and open field exposure)
were performed. After these tests, the animals were decapitated, and
the brains were removed to dissect the amygdala and hypothalamus
which were frozen at -80 °C for further high precision liquid chromatography analysis. For gene expression analysis, the animals were
submitted to the same neonatal stress procedures. However, these animals were not subjected to behavioral or feeding tests since exposure
to these tests could alter the genetic expression of the genes analyzed
(experimental design in Fig. 1).
2.1. Experimental subjects
2.2. Behavioral tests
Animal proceedings were performed in accordance to the recommendations of the Brazilian Society for Neurosciences (SBNeC) and
Brazilian Law on the use of animals (Federal Law 11.794/2008), and
were approved by the Institutional Ethical Committee. Wistar rats from
our own colony were used. The animals were kept at room temperature
(23 ± 1 °C), water and food were available ad libitum, and 12-h light/
12-h dark cycle was maintained throughout the experiments (lights on
2.2.1. Feeding behavior
The consumption of standard lab chow was measured daily during 7
days in the housing cage. The measurements were performed in the
morning and in the evening. The food consumed (in grams) was divided
by the number of animals in each box, and the sum of day and night
consumption was used as the total amount consumed for 24 h consumption [15].
promote hyperphagia (evaluated considering standard chow) and
weight gain during the development until adulthood [14]. On the other
hand, studies with maternal deprivation for 24 h have shown slow
growing rate and reduced intake of standard chow as well as reduction
of neuropeptide Y (NPY) in the arcuate nucleus of hypothalamus [15].
However, the mechanisms involved in the difference between these
results still need a better understanding.
The hypothalamus is an important region for regulating food intake
[16]. This region has two groups of neuropeptides involved in the
regulation of food intake: the first group is composed of anorexigenic
neuropeptides pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART), and the second group is composed of orexigenic neuropeptides neuropeptide Y (NPY) and the
agouti-related peptide (AgRP) [17]. The release of these neuropeptides
is influenced by leptin, a hormone produced by adipocytes whose action leads to a reduction in food intake and an increase in energy expenditure. Leptin functions are mediated by receptors, ObRb, located in
the arcuate nucleus of the hypothalamus. When bound to these receptors, leptin induces an increase in the expression of POMC / CART in
neurons which produces the neuropeptides mentioned above, and antagonizes the activity of NPY / AgRP neurons [18,19].
In addition to the physiological and homeostatic regulation of food
control, feeding behavior is modulated by systems involved in mood
control. One important neurotransmitter involved with this response is
the serotonin [20]. Psychological factors also play an important role in
food control. Studies indicate that levels of anxiety and depression have
a direct action on appetite and type of food preferences [21–23]. In
addition, the serotoninergic system is highly related to satiety [20].
Therefore, we propose to evaluate how different protocols of neonatal stress affect anxiety and feeding behavior in adulthood, as well as
the mechanisms involved in the body weight control of these animals.
We analyzed whether maternal deprivation and maternal separation
alter appetite and body weight, quantified the expression of leptin receptors and of the neuropeptides POMC, CART, NPY in the hypothalamus, and evaluated whether the serotonergic system in the amygdala
and in the hypothalamus could be affected. Throughout the experiments, we evaluated sex-specific effects for all analyzed parameters.
Fig. 1. Experimental design. At postnatal day 0 (PND0), the litters were standardized (with 4 males and 4 females each) and divided in control, maternal separation
(MS) and maternal deprivation (MD). The control animals remained with the mothers until weaning (PND 21). Maternal Separation was performed for 14 consecutive days. Maternal deprivation was performed on days 9 and 11 after birth. After weaning, body weight was measured once a week. At PND 60, one set of
animals was submitted to behavior tests, and other set of animals was killed and amygdala and hypothalamus collected to PCR analyses. The behavioral tests used
were elevated plus maze, open field, and food consumption, in that order. These animals were killed by decapitation and the brains were removed, amygdala and
hypothalamus were dissected for further high precision liquid chromatography (HPLC) analysis.
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Behavioural Brain Research 379 (2020) 112399
R.M.S. de Lima, et al.
pump (Shimadzu LC-10). Chromatography was performed with a C18
(Shim-pack CLC-ODS) analytical column (150 mm X 4.6 mm, ID) with
3 μm particles. An integrator (Shimadzu C-R7 Ae plus) was used to
analyze the chromatographic data. The mobile phase consisted of a
solution of 0.194 M citric acid monohydrate, 0.243 M sodium acetate
trihydrate, and 0.295 mM EDTA. The flow was 1.0 ml/min and the
potential of the electrode was 0.85 V. The tissue concentrations of 5-HT
and its main metabolite, 5-HIAA, were calculated by the interpolation
of the respective standard curves. The values obtained were expressed
as ng/g wet tissue. The 5-HIAA / 5-HT ratio was used as an index of the
turnover rate of 5-HT [28].
2.2.2. Palatable food test
The palatable food test consisted of 7 days exposure to sweet food
for two hours daily in the housing cage. Exposure to sweet food occurred between 3 PM and 6 PM, where the animals were able to choose
between standard rat chow and the sweet food. The sweet food consisted of pellets of a mixture of condensed milk (395 g), sugar (220 g)
and milk powder (480 g) (modified from [25,26]) and was offered ad
libitum for 2 h/day. The food ingested (in grams) was divided by the
number of animals in each cage.
2.2.3. Elevated plus-maze test (EPM)
The elevated plus maze test was conducted after PND 60, using a
standard plus maze apparatus kept 50 cm above the floor, consisting of
four arms arranged in the shape of a cross (arms measured 50 × 10 cm).
The four arms were joined at the center by a 10 cm square platform.
Two of the arms, opposite to each other, had no walls (open arms); the
two other arms (closed arms) had 40 cm high walls. The test was performed in an illuminated room with fluorescent overhead lights that
produced light intensities of 90 lx. The EPM tests the anxiety state of the
animal, based on the principle that exposure to an elevated and open
arm leads to an approach conflict that is stronger than that evoked by
exposure to an enclosed arm maze [27]. The animal was placed on the
center of the maze, facing one of the open arms. A rat was considered to
have entered one arm of the maze when all four feet were within the
arm. Conventional parameters of anxiety-like behavior were monitored:
entries into open and closed arms, time in open and closed arms and
percentage of time in open arms (determined by the time in the open
arms divided by total time in the arms and multiplied by 100).
2.2.7. Statistical analysis
Statistical analysis was performed using the IBM SPSS Statistics
software. Data are expressed as mean ± SE of the mean, and analyzed
using two-way ANOVA for comparation between males and females,
ANOVA for the comparisons among groups, repeated measures ANOVA
and three-way ANOVA for body weight. When necessary, Duncan’s
post-hoc test was applied. The significance level was p ≤ 0.05 for all
analyses. Outliers (more than two standard deviations from the mean)
were excluded from the analysis.
3. Results
3.1. Early life stress changes body weight
Fig. 2 shows the effects of maternal separation and maternal deprivation on body weight. Three-way ANOVA indicated main effect of
the sex (F (5,69) = 10489.64, p < 0.0001), and interaction between
sex and time (F (5,69) = 92.70, p < 0.0001). As expected for both
main effect and interaction, females weighed less than males. No interaction between group and time or group, time and sex were observed
(p > 0.05). For males, there was an effect of time (repeated measures
ANOVA, F (2,38) = 1239.39, p < 0.0001) and an interaction between
group and time(repeated measures ANOVA, F (2,38) = 4.60,
p < 0.0001). The Duncan's post hoc analysis indicated that the MD
group was different from the other groups, showing a decrease on body
weight (Fig. 2). For females, repeated measures ANOVA also indicated
the effects of both the time and the interaction among groups and time
(time [repeated measures ANOVA, F (2,35) = 53.44; p < 0.0001]; Interaction Group*time [repeated measures, F (2,35) = 4.32;
p < 0.0001]). The Duncan's post hoc analysis indicated that both the
MS and MD groups weighed less than the control group.
2.2.4. Open field
Open field (OF) test was used to evaluate motor activity. To perform
the test, the animals were individually exposed to an open field, in a1 m
square box with 30 cm high walls and their behavior was recorded on
video for 5 min (Logitech HD C270, Switzerland). For the behavioral
analysis, locomotor activity was observed in the center and periphery of
the apparatus. The EPM and OF were performed between 1 PM and
6 P M. Behavior was recorded and analyzed using the ANY-Maze videotracking system (Stoelting, CO). Between trials, apparatuses were
cleaned with 70 % ethanol.
2.2.5. Analysis of gene expression
Total RNA was extracted using TRIzol® Reagent (Life Technologies,
USA) and following the manufacturer's recommendations. The concentration and quality of the extracted RNA were verified using the
NanoDrop™ equipment (ThermoScientific, Wilmington, USA) and by
agarose gel electrophoresis, respectively. The cDNA synthesis was
performed using iScript Reverse Transcription Supermix for RT-qPCR
(Biorad, CA, USA) using the S1000 Thermal Cycler (Biorad, CA, USA).
Real-time PCR reaction was performed using CFX96 Real Time PCR
(Biorad, CA, USA) and iQ SYBR Green Supermix (Biorad, CA, USA). The
relative quantification of gene expression was done by the 2−ΔΔCt
method, using the cyclophilin gene as normalizer. The primers were
designed in PRIMER BLAST software. The genes, as well as the primers
used, are described in Table 1.
3.2. Early life stress changes feeding behavior in a sex-specific way
First, we analyzed the effect of neonatal stress and sex on rat chow
consumption. The two-way ANOVA indicated a main effect of the sex
on consumption of rat chow, showing that males ate more than females
(F (1, 134) = 344.20; p < 0.001). Then, we run an analysis separately
for each sex. For males, one-way ANOVA indicated a difference among
the groups (F (2,63) = 8.22; p < 0.001]. A post-hoc test showed that
the MD group was different from the other groups since the MD group
consumed lower amounts of rat chow in comparison to others (Fig. 3).
No difference was found in the same test when analyzing females
(ANOVA, p > 0.05).
2.2.6. High precision liquid chromatography analysis
The samples (amygdala and hypothalamus) were weighed and
homogenized with 290 μl of 0.1 M perchloric acid (HClO4), 5 μl of
0.1 mM ethylenediaminetetraacetic acid (EDTA), and 5 μl of 0.4 mM
sodium metabisulfite (Na2S2O5). They were then centrifuged at 4 °C at a
speed of 10,000 rpm for 30 min. The supernatant from the individual
samples (200 μl) was maintained between 0 °C and 4 °C until analyzed
by high performance liquid chromatography (HPLC). For this, a
Shimadzu HPLC (LC10CE, Tokyo, Japan) was used with 200 μl and
10 μl loops (Rheodyne 7725-I, California, USA) and coupled carbonglass electrochemical detector (Shimadzu LECD-6A) to the pressurizing
3.3. Palatable food test
In the palatable test, none of the groups ate rat chow. Therefore, we
only analyzed the consumption of palatable food. We observed main
effects of neonatal stress, and of the sex, and an interaction between
neonatal stress and sex on palatable food consumption (two-way
ANOVA: Neonatal stress, F(1, 96) = 19.46; p > 0.0001; Sex, F
(1,96) = 20.79; p > 0.0001; Interaction neonatal stress*sex F(1,
96) = 20.32, p = 0.00002). One-way ANOVA indicated a difference
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R.M.S. de Lima, et al.
Table 1
Sequence of the primers used for gene expression.
mRNA
Gene
Sequence
Ciclofilin
CYPA
Leptin Receptor
LEPR
Pró-opiomelanocortin
POMC
Neuropeptide Y
NPY
Cocaine and amphetamine-regulated transcript
CARTPT
F-5′-TGGCAAGCATGTTGGGTCTTTGGGAG-3’
R- 5′-GGTGATCTTCTTGCTGGTCTGCCATTC-3’
F-5′-GTGCTTCCTGGGTCTTCATAC-3’
R- 5′-CAACACTCGTCAGAATTTTGGG-3’
F-5′-CCACTGAACATCTTCGTCCTC-3’
R- 5′-GAATCTCGGCATCTTCCAGG-3’
F-5′-GTGTGTTTGGGCATTCTGGC-3’
R- 5′-TGTCGCAGAGCGGAGTAGTA-3’
F-5′GCGCTGTGTTGCAGATTGAA-3’
R- 5′-CAGTCACACAGCTTCCCGAT-3’
females showed higher total distance traveled, and both distance traveled on the periphery and center of the apparatus [Sex (Total distance
traveled F (1, 72) = 54.09; p < 0.001); Periphery (F(1,72) = 47.60;
p < 0.001); Center F (1, 72) = 26.20; p < 0.001)]. However, we did
not observe effects of neonatal stress or interaction between neonatal
stress and sex (p > 0.05).
among groups. Both the MS and the MD male groups were different
from the control group, presenting an increase in palatable food consumption (one-way ANOVA, F (2, 46) = 32.5; p < 0.0001]. In females,
the test showed a difference among all the groups, since MD and MS
were different from each other and different from the control group
(one-way ANOVA, F (2,45) = 12.65; p < 0.0001] (see Fig. 4). We
observed that females submitted to MS showed increased consumption
of palatable food, and females submitted to MD showed a decrease in
the amount of palatable food consumed.
3.5. Hypothalamic peptides known to influence feeding behavior have
altered expression in male animals subjected to early life stress
Since our results pointed to altered feeding behavior in MS and MD
animals, we evaluated hypothalamic neuropeptides involved in feeding
regulation. The two-way ANOVA pointed to sex differences in neuropeptide Y expression. We observed decreased NPY gene expression in
females compared to males (F (1,26) = 8.18 p = 0.009). No other sexdifferences were observed (p > 0.05). In males, we observed a significant effect of early stress decreasing the pro-opiomelanocortin
(POMC) expression in the hypothalamus [One-way ANOVA F (2,
14) = 7.39; p = 0.01]. There was also a trend towards an effect of
leptin receptor expression in males [one-way ANOVA, F (2,14) = 3.14;
p = 0.08]. However, no differences in gene expression of the cocaineand amphetamine-regulated transcript (CART) and neuropeptide Y
(NPY) were detected (Fig. 7A-D). In females, no differences in genes
expression were observed (Fig. 8).
3.4. Evaluations suggest sex-specific effects of early life stress on anxietylike behavior
We analyzed the effect of the neonatal stress and the sex on percentage of time in the open arms. The two-way ANOVA indicated effect
of the sex on percentage of time in the open arms (Sex F (1,
82) = 20.33; p < 0.001) and we observed a trend in both groups on
neonatal stress (Neonatal stress F(1, 82) = 3.10; p = 0.08). We also
analyzed sex-specific differences in other parameters of the test. In
males, we observed that both groups subjected to early stress showed
different percentage of time in the open arms when compared to the
control group [one-way ANOVA, F (2,35) = 5.21; p < 0.01, followed
by Duncan].In the time spent in the open arm, the groups MS and MD
were different from the control group, both decreased the time in open
arms[one-way ANOVA, F (2,35) = 5.65; p < 0.007]. We also observed
differences among the groups in the number of entries in open arms, in
which the MS group showed a decrease in number of entries compared
to the MD group [One-way ANOVA, F (2,35) = 3.35; p < 0.04, followed by Duncan]. No differences were observed in the number of
entries in the closed arms or in the percentage of entries in the open
arms [one-way ANOVA, p > 0.05)]. We observed a difference among
the MS and the other groups [one-way ANOVA, F (2,30) =3.32;
p < 0.04] where the MS group spent more time in the closed arms. In
females, no differences were found for the analyzed parameters (p
> 0.05). Results are presented in Figs. 5 and 6.
Results from the open field test are shown in Table 2. The two-way
ANOVA shows the effect of the sex in all parameters. We observed that
3.6. Early life stress alters serotonergic activity
Serotonin has been reported to be involved in the control of feeding
behavior [20], and anxiety [29]. Since our animals presented altered
anxiety-like behavior, which may affect feeding [30], we also assessed
the serotonergic activity in the amygdalaand in the hypothalamus,
through the evaluation of serotonin and its metabolite 5HIAA contents.
HPLC measurements were performed on different days for males and
females, and were therefore analyzed separately. In the amygdala of
males, we observed that serotonin content was decreased in the MS
group [one-way ANOVA, F (2,19) = 3.84; p = 0.04, followed by
Duncan; see Fig. 9A]. The serotonin metabolite 5HIAA content was
Fig. 2. Effect of maternal separation and maternal deprivation on body weight (in grams). Data expressed as mean ± SEM. A: Males. B: Females. N = 11–15 per
group. *Significantly different from all other groups (Duncan, p < 0.05), #: Significantly different from control group (Duncan, p < 0.05).
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R.M.S. de Lima, et al.
Fig. 3. Effect of maternal separation and maternal deprivation on standard chow consumption. Data represent the average consumption/rat/24 h over a 7 days
period, and it is expressed as mean + SEM. N = 11–15 per group. *: Different from all other male groups (Duncan test, p < 0.05).
Fig. 4. Effect of maternal separation and maternal deprivation on palatable food consumption. Data expressed as mean + SEM. N = 13-21. *: Different from control
group male, #: Different from all other female groups (Duncan test, p < 0.05).
5HIAA, which was decreased in the MD group [one-way ANOVA, F
(2,19) = 4.59; p = 0.02, followed by Duncan’s test; Fig. 9B], with no
differences in the other parameters analyzed (p > 0.05).
Serotonin content in the hypothalamus was decreased in both male
increased in the maternal deprivation group [one-way ANOVA, F
(2,17) = 7.10; p = 0.007, followed by Duncan]. No differences were
observed on serotonin turnover, evaluated by the ratio 5HIAA/5 H T
(p > 0.05). In females, we observed a difference among the groups on
Fig. 5. Effect of maternal separation and maternal deprivation on the behavior of males in
the elevated plus maze. (A) Percentage of time
in the open arms, (B) number of entries in the
open arms, (C) number of entries in the closed
arms. Data expressed as mean + SEM. Males,
N = 12–14 per group. *: Different from the
control group (Duncan test, p < 0.05).
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R.M.S. de Lima, et al.
Fig. 6. Effect of maternal separation and maternal deprivation on the behavior of female animals in the elevated plus maze. (A) Percentage of time in the open arms,
(B) number of entries in the open arms, (C) number of entries in the closed arms. Data expressed as mean + SEM. Females, N = 13–15 per group.
groups subjected to early life stress [one-way ANOVA, F
(2,21) = 12.87; p < 0.0001, followed by Duncan’s test; Fig. 10A].
There was no difference in 5HIAA levels [one-way ANOVA, F
(2,21) = 0.36], but both groups presented higher 5-HIAA / 5-HT ratio
[one-way ANOVA, F (2,21) = 12.48; p < 0.0001].In the females, hypothalamic serotonin levels were also decreased in MS and MD groups
(one-way ANOVA, F (2,22) = 5.27; p = 0.014; Fig. 10B]. In addition,
5HIAAwas increased in both groups [one-way ANOVA, F (2,22) = 8.09;
p = 0.003], and a trend towards an effect in the 5-HIAA / 5-HT ratio
was observed [One-way ANOVA, F (2,22) = 2.92; p = 0.07].
Table 2
Effect of maternal separation and maternal deprivation on behavior in the open
field of adult rats.
Group
Sex
Total distance traveled
Periphery
Control
MS
MD
Control
MS
MD
Male
Male
Male
Female
Female
Female
183.24
209.59
193.67
297.31
298.44
284.90
162.48
186.72
169.92
254.33
258.73
249.27
±
±
±
±
±
±
18.22
21.94
16.96
14.10
10.68
12.32
±
±
±
±
±
±
Center
16.03
19.20
61.47
8.71
8.71
11.06
20.76
22.86
23.74
42.98
39.71
35.62
±
±
±
±
±
±
3.67
3.80
3.38
4.95
3.82
5.72
Data are expressed as mean ± SEM, N = 11–15/group.
Fig. 7. Effect of maternal separation and maternal deprivation on gene expression of the
Leptin
Receptor,
Pro-opiomelanocortin
(POMC), cocaine- and amphetamine-regulated
transcript (CART), and neuropeptide Y in the
hypothalamus of male animals. Data expressed
as mean + EPM, N = 4–5 per group. *:
Different from the control group (Duncan's
test, p < 0.05).
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Fig. 8. Effect of maternal separation and maternal deprivation on the gene expression of the Leptin Receptor, Pro-opiomelanocortin, cocaine- and amphetamineregulated transcript (CART), and neuropeptide Y in the hypothalamus of female animals. Data expressed as mean + EPM, N = 4–5 per group.
4. Discussion
hypothalamic POMC levels in adult males. Serotonergic activity in
hypothalamus resulted in an increase in serotonin turnover by both
stress models in males and females. In the amygdala, males were more
affected, with MS decreasing serotonin content and MD increasing its
metabolite content as well as serotonin turnover.
The effects of early life stress on body weight were sex-specific: in
males, both early life stress models affected body weight, while MS
animals were able to catch-up and reach a body weight similar to the
control group after puberty. The effect on MD animals were longer
lasting and present in adulthood. The decrease in chow consumption
caused by maternal deprivation may be associated with a decrease in
The present study demonstrated that different protocols of neonatal
stress cause distinct and sex-specific effects on feeding behavior and
body weight. We observed a decrease in standard rat chow consumption in males that had been exposed to MD. At the same time, both
neonatal stress protocols increased the consumption of palatable food,
and led to anxiogenic behavior in male animals. In females, we did not
observe effects on behaviors related to standard chow consumption or
anxiety, but we did see that distinct early life stress differently alters the
consumption of palatable food. MD induced a decrease in the
Fig. 9. Effect of maternal separation and maternal deprivation on the contents of serotonin (5 H T) and its metabolite (5HIAA) and on the ratio 5HIAA/5 H T in the
amygdala. Data expressed as mean + EPM. N = 6–7 per group. A: Males., B: females. *: Different from the respective control group (ANOVA, followed by Duncan's
test, p < 0.05).
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R.M.S. de Lima, et al.
Fig. 10. Effect of maternal separation and maternal deprivation on hypothalamic levels of serotonin and its metabolite and on the 5HIAA/5 H T ratio. Data expressed
as mean + EPM. A: Males, N = 6–9 per group. B: Females, N = 7–9 per group. *: Different from the respective control group (Duncan's test, p < 0.05).
appetite, which could help explain the findings of lower body weight in
the MS group. In females, body weight was also reduced by neonatal
stress, but they were able to catch-up with no effect observed in
adulthood. These results are in agreement with early studies concerning
neonatal undernutrition, which have reported that catch-up growth in
weight was complete in females, but not in male rats [31].
Satiety and appetite are processes related to the control of feeding,
and the appetite process is related to the acquisition of energy substrates. The homeostatic processes necessary to balance energetic needs
and energy expenditure are regulated by hormones and neurotransmitters. The hypothalamus receives peripheral leptin information
concerning the energy store [16]. However, the feeding process is also
associated with emotion, such as anxiety [32], and with pleasure, since
the taste, texture and smell of food can cancel satiety and stimulate the
appetite. The hedonic component of this behavior is mediated by central mechanisms with principal action of the limbic system [33]. In the
present study, changes in palatable food intake caused by both neonatal
stress protocols suggest that the stressful environment can change the
hedonic component of eating behavior. These results corroborate the
bidirectional theories of the influence of stress on food consumption
[6]. Maternal separation in early life may be related to stress and increased food intake, resulting in a weight catch-up. On the other hand,
maternal deprivation would be a model more related to reduced food
intake. The consumption of palatable food is related to the hedonic
aspect of feeding behavior, and may be affected by different factors,
including anxiety [32,34]. Sex-specific-results were found when evaluating sweet food consumption, which involves motivation for food
[35]. Our results showed that both neonatal stress protocols caused
increased consumption of sweet foods in males. Similarly, in MS female
animals, we observed an increase in the consumption of this type of
food while MD had the opposite effect. Therefore, the results demonstrate that adversities in the postnatal period can change not only appetite control and satiety, but also the pleasure and motivation to
eating.
Both MS and MD increased anxiety-like behavior in adult males.
However, females presented no effect. The effects of neonatal stress on
anxiety are still controversial, since numerous studies have found an
increase in anxiety, while others did not observe robust effects on anxiety-related behaviors [36–38]. The differences in these results can be
explained by the time and duration in which neonatal stress is applied,
with maternal separation being performed from 15−360 min per day
[39–41], while maternal deprivation protocols can occur on different
postnatal days [42,12,11,43–45]. Increased anxiety levels could be involved in the increased consumption of sweet food, as observed here in
the animals subjected to early life stress. It has been shown that chronic
stress leads to increased consumption of sweet food, an effect that has
been shown to be reversed by anxiolytic drugs [46,30]. This interplay
between anxiety and sweet food consumption possibly functions in a
two–way fashion, since palatable food may reduce stress levels [47]. In
this scenario, it would be interesting to point out that our animals had
only a short-term access to palatable food. It would be interesting to
know the effects of chronic access to this type of food (similar to what
happens in the human population) when animals are subjected to early
life stress.
Furthermore, we observed that in males, the MS group showed a
decrease in serotonin in the amygdala, and maternal deprivation increased its metabolite. Serotonin is a neurotransmitter associated to
physiological and behavioral functions, such as motor activity, hormone secretion, mood, and cognition [48,49]. In fact, the serotonergic
system has an important role in the regulation of fear and anxiety,
especially in the amygdala [50–53]. Several studies have associated the
levels of serotonin in amygdala with variations in anxiety-like behavior.
The decrease in serotoninergic activity in the entire amygdala is associated with an increase in anxiety-like behavior [54,55]. In our data, we
observed a decrease in serotonin in amygdala induced by MS in males,
which could help explain the increased anxiety-like behavior observed
in these animals. However, different results concerning serotonergic
activity in the amygdala and its effects on anxiety have been reported
when distinct 5 H T receptors or post-synaptic targets are studied
[50,51,53], and further studies are necessary to better understand the
relation between serotonin in the amygdala and anxiety-like behavior
in MS male rats.
Interestingly, we observed that in females, none of the neonatal
stress protocols altered anxiety-related behaviors. However, maternal
separation increased consumption of palatable foods and maternal deprivation decreased consumption of the same food. We observed that
MD females showed decreased consumption of palatable food and decreased 5HIAA in the amygdala. The decrease in serotonin metabolite is
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Behavioural Brain Research 379 (2020) 112399
R.M.S. de Lima, et al.
these differences may be related with the results observed. Psychiatric
disorders are characterized by sex differences in prevalence, symptomatology, and response to treatment [80].These results open a window
to study the influence of postnatal adversity on males and females, since
we observed that stress environments before sexual differentiation may
induce different phenotypes and neurochemical changes in males and
females.
Maternal separation and maternal deprivation are well-established
protocols used to investigate stress-related neurobiological and behavioral changes [81–83]. Extensive literature investigates the short- and
long-term consequences of neonatal stress [14,15,84,85]. However, the
effect of the time and extension of the neonatal stress protocols are
associated to different neurobiological and behavioral consequences.
The maternal separation paradigm is a model to investigate the consequences of stressors in a short, repeated period in early life [86].
Usually, MS protocols are applied in the first days of life, varying from 3
to 6 h per day in PND 14 to PND 21 [87]. Maternal deprivation, on the
other hand, represents a combination of stressors related to maternal
care and protection, including metabolic variations and hormonal
changes [88]. The animals are deprived of maternal care for 24 h.
However, when reunited with the mother, they receive greater care and
protection, which in itself could offset the effects of MD [85,89]. The
present maternal deprivation paradigm was chosen to propose a model
that represents an early life stress protocol closer to that observed in the
clinic. Children in pediatric intensive care units, for example, usually
remain away from maternal presence for a long time, ranging from days
to months [90,91]. Considering the importance of parental contact in
the early years of life, neonatal stress is a great model to understand the
long-term effects of exposure to early life adversity similar to those
observed in the clinic.
In conclusion, our findings showed the effects of distinct types of
neonatal stress on behavioral aspects, as well as on body weight, and we
propose that these outcomes may be related to central alterations of
serotonergic activity and of the levels of neuropeptides involved in
eating behavior. To our knowledge, our study is the first in the literature that compares different protocols of neonatal stress (maternal separation and maternal deprivation), analyzing eating behavior and
anxiety in males and females. We observed that males are more vulnerable and females are more resilient to the effects of neonatal stress
on anxiety-like behavior, as well as on food consumption, and on the
central changes observed. This data together supports the concept that
the environment contributes to the development of eating disorders.
Postnatal adversity can trigger numerous behavioral changes, as we
have observed in our work. Therefore, studies concerning the susceptibility of different individuals to early stress effects on feeding behavior
are warranted.
associated to increased depression like-behavior, which could explain
the decrease in palatable food as anhedonia [56]. In agreement with
these results, Goodwill et al., 2019 observed that early life stress leads
to a depressive-like behavior and anhedonia phenotype in females [57].
These results together suggest that, in females, MD may lead to depressive behavior problems, while in males the effects are more related
to anxiety behavior problems.
The involvement of serotonin in satiety control has been studied for
several years. 5-HT is a neurotransmitter widely distributed in the
central nervous system with important actions to control satiety in both
the amygdala and the hypothalamus [58]. Studies indicate that drugs
that increase the availability of 5-HT in the synapse increase satiety and
decrease appetite, which explains why serotonergic drugs are used in
treating eating disorders [20]. Our results indicate that both protocols
increased 5-HT turnover in the hypothalamus, most notably in MD
males. These results agree with those of Cabbia and colleagues, who
reported an increase in 5-HT levels in the hypothalamus in animals
deprived on PND11 [55]. Corroborating the literature, we observed a
decrease in the consumption of standard chow caused by MD [14]. A
primary mechanism through which serotonin is able to regulate appetite and body weight involves activation of pro-opiomelanocortin
(POMC) neurons in the arcuate nucleus (ARC) of the hypothalamus
[59–62]. It is interesting that our analysis of the expression of hypothalamic neuropeptides involved in the appetite regulation pointed
POMC as the most affected one in MD males. POMC is an anorexigenic
neuropeptide in ARC of the hypothalamus. It produces the anorectic
peptide MSH. Its function is related to decreased food intake and body
weight [63–65], and its gene deletion is related to increased fat mass
and body weight [66,67]. However, in situations of low body weight or
low glucose availability, its expression is decreased as a compensatory
way to stimulate food consumption [68].Thus, the decrease in POMC
mRNA, in the same group that we observe a decrease in food consumption, may be related to compensatory effects in an attempt to increase dietary intake, since the body weight of MD is below the weight
of the control group during development.
Leptin actions occur mainly in its receptors located in the hypothalamus [69], where it affects the activity of neurons producing
anorexigenic and orexigenic neuropeptides [16,70,71]. In our study, we
observed variations in the expression of LepR in MS and MD animals,
possibly in response to the changes in body weight caused by these two
treatments. According to leptin's functions of modulating these neuropeptides, their expression was expected to be altered. One limitation in
our study is that we did not analyze the circulating leptin, and the
expression of leptin receptors could be a response to altered levels of
this hormone. Other studies have analyzed the effect of neonatal stress
on circulating leptin and found that stress conditions are associated
with increased levels of circulating leptin [72,73]. Studies analyzing
chronic stress exposure in early life have observed altered peripheral
metabolic parameters related to adipose tissue and plasma leptin levels
[74], while our study focused on hypothalamus.
It is interesting to note that we observed sex-specific differences in
all analyzed parameters. Although stress was applied early in the
postnatal period, we observed different responses in adulthood. Few
studies have explored sex-specific differences in eating behavior after
early life stress. However, the responses to stress are considered sexually dimorphic [69,72–76].The effects of stress on the consumption of
standard lab chow were attenuated in females compared to males. But,
the effect on palatable food intake was changed in both neonatal stress
groups, in males and females. At the same time, females exhibited less
anxiety-type behaviors than males. Furthermore, female also exhibited
lower level of NPY compared to males. Previous studies that analyzed
the difference between sexes caused by neonatal stress suggest that
neurochemical and behavioral alterations observed in response to
neonatal stress may be associated with endocrinological characteristics,
considering that stress reactivity is influenced by sex [77,78]. Sexual
differences in the brain structure are important early in life [79], and
CRediT authorship contribution statement
Randriely Merscher Sobreira de Lima: Conceptualization,
Investigation, Formal analysis, Writing - original draft. Lucas Victor
dos Santos Bento: Investigation. Marcelo di Marcello Valladão
Lugon: Investigation. Valerio Garrone Barauna: Investigation,
Resources. Athelson Stefanon Bittencourt: Investigation, Resources.
Carla Dalmaz: Conceptualization, Methodology, Visualization, Writing
- original draft, Supervision. Ana Paula Santana de Vasconcellos
Bittencourt: Conceptualization, Methodology, Visualization, Writing original draft, Supervision.
Acknowledgments
This research was supported by National Research Council of Brazil
(CNPq), Coordination of Improvement of Higher Level Personnel
(CAPES), Foundation for the protection of research and innovation in
Espírito Santo (FAPES) and National Institutes of Science and
Technology (INCT). In addition, we acknowledge the Laboratório
9
Behavioural Brain Research 379 (2020) 112399
R.M.S. de Lima, et al.
Multiusuário de Análises Biomoleculares (LABIOM) of the Universidade
Federal do Espírito Santo, and the professors Dr. Vanessa Beijamini
Harres and Dr. Luiz Carlos Schenberg for their support.
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