NEUROSCIENCE

RESEARCH ARTICLE
L. C. do Amaral Almeida et al. / Neuroscience 463 (2021) 254–263

Blockade of Opiodergic System During Early Weaning Reverts

Feeding Behavior Altered Patterns
Larissa Cavalcanti do Amaral Almeida, a Julliet Araújo de Souza, a Gabriel Araújo Tavares, b Matilde Cesiana da Silva, c
Bruna Times Silva, d Paula Luiza Menezes Cruz, d Felipe Leitão de Souza, e Elizabeth do Nascimento, e
Taisy Cinthia Ferro Cavalcante, f Amanda Alves Marcelino da Silva f and Sandra Lopes de Souza g*
a
Programa de Pós-graduação em Neuropsiquiatria e Ciências do Comportamento (Federal University of Pernambuco), Brazil
b
Programa de Pós-graduação em nutrição (Federal University of Pernambuco) and Nantes Université, INRAE, UMR 1280, PhAN, Nantes, France
c
Department of Nutrition (Academic Center of Vitória de Santo Antão – Federal University of Pernambuco), Brazil
d
Department of Nutrition (Federal University of Pernambuco), Brazil
e
Programa de Pós-graduação em nutrição (Federal University of Pernambuco), Brazil
f
Programa de pós-graduação em ciências da saúde – Faculdade de Ciências Médicas (Universidade de Pernambuco), Brazil
g
Programa de Pós-graduação em Neuropsiquiatria e Ciências do Comportamento (Posneuro), and Department of Anatomy (Federal University
of Pernambuco), Brazil

Abstract—Adverse experiences that occur during the early stages of life can have permanent repercussions in
adulthood. Among these experiences, early weaning is one that can alter the molecular, cellular, and behavior pat-
terns in later life. Centered on this fact, the objective of the current study was to evaluate the effect of early wean-
ing at 15 days of life of Wistar rats on their feeding behavior and if the opioidergic system blockade would cause a
reversal of these outcomes. Experimental groups were formed based on the weaning period of each litter. On
postnatal day 15, the group D15 was weaned and, on postnatal day 30 (natural weaning), the group D30 was
weaned. The rats weaned on postnatal day 15, and administered subcutaneous Naltrexone (3 mg/kg) were from
group D15 + NTX. Those weaned at 15 days of age exhibited higher depressive-like behavior, lesser reactivity
time to sucrose, and higher intake of palatable food than the control group. The Naltrexone administration was
observed to reverse some outcomes, such as increasing the reactivity time to sucrose and decreasing the quan-
tity of palatable food consumed, to levels similar to those of the control group. Together, the findings of the pre-
sent study are indicative of the vital role played by the opioidergic system in inducing the changes noted in the
eating behavior patterns during adulthood, post early weaning. Ó 2021 IBRO. Published by Elsevier Ltd. All rights
reserved.

Key words: early weaning, eating behavior, naltrexone.

INTRODUCTION ment. With an ideal and perfectly balanced composition of

The beginning of life is characteristically a rapid
developmental period, and all the adverse experiences
during this phase can exert enduring and ongoing
results in adulthood (Tierney and Nelson, 2009). One
such experience is early weaning, which occurs when
the child is not exclusively breastfed until 6 months of
age, in accordance with the World Health Organization
recommendations **(WHO, 2017). Breast milk is the best
nutritional source required for proper growth and develop-
*Corresponding author. Address: Av. Prof. Moraes Rego,
1235 – Cidade Universitária, Recife – PE – CEP 50670-901, Brazil.
E-mail addresses: sanlopesufpe02@gmail.com, Sandra.lsouza2@
ufpe.br (S. L. de Souza).
Abbreviations: AgRP, Agouti Related Protein; MOR, Mu-opioid
receptor agonist; POMC, pro-opiomelanocortin.
macronutrients, micronutrients, hormones, and antibod-
ies, it is the best method of support to provide both nutri-
ent needs and immunoprotection to the offspring
(Andreas et al., 2015). Furthermore, the maternal physi-
cal contact is the stimulation which warrants the survival
and adaptation to the environment of the newborn. It also
affords social protection and facilitates maturation of the
neural characteristics (Kojima et al., 2012).
In the standard experimentation environment with
rats, the mothers were observed to spend 12 h on
average with their young until the weaning period was
completed (Weissman et al., 2004). Until the second
week of life, the milk intake is exclusively under the con-
trol of the mother, as the litter still lacks the mature neuro-
motor control for solid food intake (which commences
only on day 14). The weaning phase begins from day

https://doi.org/10.1016/j.neuroscience.2021.02.025
0306-4522/Ó 2021 IBRO. Published by Elsevier Ltd. All rights reserved.

254
 L. C. do Amaral Almeida et al. / Neuroscience 463 (2021) 254–263
21 and extends until day 30 (Weissman et al., 2004).
Hence, any break in the maternal bond prior to the 21st
day of life may be indicative of early weaning in rats.
There is evidence to link early weaning to the
consumption of a fat-rich diet associated with an
increased preference for palatable food (dos Santos
Oliveira et al., 2011). Furthermore, early weaning is
related to an increased expression of the Drd1 gene
receptor in the hypothalamus and brainstem, an
increased expression of the Drd2 in the brainstem, and
higher intake of palatable food in the rats in their
middle-age (Tavares et al., 2020). Therefore, it can be
stated that early weaning alters the long-term eating pat-
terns, particularly the hedonic control of eating behavior,
which is correlated to the pleasure of eating and not to
the energy need.
Largely, the hedonic control of the eating behavior is
the responsibility of the mesolimbic system, and the
opioidergic transmission is critical as it encodes the
hedonic impact of food. The opioid peptides and their
receptors which are extensively noted in the neural
circuitry, regulate the eating behaviors (Le Merrer et al.,
2009). Opioid administration in the nucleus accumbens
raises the consumption of the sucrose-rich solution and
the preference for high-fat foods (Zhang and Kelley,
2002). Thus, opioids directly affect and amplify the hedo-
nic perception of a food reward (Havermans, 2011).
As mentioned earlier, early weaning alters eating
behaviors, which are under the control of the opioidergic
system. Thus, an investigation is required into observing
whether the behavioral changes occur via the
opioidergic system. The present study, using Wistar
rats, assessed the effects of early weaning at 15 days of
life on the feeding behavior, and whether an opioidergic
system block would reverse these outcomes. The
hypothesis we put forth is that any changes in the
eating behavior which occur due to early weaning take
place through alterations in the opioidergic system, and
that blocking the system during early weaning will
induce a reversal of these effects on the eating
behavior. The results show that the early weaning leads
to increased depressive-like behavior, decreased
reactivity to sucrose, and increased palatable food
intake. Interestingly, the treatment with naltrexone
during the early weaning period reversed the decreased
reactivity to sucrose and the increased palatable food
intake, demonstrating the involvement of the opioidergic
system with the modulations of the feeding behavior
promoted by the early weaning.
EXPERIMENTAL PROCEDURES
Animals
The Wistar albino rats were supplied by the animal facility
of the Department of Nutrition of the Federal University of
Pernambuco (UFPE). Mating was observed to take place
in the ratio of two females to one male. Pregnancy was
diagnosed by the presence of sperm in the vaginal
smear and confirmed by body weight gain. Post
diagnosis, the pregnant rats were placed in individual
cages, and standard diet (Labina, PresenceÒ) was
255
provided during pregnancy and lactation. The litters
were culled at birth, to 8 pups. In this study we used 36
litters, which were adjusted to include an identical
number of males and females (4:4). After weaning, the
males alone (101 animals) were selected for this study.
The animals were maintained under standard conditions
(temperature of 22 ± 10 °C, inverted light/dark cycle of
12 h, lights on at 6 pm) throughout the experiment, and
were provided with food and water ad libitum. The
Ethics Committee on the Use of Animals (CEUA) from
the Federal University of Pernambuco evaluated and
approved all the procedures done with the animals in
this study.
Early weaning
Experimental groups were constituted based on the
weaning period of each litter. On postnatal day 15, the
litters from group D15 were weaned and, on the
postnatal day 30 (natural weaning), those of the group
D30 were weaned. The D15 animals, separated from
their dams, were fed on small pellets of a standard diet
(Labina, PresenceÒ). From day 30, all the groups were
given the same standard diet.
Experimental groups
The animals were segregated, based on the day of
weaning and neonatal treatment, into the following
categories: Control Group (D30) which included animals
weaned on postnatal day 30, Early Weaning Group
(D15) which included the animals weaned on postnatal
day 15, Early Weaning + Naltrexone Group (D15
+ NTX) which included the animals weaned on
postnatal day 15 and administered subcutaneous
naltrexone (3 mg/kg). Only two or three animals per litter
were used to form each group and for each behavioral
analysis.
Drug
The non-specific opioid antagonist Naltrexone
(C20H23N04, 341,401 g/mol; RovalÒ, Recife, Brasil, lot
27042017) was administered for the neonatal treatment
and in adulthood, using sterile saline as the dilution
medium. A subcutaneous dose of 3 mg/kg was given
(Michaels and Holtzman, 2007). Based on the drug
administration, we segregated two groups. One group
received a single dose during adulthood (120 days) prior
to food consumption, in order to decrease the food intake.
The second group received two doses, at different devel-
opmental periods, the first dose being given from day 15
to 30 of early weaning and the second dose in adulthood
(120 days) prior to food consumption. The dose applied is
suitable for obtaining the pharmacological effects, which
is to decrease consumption.
Body weight
The body weight of each rat was measured on a digital
electronic scale (Marte XL 500, class II, with 500 g
maximum capacity and lowest division of 0.001 g, São
Paulo, Brazil). The weighing was done on the days of
256 L. C. do Amaral Almeida et al. / Neuroscience 463 (2021) 254–263
life of 1, 7, 14, and 21, as well as on days 30, 60, 90, and
120 (D30, n = 08; D15, n = 08; D15 + NTX, n = 08).
Tail suspension test
The tail suspension test was performed at 60 days of age
(D30, n = 08; D15, n = 08; D15 + NTX, n = 08). In this
test, the animal is suspended by the tail, using a metallic
apparatus elevated at a height of 1.5 m from the floor.
This hemodynamic stress is one of being hung in a
condition that cannot be circumvented (Cryan et al.,
2005). The time the animal spends in an immobile posture
surrendering to a no-escape mode in an aversive situation
is recorded. Two minutes prior to the test, the distal por-
tion of the animal’s tail was wrapped with adhesive foam
(Reston Ò, São Paulo, Brazil), to prevent skin lesions.
Each animal remained suspended for 6 min, and the
immobility time, in seconds, was recorded.
Eating behavior tests
Eating palatable food involves eating for pleasure and not
to satisfy an energy need. This pleasure-motivated
consumption is a striking characteristic of hedonic
eating behavior, supported by three ‘pillars’: ‘Wanting’,
‘Liking’, and ‘Learning’ (Berridge et al., 2009). In the pre-
sent study, specific tests were done to assess each of
these pillars. The Runway Task incentive was performed
to assess the ‘Wanting’ and ‘Learning’. The taste reactiv-
ity test was done to evaluate the ‘Liking’. Both tests were
conducted prior to food consumption (at 120 days of age)
as they clearly indicated whether early weaning induced
alterations in these three pillars, which would directly
affect the food intake.
Runway Task
At 60 days of age, the animals were subjected to the
Runway Task to assess the ‘Learning’ and ‘Wanting’
standards after 4 h of food deprivation (D30, n = 07;
D15, n = 08; D15 + NTX, n = 08) (Peciña et al., 2003;
Silveira et al., 2010; da Silva et al., 2016). The structure
of the apparatus included a corridor (polypropylene,
140  14  30 cm) with two boxes, one initial and another
target (acrylic 19  14  30 cm), placed at opposite ends.
The tests involved 11 sessions on alternate days
(22 days), each of which lasted for 5 min. In the first three
sessions, the rats were placed directly in the target box
(with the doors closed) for 5 min, with free access to the
reward (Chocolate Cookies from BauduccoÒ, São Paulo,
Brazil). The animals adapted to eating the Cookies (Bau-
duccoÒ, São Paulo, Brazil) during the first three sessions.
In the fourth training session, the initial box was placed at
15 cm distance from the target box. When the animal was
in this box for 30 s, with the door closed, a guillotine type
door was raised, and the animal continued to move on,
through the running center. If the animal failed to leave
the initial box within 3 min, the researcher would gently
push it to the target box. In the fifth session, the initial
box was placed at 30 cm distance from the target box.
In the sixth session, the distance was increased to
60 cm and in the seventh session, it was 75 cm. In the
eighth and ninth sessions, the distance was further
increased to 90 and 120 cm, respectively. In sessions
10 and 11, the distance was maintained at 140 cm. In
each case, a record was kept of the distance covered
and the time taken, in centimeters and seconds respec-
tively, by the animal to leave the initial box and reach
the reward in the target box. The speed of the travel to
the food reward was noted in centimeters/second (cm/s).
Taste reactivity test
The taste reactivity test evaluates the ‘Liking’ from the
reaction time against different substances (Grill and
Norgren, 1978; da Silva et al., 2016). At 90 days of life,
the animals were placed individually in an arena (25 cm
height  25 cm front  20 cm bottom), that had a trans-
parent floor and walls (D30, n = 08; D15, n = 08; D15
+ NTX, n = 08). One milliliter (1 ml) of sucrose solution
at 30% or quinine solution (C20H24N2O2
HCl
2H2O,
SigmaÒ) was offered at 0.3 M, in the left corner of the
arena and directly on the floor. The animals were placed
in the test arena for 2.5 min prior to the test. In each trial,
using either sucrose or quinine, the animals were permit-
ted to ingest the solutions for 2.5 min. During this time, the
time the animal spent drinking each solution was
recorded, for subsequent analysis. The video camera
(Sony Handycam DCR-DVD650, with night shot function,
Tokyo, Japan) was positioned under the transparent floor
to capture the precise moment when the animals were
ingesting the solutions. The analyses between one type
of solution and the other had a minimum interval of 48 h.
Palatable food intake
At 120 days of age, the animals were submitted to a one-
hour evaluation, for three consecutive days, to calculate
the average consumption of palatable food (Chocolate
Cookies from BauduccoÒ, São Paulo, Brazil) as shown
in Table 1 (D30, n = 09; D15, n = 08; D15 + NTX,
n = 08). It always occurred at the same time,
established between 11:00 am and 12:00 pm. Food
intake was estimated as the difference between the
amount offered and the quantity rejected, after 1 h. The
weights were measured using a digital electronic scale
(Marte XL 500, class II, São Paulo, Brazil), maximum
capacity 500 g, (lowest division 0.001 g).
Table 1. Nutritional composition of the Chocolate Cookie
Nutrition facts (portion size: 30 g)
Calories 141 kcal (592 Kj)
Carbohydrate 19 g
Protein 2.1 g
Total fat: 6.3 g
 Saturated fat 3.0 g
 Monounsaturated fat 1.1 g
 Polyunsaturated fat 1.8 g
 Cholesterol 0.8 g
Dietary fiber 1.3 g
 L. C. do Amaral Almeida et al. / Neuroscience 463 (2021) 254–263 257

Palatable food intake after acute stress same time (application at 10:30 min and consumption
between 11 am and 12 pm). Food intake was the
At 120 days of age, the animals were subjected to a one-
difference between the quantity offered and amount
hour evaluation, for three consecutive days, after acute
rejected, after the one-hour period. The weights were
stress, to arrive at the average consumption of palatable
measured using a digital electronic scale, Marte XL 500,
food (Chocolate Cookies from BauduccoÒ, São Paulo,
class II, (São Paulo, Brazil) maximum capacity 500 g
Brazil), (D30, n = 09; D15, n = 08; D15 + NTX,
(lowest division 0.001 g).
n = 10). Feeding stress included the introduction of a
palatable diet in a container that prevented the animal
from feeding. Thus, the fasting animal could only obtain Statistical analysis
visual and olfactory information regarding the food for
All the data were analyzed for normality of distribution and
20 min. The experiment was always conducted at the
equality of variances employing the Shapiro-Wilk test and
same time (acute stress between 10:40 and 11 am, and
the Brown-Forsythe test, respectively, which revealed
food intake between 11 am and 12 pm). Food intake
normal distribution and equal variances of the data. As
was the difference between the quantity offered and
the data presented equal distribution, we performed the
amount rejected, after a one-hour evaluation. The
fixed-model ANOVAs and corrected P values for
weights were measured using a digital electronic scale,
multiple comparisons using statistical hypothesis testing
Marte XL 500, class II, (São Paulo, Brazil), maximum
and presented the multiplicity P values for multiple
capacity 500 g (lowest division 0.001 g).
comparisons. Additionally, as we used a fixed effect
model, the data presented here only applies to the
Standard diet intake after 8 h of food deprivation sampled animals and cannot be extrapolated to the
population of animals. The ordinary one-way ANOVA
At 120 days of age, the animals were assessed for a
was performed for the analyses of palatable food intake,
standard diet food intake (PurinaÒ, São Paulo, Brazil),
palatable food intake after acute stress, standard food
for one hour, following an eight-hour period of food
intake after 8 h of food deprivation, tail suspension test,
deprivation (D30, n = 09; D15, n = 08; D15 + NTX,
intergroup palatable food intake after acute dose of
n = 08). The experiment was always performed at the
naltrexone and taste reactivity test, followed by the
same time (fasting from 7 am to 3 pm and consumption
Bonferroni’s multiple comparison test, with a single
from 3 pm to 4 pm). Food intake was the difference
pooled variance. On the other hand, a full model
between the quantity offered and amount rejected, after
ordinary two-way ANOVA was performed for intragroup
one hour. A digital electronic scale, Marte XL 500, class
palatable food intake after an acute dose of naltrexone,
II, maximum capacity 500 g (lowest division 0.001 g),
body weight and the Runway Task incentive. For the
was used (São Paulo, Brazil). The fasting period was
intragroup palatable food intake after the acute dose of
commenced at the beginning of the dark phase, when
naltrexone, the main factors included (1) weaning age
the animal normally ingests the greatest quantity of its
+ NTX treatment and (2) acute dose of NTX or saline.
needs, approximately 80% (Strubbe et al., 1986;
For the body weight, the main factors were (1) weaning
Rowland, 2007). The 8-hour period was selected for the
age + NTX treatment and (2) age. For the Runway
reasons listed: (1) because the animal feeds more in the
Task incentive, the main factors included (1) weaning
dark and, therefore, it is more aggravated when it is
age + NTX treatment and (2) sessions. After the
deprived of food in the dark; (2) because after 6 h the rats’
ordinary two-way ANOVA was done, the Bonferroni’s
stomachs are already empty, so no differences are evi-
multiple comparison test was performed, with individual
dent between fasting for 6 or 18 h, in terms of intestinal
variances computed for each comparison. Data were
emptying (Vermeulen et al., 1997); (3) because, after a
presented as ‘box-and-whiskers’, where the ‘box’
3-hour period, metabolic changes are already present to
depicts the median and the 25th and 75th quartiles and
indicate that the animal has fasted (Palou et al., 1981)
the ‘whisker’ indicates the 5th and 95th percentiles. The
and (4) because a sudden and unexpected removal,
results were considered significant when p < 0.05. The
especially of the animal’s basic needs, like food, can be
software GraphPad Prism v9 was employed, for
particularly distressing and can raise the corticosterone
carrying out the analyses.
levels in several vertebrates (Wingfield and Kitaysky,
2002).
RESULTS
Palatable food intake after an acute dose of Body weight
naltrexone
The two-way ANOVA revealed group effect
At 120 days of age, the animals were evaluated for (F2, 84 = 7.651, p = 0.0009) and effect of age
palatable food intake (Chocolate Cookies from (F3, 84 = 3469, p  0.0001) for body weight during the
BauduccoÒ, São Paulo, Brazil), for one hour, after an pre-weaning ages, but no interaction between both the
acute dose of saline solution (3 mg/kg) (D30, n = 08; factors (F6, 84 = 0.1245, p = 0.9931). During the post-
D15, n = 08; D15 + NTX, n = 08). One week later, the weaning period, the two-way ANOVA demonstrated the
same process was done, with an acute administration of effect of age (F3, 84 = 449.2, p  0.0001), no effect of
Naltrexone (3 mg/kg), an inhibitor of the opioidergic group (F2, 84 = 0.03928, p = 0.9615) and no interaction
receptors. The experiment was always performed at the between both factors (F6, 84 = 1.019, p = 0.4185). The
258 L. C. do Amaral Almeida et al. / Neuroscience 463 (2021) 254–263
Bonferroni post-hoc test revealed no differences among
the groups at the pre- or post-weaning ages.
Tail suspension test
Regarding the tail suspension test, the one-way ANOVA
showed differences between the groups (F2, 21 = 39.28,
p  0.0001), while the Bonferroni post-hoc test showed
increased immobility time in D15 (p  0.0001) and D15
+ NTX (p  0.0001) when compared to D30 (Fig. 1).
Runway Task
The two-way ANOVA performed for the Runway Task
incentive test showed significant effects for the groups
(F2, 160 = 4.599, p = 0.0114) and sessions (F7,
160 = 135.2, p  0.0001), but no interactions were
evident between both factors (F14, 160 = 0.9098,
p = 0.5495). On the other hand, the Bonferroni post-
hoc test revealed decreased speed in D15 (p = 0.0143)
during session 10 and decreased speed in D15
(p = 0.0313) and D15 + NTX (p = 0.0372) during
session 11, both in comparison to D30 (Fig. 2).
Taste reactivity test
To estimate the taste reactivity test for sucrose, the one-
way ANOVA presented significant differences among the
groups (F2, 21 = 28.94, p  0.0001). The Bonferroni post-
hoc test showed decreased reactivity to sucrose in D15
(p  0.0001), when compared to D30, but increased
reactivity to sucrose in D15 + NTX (p  0.0001), when
compared to D15 (Fig. 3A). On the other hand, to
evaluate the taste reactivity to quinine, the one-way
Fig. 1. Effects of early weaning and post-natal treatment with
Naltrexone on depressive-like behavior. Rats were weaned naturally
(D30) or early (D15) and the rats weaned early were either submitted
or not to the naltrexone treatment, from D15 to D30. At D 60, the tail
suspension test was performed to assess the depressive-like
behavior, evaluated using the immobility time (n = 08 per group).
Data are presented as ‘box-and-whiskers’, where the ‘box’ depicts the
median and the 25th and 75th quartiles and the ‘whisker’ shows the
5th and 95th percentiles, ****p  0.0001. (h) D30, ( ) D15, (jh)
D15 + NTX.
ANOVA also presented significant differences among
the groups (F2, 21 = 8.79, p = 0.0017). The Bonferroni
post-hoc test showed decreased reactivity to quinine in
D15 + NTX (p = 0.0012) when compared to the D30
(Fig. 3B).
Food intake
For the various analyses of food intake, the one-way
ANOVA highlighted significant differences among the
groups for palatable food intake (F2, 22 = 6.56,
p = 0.0058) (Fig. 4A) and standard food consumption
after an 8-hour food deprivation period (F2, 22 = 6.101,
p = 0.0078) (Fig. 4C); however, no differences were
evident among the groups for palatable food intake, post
acute food stress (F2, 24 = 1.421, p = 0.2610). The
Bonferroni post-hoc test showed increased palatable
food intake in D15 (p = 0.0157), when compared to
D30, and decreased palatable food intake in D15
+ NTX (p = 0.0123), when compared to D15 (Fig. 4A).
Further, the post-hoc analyses demonstrated increased
standard food intake after an 8-hour period of food
deprivation in D15 + NTX (p = 0.0065), when
compared to D30 (Fig. 4B).
Palatable food intake after an acute dose of
naltrexone
The one-way ANOVA demonstrated significant
differences among the groups when submitted to the
NTX acute treatment (F2, 21 = 22.18, p  0.0001). The
Bonferroni post-hoc test showed increased food
consumption in response to the NTX treatment in both
the D15 + NTX when compared to the D30
(p  0.0001) and D15 (p = 0.0017) (Fig. 5A). For the
intra-group analyses of palatable food intake in
response to NTX or saline, the two-way ANOVA
revealed the effects of drug treatment (F1, 42 = 39.05,
p  0.0001) and groups (F2,42 = 15.47, p  0.0001), but
no interaction between both the factors (F2, 42 = 2.450,
p = 0.0985) (Fig. 5B). The post-hoc analyses showed
decreased palatable food intake in the D30 + NTX
(p  0.001) and D15 + NTX (p = 0.0075), when
compared to the D30 + Sal and D15 + Sal,
respectively (Fig. 5B). Further, (D15 + NTX) + NTX
revealed increased food intake when compared to the
D15 + NTX (p = 0.0261) and D30 + NTX (p  0.0001)
(Fig. 5B).
DISCUSSION
The present study demonstrated the effects of early
weaning at 15 days of age in Wistar rats on the eating
behavior observed during adulthood and the part played
by the opioid system in the eating behavior adaptations
seen in response to early weaning. Early weaning alone
was linked to depressive-like behavior, shorter sucrose
exposure time, and higher consumption of a palatable
diet. Early weaning, with the drug naltrexone, was
related to depressive-like behavior, and shorter time of
exposure to quinine. Other results included the
normalization of the palatable diet intake, normalization
 L. C. do Amaral Almeida et al. / Neuroscience 463 (2021) 254–263 259

the social protection provided by

the mother. Several studies have
shown that early weaning implies
several alterations in the neurobe-
havioral, hormonal and ontological
patterns of mammals (Ito et al.,
2006; Kikusui et al., 2007;
Townsend and Pitchford, 2012).
Thus, the changes resulting from
early weaning, as shown here,
depend on the deprivation of both
nutritional sources and social fac-
tors, in which the mother exerts a
primary influence, early in life.
From the tail suspension test it
is evident that early weaning is an
early life stress. Stress is a causal
factor for the development of
anxiety and depression. Both
disorders of emotional behavior,
also termed ‘‘stress-related
disorders” occur due to an
inappropriate adaptation to stress
(Bali et al., 2015). In the present
study, the weaned animals spent
more time staying immobile while
Fig. 2. Effects of early weaning and post-natal treatment with Naltrexone on seeking for food reward. suspended by the tail. Immobility
Rats were weaned naturally (D30) or early (D15) and the rats weaned early were submitted or not to
can be indicative of the absence
the naltrexone treatment, from D15 to D30. At D 60, D60 the runaway incentive task was performed to
access the seeking for food reward (D30, n = 07 per group; D15 and D15 + NTX, n = 08 per group). of escape behavior and the expres-
Data are presented as ‘box-and-whiskers’, where the ‘box’ depicts the median and the 25th and 75th sion of a depressive-like behavior
quartiles and the ‘whisker’ shows the 5th and 95th percentiles, *p  0.05. (h) D30, ( ) D15, (jh) (Can et al., 2011). It is also note-
D15 + NTX. worthy that until the tail suspension
test, performed at 60 days of life,
of reactivity to sucrose, and increase in the intake of the the animals were maintained under
palatable diet after the administration of naltrexone, in standard facility conditions. This reveals that the
later life. depressive-like behavior, observed during the tail suspen-
Early weaning is a sudden break in the maternal sion test, is induced by early life stress, performed
presence experienced by the litter. Maternal influences 45 days prior. Thus, these results can imply that early
arise from two main perspectives, namely, nutritional weaning is an early life stress and that the depressive-
and physical contact between the dam and pups. Thus, like behavior is a stress-related disorder.
early weaning deprives the litter of these significant In the Runway Task, the early weaning decreased the
influences, which are vital for proper development. speed with which the animals sought the food reward,
Under standard conditions, laboratory rats are which can be interpreted as a minor or lower motivation
exclusively dependent on the mother for food acquisition (‘Wanting’) to obtain a food reward. However, this lower
during the first two weeks of lactation, and the dam motivation was evident only in one session (session 10),
devotes upwards of 12 h of intense, daily physical of the eleven of which the test is a part and, therefore, a
contact, until the weaning period, approximately transitory effect. Thus, we can propose that this
between days 23–33 postpartum (Plaut and Davis, transient effect is inadequate to state that animals
1972; Cramer et al., 1990). Besides this dependence for weaned earlier are less motivated by the food reward. In
food acquisition, the physical contact between mother this test, sessions 7–9 are learning sessions. The early
and offspring plays a crucial role in conveying information weaned group exhibited no deficit in learning the task,
from the environment to the litter. Important information which is revealed by the similar speed between the
regarding the environment in which the nursing mother groups in the learning curve. Therefore, we suggest
is placed is passed on to the litter through physical con- that, also in terms of ‘Learning’, no loss can be
tact, which is one of the main sensory stimuli used to attributed to early weaning.
transfer this data to the offspring (Sullivan and Holman, In the taste reactivity test, early weaning reduced the
2010; Barrett and Fleming, 2011). Early weaning, there- time the animals spent in consuming the sucrose
fore, can be understood as a break in the relationship solution. This behavior can be indicative of a diminished
between the nursing mother and her offspring, which reaction to pleasure. The amount of sucrose offered is
includes the cessation of breastfeeding and the physical not intended to assess the quantity of the solution
separation of the offspring, involving the disruption of consumed, which is merely a single drop, but rather the
260 L. C. do Amaral Almeida et al. / Neuroscience 463 (2021) 254–263
Fig. 3. Effects of early weaning and post-natal treatment with
Naltrexone on the reactivity to pleasant or aversive taste. The rats
were weaned naturally (D30) or early (D15) and the rats weaned early
were submitted or not to the naltrexone treatment, from D15 to D30.
At D90, the taste reactivity test was performed to evaluate the
reactivity to sucrose or quinine (n = 08 per group). Data are
presented as ‘box-and-whiskers’, where the ‘box’ depicts the median
and the 25th and 75th quartiles and the ‘whisker’ shows the 5th and
95th percentiles, **p  0.01, ****p  0.0001. (h) D30, ( ) D15, (jh)
D15 + NTX.
time of exposure to that drop. The sucrose solution is a
pleasurable food substance for rats, and therefore, the
decrease in the time of exposure to this solution can
imply lower ability to obtain pleasure through consuming
it. It is interesting to note that diminished pleasure
reactions are recompensed by increasing the energy
consumption to achieve the food reward standards
(Bellisle et al., 2012). This pattern was also observed in
the present study, as the weaned animals ingested a
higher quantity of palatable food. A decreased feeling of
pleasure could initiate a higher threshold of the perception
of food pleasure, resulting in a higher intake of the palat-
able diet. Studies demonstrate that those individuals sub-
jected to food injuries and/or the deficit in social protection
and maturation of the sensory system provided by the
mother are more likely to consume palatable foods in
Fig. 4. Effects of early weaning and post-natal treatment with
Naltrexone on the food intake parameters. The rats were weaned
naturally (D30) or early (D15) and the rats weaned early were
submitted or not to the naltrexone treatment, from D15 to D30. At
D120, the rats were submitted to the tests of palatable food intake
(n = 08–09 per group) (A) and standard food intake in response to an
8-hour period of food deprivation (n = 08–09 per group) (B). Data are
presented as ‘box-and-whiskers’, where the ‘box’ depicts the median
and the 25th and 75th quartiles and the ‘whisker’ shows the 5th and
95th percentiles, *p < 0.05, **p < 0.01. (h) D30, ( ) D15, (jh)
D15 + NTX.
adulthood (dos Santos Oliveira et al., 2011; da Silva
et al., 2014; Rocha et al., 2014). Thus, the higher intake
of the palatable diet may be suggestive of a lower capac-
ity of the animals weaned earlier to perceive the pleasure
of food, causing them to compensate by increasing their
intake.
When palatable food consumption followed the acute
stress, this effect disappeared, which can be one of
many effects of acute stress on food intake. These
animals, under baseline conditions, demonstrated an
increase or a higher quantity of consuming a palatable
diet; however, in a stressful situation, the body inhibits
the food intake to encourage the development of
appropriate behaviors to cope with the stressor (Yau
and Potenza, 2013). Thus, the higher food consumption,
evident in the animals weaned earlier and without stress,
may have been inhibited by the application of a stressor
prior to this intake, resulting in an absence of any differ-
 L. C. do Amaral Almeida et al. / Neuroscience 463 (2021) 254–263
Fig. 5. Effects of early weaning and post-natal treatment with
Naltrexone on palatable food intake in response to an acute dose
of naltrexone. The rats were weaned naturally (D30) or early (D15)
and the rats weaned early were submitted or not to the treatment with
naltrexone, from D15 to D30. At D120, the animals were submitted to
an acute injection of saline solution or naltrexone (n = 08 per group),
and the data were analyzed as intergroup (A) and intragroup (B).
Data are presented as ‘box-and-whiskers’, where the ‘box’ depicts the
median and the 25th and 75th quartiles and the ‘whisker’ shows the
5th and 95th percentiles, *p  0.05, **p  0.01, ***p  0.001,
****p  0.0001. (h) D30, ( ) D15, (jh) D15 + NTX.
ence when compared to the control group. This result
would also explain the nonappearance of any difference
regarding the standard diet consumed, after the 8-hour
food deprivation stress.
Another group was subjected to early weaning with
the concomitant administration of naltrexone, an
opioidergic receptor antagonist. The blockade of the
opioidergic system during early weaning has revealed
the vital part played by this system in the behavioral
adjustments that are due to a stressful event earlier in
life. The D15 + NTX group normalized the higher
palatable food intake lowering to a control group similar
261
amount. This result suggests that the opioid system
contributes to the higher palatable food intake once the
naltrexone eliminated this coping behavior. Evidence
suggests beta-endorphin, an endogenous opioid, plays
a critical role in the coping behavior. The activation of
the opioidergic afferences tends to suppress the actions
of the Corticotrophin Releasing Hormone (CRH) and
contributes to the restoration of the normal pre-stress
levels (Bali et al., 2015). Thus, these data suggest that
the blockade of the opioidergic system during early wean-
ing impedes the development of behavioral adjustments
in adulthood, such as a higher intake of the palatable diet.
It is noteworthy that the time of exposure to sucrose was
also normalized, suggesting that a possible normalization
in the perception of pleasure is reflected in the consump-
tion of the palatable diet, enabling its normalization. Fur-
thermore, the animals weaned earlier with the
naltrexone administration were more sensitive to the aver-
sive substance quinine. This result suggests a possible
link between the ability to restore the perception of plea-
sure and the ability to perceive aversive substances.
This study also assessed whether individuals exposed
to an opioidergic system blockade earlier in life retained
the functions of this system intact in adulthood. To
accomplish this, at 120 days of life, the animals were
given naltrexone prior to offering them palatable food.
The group weaned earlier decreased the intake level, as
did the control group. On the other hand, the group
weaned earlier with naltrexone did not lower the intake
of palatable food. Opioids contribute to the consumption
of palatable foods. In laboratory animals, injections of an
exogenous opioid, like morphine raise the carbohydrate
consumption in mice that prefer carbohydrates, while in
animals that prefer fat, they increase the intake of high-
fat diets (Gosnell et al., 1990). Similar to the findings of
this study, the infusion of a Mu-opioid receptor agonist
(MOR) directly into the nucleus accumbens stimulated
the ingestion of a high-fat diet in animals showing a pref-
erence for fat (Naleid et al., 2007). Thus, the lower intake
of palatable food, demonstrated in the present study, can
be induced by the opioidergic receptor blockade, pro-
moted by the drug. The early weaning group with naltrex-
one showed no decrease in the intake of palatable food
after a new administration of the drug in adulthood, sug-
gesting that the precocious administration of naltrexone
in a stressful situation permanently alters the working of
this system. Besides this, although the early weaning
group without the drug administration lowered the intake
of the palatable diet, this decrease was not as efficient,
thereby causing this intake to be even more than that of
the control group with naltrexone. This finding may sug-
gest that the stress of early weaning itself is already ade-
quate to induce some disruption in the proper functioning
of the opioid system in adulthood.
The opioidergic system and its receptors are highly
significant in modulating food consumption (Fields and
Margolis, 2015). Opioidergic antagonists, such as nalox-
one and naltrexone lower the food intake, while agonists,
such as synthetic morphines and enkephalins, raise the
food intake level (Statnick et al., 2003; Ferenczi et al.,
2010; Mason et al., 2015). Furthermore, the opioidergic
262 L. C. do Amaral Almeida et al. / Neuroscience 463 (2021) 254–263
receptors are widely distributed in the vital regions of the
central nervous system responsible for the control of food
intake and energy homeostasis (Bodnar and Klein, 2004).
In fact, b-endorphin, derived from the anorexigenic neu-
ropeptide pro-opiomelanocortin (POMC), expressed in
the cells of the Hypothalamic Arcuate Nucleus and in
the brain stem, acts on eating behavior via the MOR opi-
oidic receptor (Bodnar and Klein, 2004). The POMC neu-
rons have MOR and respond to selective agonists of this
receptor with hyperpolarization, inhibiting the release of
POMC and, consequently, its anorectic action (Pennock
and Hentges, 2011). An antagonist of the POMC deriva-
tive receptors is the orexigenic neuropeptide AgRP
(Agouti Related Protein). The AgRP orexigenic effect is
inhibited after antagonism of the opioidergic system with
naloxone (Hagan et al., 2001). Another crucial orexigenic
peptide intrinsically linked to the opioids is orexin. When
orexin is administered in the hypothalamus it increases
the enkephalin gene expression in the Ventral Tegmental
Area and in the Amygdala (Karatayev et al., 2009). Fur-
thermore, this orexigenic action of orexin A is blocked
by naltrexone (Sweet et al., 2004), which implies that
the effects of early weaning in the long term may be
caused by the modulatory action of the opioids in these
vital neuronal populations and brain regions related to
eating behaviors.
The present study includes a few limitations. One
such limitation is the inability to measure the
corticosterone during early weaning. Other studies,
however, have demonstrated that this hormone is
elevated and its levels can remain unchanged, until 48 h
after (Kikusui et al., 2009; Ghizoni et al., 2014). Thus, it
can be assumed that, similar to the demonstration by
other studies, and employing the same protocol, early
weaning is an early life stress. Besides, we performed
the tail suspension test, which revealed depressive-like
behavior in the animals weaned early. This outcome is
indicative of a stress-related disorder. Another limitation
in this study was the non-use of females. This is because
it is well known that males and females reveal different
results and the data shown here cannot be generalized
for both sexes. Finally, we were unable to perform molec-
ular and/or morphological analyses, such as c-fos saining,
to confirm the changes in the components of the opioider-
gic system in specific brain areas, which gives us per-
spectives for future studies to fill this gap. However,
pharmacological data presented here strongly supports
the association of the opioidergic system with the behav-
ioral data observed in the context of the early weaning.
On the whole, the findings of the present study
indicate that early weaning is an early life stress. It has
permanent consequences in later life, causing
alterations in emotional behavior, favoring the
appearance of stress-related disorders, and eating
behaviors, facilitating the development of
‘coping’behaviors. These ‘coping’ behaviors can trigger
the development of some disturbances, like obesity.
Furthermore, the results indicate that the opioidergic
system is indispensable for the adjustments that are
evident in the eating behaviors during adulthood, after a
stressful event in early life, such as early weaning.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
ACKNOWLEDGEMENTS
This work was supported by the Foundation for Science
and Technology of the State of Pernambuco (FACEPE),
the National Council of Scientific and Technological
Development (CNPq) (Universal 421752/2016-5). This
study was also financed, in part, by the Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior – Brasil
(CAPES) – Finance code 001. The authors also thank
the Neuroplasticity and Behavior research group.
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