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Psychoneuroendocrinology. Author manuscript; available in PMC 2022 July 01.
Published in final edited form as:
Psychoneuroendocrinology. 2021 July ; 129: 105249. doi:10.1016/j.psyneuen.2021.105249.
Gonadal steroid hormone receptors in the medial amygdala
contribute to experience-dependent changes in stress
vulnerability
Matthew A. Coopera,*, Catherine T. Clinardb, Brooke N. Dulkac, J. Alex Grizzelld, Annie L.
Loewena, Ashley V. Campbella, Samuel G. Adlera
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aDepartment
of Psychology, University of Tennessee, Knoxville, TN, United States
bDepartment
of Social Sciences, Dalton State College, Dalton, GA, United States
cDepartment
of Psychology, University of Wisconsin, Milwaukee, WI, United States
dDepartment
of Psychology and Neuroscience, University of Colorado, Boulder, CO, United
States
Abstract
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Social experience can generate neural plasticity that changes how individuals respond to stress.
Winning aggressive encounters alters how animals respond to future challenges and leads to
increased plasma testosterone concentrations and androgen receptor (AR) expression in the social
behavior neural network. In this project, our aim was to identify neuroendocrine mechanisms that
account for changes in stress-related behavior following the establishment of dominance
relationships over a two-week period. We used a Syrian hamster model in which acute social
defeat stress increases anxiety-like responses in a conditioned defeat test in males and in a social
avoidance test in females. First, we administered flutamide, an AR antagonist, via intraperitoneal
injections daily during the establishment of dominance relationships in male hamsters. We found
that pharmacological blockade of AR prevented a reduction in conditioned defeat in dominant
males and blocked an upregulation of AR in the posterior dorsal medial amygdala (MePD) and
posterior ventral medial amygdala (MePV), but not in the ventral lateral septum. Next, we
administered flutamide into the posterior aspects of the medial amygdala (MeP) prior to acute
social defeat stress or prior to conditioned defeat testing in males. We found that pharmacological
blockade of AR in the MeP prior to social defeat, but not prior to testing, increased the
conditioned defeat response in dominant males and did not alter behavior in subordinates. Finally,
we developed a procedure to establish dominance relationships in female hamsters and
investigated status-dependent changes in plasma steroid hormone concentrations, estrogen receptor
alpha (ERα) immunoreactivity, and defeat-induced social avoidance. We found that dominant
female hamsters showed reduced social avoidance regardless of social defeat exposure as well as
increased ERα expression in the MePD, but no status-dependent changes in the concentration of
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*
Corresponding author Department of Psychology, University of Tennessee Knoxville, Knoxville, TN 37996, USA,
mcoope10@utk.edu, Phone: +1-865-974-8458.
Declarations of Interest: none
Appendix A. Supplementary materials
Supplementary data related to this article can be found in the online version.
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plasma steroid hormones. Overall, these findings suggest that achieving and maintaining stable
social dominance leads to sex-specific neural plasticity in the MeP that underlies status-dependent
changes in stress vulnerability.
Keywords
social dominance; aggression; stress; social defeat; medial amygdala; androgen receptors; estrogen
receptors
1.
Introduction
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There is considerable variation in how humans and other animals respond to stress.
Traumatic stress is a clear and well-known risk factor for the development of post-traumatic
stress disorder (PTSD), yet the majority of people exposed to traumatic stress do not develop
stress-related mental illnesses and are considered resilient (Yehuda and LeDoux, 2007).
Understanding the neurobiological mechanisms underlying stress resilience should help
identify targets for novel treatments in stress vulnerable populations. While genetic
differences no doubt create variation in stress vulnerability, experience-dependent neural
plasticity also contributes to the emergence of resilient responses to stress. Several types of
experiences are known to generate neural plasticity that promotes proactive coping and
stress resilience (Baratta and Maier, 2019; Christianson and Greenwood, 2014; van Praag et
al., 2000), including winning competitive interactions and maintaining social dominance in a
stable hierarchy (Davis et al., 2009; Kohn et al., 2016; Sapolsky, 2005).
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The challenge hypothesis, which was originally developed studying birds and extends to a
wide variety of animals, predicts that testosterone levels rise and facilitate aggression during
social challenges that occur during territory formation, dominance disputes, and mate
competition (Ball and Balthazart, 2020; Hirschenhauser and Oliveira, 2006; Wingfield et al.,
1990). This body of research indicates that testosterone prepares individuals for future
competitive interactions. Accordingly, winners of aggressive encounters exhibit a rapid rise
in circulating testosterone compared to losers (Apfelbeck et al., 2011; Oyegbile and Marler,
2005; Yang and Wilczynski, 2002). Furthermore, winning aggressive encounters increases
the probability of winning future contests. This phenomenon is called the winner effect and
is critically dependent on contingent activity of gonadal steroids during and/or following
victory (Hsu et al., 2006; Oliveira et al., 2009). The neuroendocrine mechanisms regulating
the winner effect have been well-delineated in the male California mouse (Marler and
Trainor, 2020), which show a robust winner effect if at least three victorious territorial
disputes are followed by a testosterone pulse about 45 minutes later (Fuxjager et al., 2011b).
Interestingly, male white-footed mice do not exhibit such a surge in testosterone following
victory and thus fail to show a winner effect, although this phenomenon is robustly observed
if animals are administered post-victory testosterone (Fuxjager et al., 2011a). Male
California mice also show an increase in androgen receptor (AR) expression in several
regions of the social behavior network, though this effect is found only after winning
encounters in their home territory and not when the arena is unfamiliar (Fuxjager et al.,
2010). Altogether, these findings indicate that winning aggressive encounters increases
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circulating testosterone concentrations and upregulates AR receptors, which likely prime
neural circuits for future competitive social interactions to increase the probability of future
victories.
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Consistent with the winner effect and in line with the challenge hypothesis, achieving high
social rank is characterized by a series of victories and is often associated with a rise in
plasma testosterone (Hardy et al., 2002; Rose et al., 1975; Williamson et al., 2017). In
addition to altering future agonistic behavior with familiar individuals, establishing social
dominance also modulates neuroendocrine and neuropeptide systems as well as neural
circuits that regulate other types of social behavior and responses to environmental
challenges. Rats that gain social dominance in a visible burrow system show increased
operant responses for food alongside elevations of orexin mRNA in the medial prefrontal
cortex (Davis et al., 2009). In a social group of mice, achieving high dominance rank is
associated with coordinated neural activity within a social behavior neural network
(Williamson et al., 2019a). Mice that achieve dominant social status after repeated
aggressive encounters exhibit reduced anxiety responses in an open field and less visceral fat
than subordinate mice (Bartolomucci et al., 2001; Bartolomucci et al., 2005). Further,
establishing social dominance in a tube test has been shown to reduce anxiety-like behavior
following chronic mild stress in female, but not male, mice (Karamihalev et al., 2020). In
contrast, social dominance increases vulnerability to chronic social defeat stress in male
mice and alters the concentration of energy-related metabolites in the nucleus accumbens
(Larrieu et al., 2017). Interestingly, beneficial effects of high social status have also been
demonstrated in humans. People with high-level leadership positions in the workplace
exhibit lower salivary cortisol and reduced anxiety on a trait anxiety inventory compared to
non-leaders (Sherman et al., 2012). Similarly, when hierarchies are stable, high social status
buffers stress reactivity and improves performance in a mock job interview (Knight and
Mehta, 2017). Altogether, these studies suggest that winning competitive interactions and
establishing high dominance rank does not simply alter future aggressive behavior, but also
leads to changes in neural circuits that regulate stress responsivity.
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While winning can generate androgen-dependent neural plasticity that regulates future social
behavior in males, the mechanisms underlying potential effects of winning in females
remain elusive. One possibility is that activation of estrogen receptor alpha (ERα) regulates
aggressive behavior and contributes to the effects of winning in females. In female darkeyed juncos, expression of AR, ERα, and aromatase each predict individual variation in
aggressive responses to territorial intrusions, but testosterone does not (Rosvall et al., 2012).
Similarly, in white-throated sparrows, the more aggressive, white-striped morph shows
greater ERα expression throughout the social behavioral neural network compared to the
less aggressive, tan-striped morph (Horton et al., 2014). However, the effects of ERα activity
on aggressive behavior in female rodents is mixed. In mice, systemic activation of ERα
restores aggressive behavior following ovariectomy (Clipperton-Allen et al., 2011), yet
global knockdown of ERα increases aggression in intact females (Ogawa et al., 1998).
Interestingly, selective knockdown of ERα in the posterior aspects of the medial amygdala
(MeP), specifically the posterior dorsal MeP (MePD), reduces anxiety in a light/dark
transition test and impairs performance on a social recognition task, but does not alter
aggressive behavior (Spiteri et al., 2010). Overall, the role of ERα in female-female
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aggression and other types of social behavior appears to depend on specific cell types and
neural circuits.
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In male Syrian hamsters, acute social defeat stress leads to elevated submissive and
defensive behavior and a loss of territorial aggression in a social interaction test with a freely
moving intruder, which is called the conditioned defeat response (Huhman et al., 2003).
However, after males that had previously established high dominance rank are acutely
defeated, they exhibit reduced submissive and defensive behavior at conditioned defeat
testing, which suggests that achieving social dominance promotes resistance to the effects of
social defeat (Morrison et al., 2014). Dominant males also show greater cFos
immunoreactivity in the posterior ventral MeP (MePV) following the stress of acute social
defeat (Morrison et al., 2014; Morrison et al., 2012) or physical restraint (Cooper et al.,
2017). In addition, dominant males show a pulse of testosterone 15 minutes after winning an
aggressive encounter and an increase in AR immunoreactivity within the MePD, as well as a
similar trend in the MePV, after maintaining their dominant status for two weeks (Clinard et
al., 2016).
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The aim of our current study was to determine whether status-dependent changes in AR
activation were essential for resistance to social defeat stress in dominant animals. In male
hamsters, we tested whether pharmacological blockade of AR during the establishment of
dominance relationships would prevent status-dependent changes in conditioned defeat.
Because the MeP plays a key role in the acquisition and expression of the conditioned defeat
response in males (Markham and Huhman, 2008), we tested whether pharmacological
blockade of AR within the MeP would increase conditioned defeat in dominants but not
subordinates. In addition, we developed a procedure for creating dominance relationships in
female hamsters to explore potential sex differences in this system. We tested whether
dominant females would show less defeat-induced social avoidance, increased steroid
hormone concentrations, and elevated expression of AR and ERα in the MeP compared to
their subordinate counterparts.
2.
2.1.
Methods
Subjects
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Subjects were male and female Syrian hamsters (Mesocricetus auratus) obtained from our
breeding colony that was originally derived from male and female hamsters from Charles
River Laboratories (Wilmington, MA). Subjects were young adults aged 9-10 weeks old
(130-170 g) at the start of the study. One week prior to an experiment, animals were housed
individually in polycarbonate cages (12 cm x 27 cm x 16 cm) with corncob bedding, cotton
nesting materials, and wire mesh tops. Because Syrian hamsters are highly territorial, lowranking animals can be wounded when housed in a social group, and adults thrive in the
laboratory when housed individually. In our breeding colony, juvenile animals are raised in
social groups with environmental enrichment, such as plastic shelters and paper cups. Food
and water were available ad libitum. Cages were not changed for one week prior to an
experiment to allow individuals to scent mark their territory. Subjects were handled daily
prior to an experiment to habituate them to the stress of human handling. Animals were
housed in a temperature controlled colony room (21 ± 2 °C) and kept on a 14:10 hr
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light:dark cycle to facilitate gonad development and aggressive behavior. All behavioral
protocols were performed during the first three hours of the dark phase of their light: dark
cycle. All procedures were approved by the University of Tennessee Institutional Animal
Care and Use Committee and are in accordance with ARRIVE guidelines and the National
Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.2.
Experimental Design
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Experiment 1 examined whether pharmacological blockade of AR during the 2-week
development of dominance relationships in male hamsters would prevent status-dependent
changes in the conditioned defeat response. We used the competitive non-steroidal AR
antagonist flutamide, which was dissolved in DMSO and diluted with sesame oil to reach a
final concentration of 5% DMSO (pH = 7.4). The primary active metabolite of flutamide is
2-hydroxyflutamide, and elimination half-lives for flutamide and 2-hydroxyflutamide are 1.9
hours and 0.9 hours in rat, respectively (Zuo et al., 2002). We treated animals with systemic
flutamide (15 mg/kg, s.c.) or vehicle injections one hour prior to each daily dominance
encounter for two weeks, and this dose was selected on the basis of previous research
(Nagypál and Wood, 2007). Dominant and subordinate animals within a dyad received
similar injections such that both animals received either daily flutamide injections or daily
vehicle injections. We also administered flutamide or vehicle injections to individually
housed animals that were not exposed to dominance encounters, termed no status (NS)
controls. After dominance relationships were established, animals were exposed to acute
social defeat stress and were tested 24 hours later for a conditioned defeat response (Fig. 1a).
Overall, this experiment had a 2 (drug) X 3 (dominance status) factorial design, and we used
7-10 animals per treatment condition.
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Experiment 2 focused on whether pharmacological blockade of AR in the MeP would
prevent status-dependent changes in the conditioned defeat response. Also, this study
addressed whether AR activity in the MeP during either social defeat stress or conditioned
defeat testing was necessary for status-dependent changes in behavior. We first implanted
guide cannula directed toward the MeP and then exposed animals to daily dominance
encounters for two weeks. To test the role of AR in the acquisition of conditioned defeat, we
injected flutamide (300 ng/500 nL, 5% DMSO in phosphate buffered saline) or vehicle two
hours prior to acute social defeat stress and tested animals for the conditioned defeat
response 24 hours later. To test the role of AR in the expression of conditioned defeat, we
exposed dominant and subordinate animals to social defeat stress and then, 24 hours later,
injected flutamide or vehicle two hours prior to conditioned defeat testing (Fig. 2a). We
selected the drug dose and the two-hour time interval between intracranial injection and
testing because of similar pharmacological approaches targeting AR in the dorsal
hippocampus and basolateral amygdala (Edinger and Frye, 2006; Naghdi et al., 2003). This
study had a 2 (drug) X 2 (dominance status) factorial design, and 8-10 animals were used
per treatment condition.
Because female hamsters show territorial aggression and establish dominance relationships
with one another (Grieb et al., 2021), in Experiment 3, we sought to determine whether
social dominance modulates stress-related behavior as well as gonadal steroid hormones and
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their receptors in female hamsters. To test whether hormone concentrations change during
the formation of dominance relationships, we collected blood plasma from female hamsters
15 minutes before and 15 minutes after their first dominance encounter. We also collected
blood samples from NS control animals at similar time points. Then, half of the animals
were exposed to acute social defeat stress, and the others were exposed to empty cages in a
no-defeat control procedure. All animals then received a social avoidance test 24 hours later
(Fig. 3a). We used a social avoidance test to assay defeat-induced changes in social behavior
instead of a conditioned defeat test because previous research indicates that female hamsters
do not show a reliable conditioned defeat response (Solomon et al., 2007). In addition,
females were defeated during the diestrus I phase of their estrous cycle and tested for social
avoidance in diestrus II. To determine whether dominance status alters expression of AR and
ERα in the MePD and MePV, we performed cardiac perfusions and collected brains 24
hours following social avoidance testing (i.e. proestrus). Thus, this experiment used a 2
(social defeat) X 3 (dominance status) factorial design, and plasma hormone data were
pooled across the social defeat condition because all blood collection occurred prior to social
defeat stress.
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2.3.
Dominance Encounters
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To establish dominance relationships in Experiments 1 and 2, males were weight-matched in
resident-intruder dyads and paired in daily social encounters for 14 consecutive days.
Animals maintained dominance relationships for 14 days because our previous research
indicates that status-dependent changes in the conditioned defeat response, as well as c-Fos
immunoreactivity in the MeP, requires two weeks of dominance encounters (Morrison et al.,
2014). Subjects were assigned randomly as a resident or intruder, and all social encounters
occurred in the resident’s home cage. Each encounter prior to the clear formation of a
dominance relationship was 10 minutes in duration and 5 minutes thereafter. We previously
determined that 10-minute encounters facilitate the clear differentiation of a winner and
loser and 5-minute encounters allow the maintenance of a dominance relationship with
minimal wounding of subordinates. Pairs that did not establish a stable dominance
relationship were excluded from the study (13 of 72 male dyads). Interestingly, residency
status did not alter the probability that male hamsters would become dominant because 32
dominants were intruders, and 27 dominants were residents, which is consistent with
previous studies (Dulka et al., 2020).
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In Experiment 3, we modified the procedure for establishing dominance relationships to
account for the estrous cycle. Female hamsters exhibit a remarkably consistent four-day
cycle that includes two days in diestrus, one day in proestrus, and one day in estrus. To
determine cycle phase, we monitored animals by gently restraining them and placing a
cotton swab against their vaginal area. The presence of a thin string of vaginal discharge on
a cotton swab indicated estrus (Wise, 1974). Because female hamsters exhibit less
aggression during estrus (Wise, 1974), we cycle-matched dyads and avoided testing them
during their estrus phase. Accordingly, dominance encounters occurred on diestrus I,
diestrus II, and proestrus and were repeated 4 times for 12 total encounters over a 16-day
period. Dyads that failed to establish a stable dominance relationship were excluded from
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analysis (8 of 28 female dyads). Residency status did not predict social dominance as 9
dominants were intruders, and 11 dominants were residents.
For all experiments, we digitally recorded daily dominance encounters and quantified the
behavior of subjects. To confirm that dominance relationships were robust and stable, we
quantified the total duration of the following categories of behavior: submissive/defensive
(flee, avoid, upright and side defensive postures, tail-up, stretch-attend, head flag);
aggressive (chase, attack including bite, upright and side offensive postures); non-agonistic
social (sniff, approach); and nonsocial (locomotion, grooming, nesting, feeding). To control
for dominance status, we used a NS treatment group in which animals received human
handling but were not exposed to daily dominance encounters.
2.4.
Social Defeat Stress
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Social defeat stress consisted of three 5-minute aggressive encounters at 5-minute intertrial
intervals. Encounters occurred in the home cage of a larger, same-sex animal with
experience winning fights. Although aggressors were pre-screened to reliability attack samesex intruders, variability existed in the latency of aggression. Consequently, the 5-minute
social defeat period did not begin until aggressors attacked and intruders submitted, which
usually occurred within the first 60 seconds of the first trial. To determine whether subjects
received similar amounts of aggression, we quantified the number of attacks and duration of
aggressive behavior. Animals with a wound extending beyond the epidermis and into the
dermis layer were treated and removed from the study (n = 2).
2.5.
Behavioral Testing
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Conditioned defeat testing involved a 5-minute social interaction in which a non-aggressive
intruder was placed in the subject’s home cage. Non-aggressive intruders were younger,
group-housed animals that displayed social and nonsocial behavior only and did not direct
agonistic behavior toward subjects. We quantified the total duration of the following
categories of behavior: submissive/defensive, aggressive, social, and non-social. We also
quantified the frequency of flees and attacks displayed by the subject.
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For social avoidance tests, animals were placed into a neutral arena (12 x 27 x 16 cm) in two
sequential 5-minute trials (trial 1, trial 2). In trial 1, an empty perforated box (7 x 14 x 7 cm)
was placed on one side of the arena and animals were allowed to freely explore. In trial 2, an
unfamiliar, same-sex, adult animal was placed inside the perforated box and the subject was
again allowed to freely explore. In both trials, the testing arena was divided into three zones:
the far zone (half of the arena not containing the target box), the interaction zone (area
within 3 cm of target box), and the near zone (half of the arena containing target box but
excluding interaction zone). The location of the subject was determined by the orientation of
its snout, which meant the subject occupied the interaction zone only when attending to the
target box. We quantified the duration of time the subject spent in each zone as well as the
frequency of flees, stretch attends, and flank marks. Data are presented as frequencies,
cumulative durations in each zone, or a ratio (trial 2/trial 1) of zone durations.
For all behavioral scoring, including dominance relationships, social defeats, conditioned
defeat testing, and social avoidance testing, researchers were blind to treatment conditions.
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Interrater reliability was established on a subset of videos by reaching 90% agreement on
relevant dependent variables. All testing sessions were digitally recorded, and the behavior
of subjects was quantified using Noldus Observer software (Noldus Information
Technology).
2.6.
Blood Collection
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We performed retro-orbital blood collection under 4% isoflurane anesthesia before and after
the first dominance encounter. Blood was collected in a rapid and counterbalanced fashion
for dominants and subordinates in a dyad, which resulted in a difference of no greater than 2
minutes. Blood samples were centrifuged at 4400xg for 15 minutes, and the plasma layer
was removed and stored at −80°C. Plasma samples were treated with an ether extraction
procedure and concentrations of testosterone, progesterone, and estradiol were determined
using commercial EIA kits according to the manufacturer’s protocol (Cayman Chemical).
Samples were run in duplicates or triplicates using 50μl per well. Inter-assay variability
between plates was 9.0% and intra-assay variability within a single plate was 5.6%.
2.7.
Stereotaxic Surgery and Histology
In Experiment 2, we anesthetized hamsters with isoflurane and bilaterally implanted 26gauge guide cannulae aimed at the MeP (0.2 mm posterior and 2.7 lateral to bregma, 3.2 mm
below dura). For microinjections, we used a 33-gauge injection needle that projected 4.5 mm
below the guide cannula for a final projection of 7.7 mm below dura. After surgery, dummy
stylets that projected 0.1 mm below the guide cannulae were inserted to maintain patency.
Animals were given 7 days to recover from surgery before beginning dominance encounters.
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Immediately following testing, animals were overdosed with isoflurane and infused with 200
nl of India ink into the MeP. Brains were removed, frozen on dry ice, and stored at −80° C.
Brains were sliced at 40 μm on a cryostat, and sections were stained with neutral red and
coverslipped. Sections were examined under a light microscope and injection sites were
plotted on images from a hamster stereotaxic atlas (Morin and Wood, 2001). Subjects with
injection sites within the boundaries of the MePD or MePV were included in analysis and
subjects with injection sites outside these regions were analyzed as anatomical controls (Fig.
2b).
2.8.
Immunohistochemistry and Cellular Quantification
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In Experiments 1 and 3, animals were anesthetized with isoflurane 24 hours after
conditioned defeat or social avoidance testing and transcardially perfused with 100ml of 0.1
M phosphate buffer (PB) followed by 100ml of 4% paraformaldehyde solution. Brains were
removed and soaked in 4% paraformaldehyde for 24 hours, followed by 0.1 M PB/30%
sucrose solution for 48 hours, and then were stored in cryoprotectant, all at 4°C. A
consecutive series of 40 μm coronal sections were sliced on a vibrating microtome, collected
into vials, and stored as free-floating sections in cryoprotectant at 4°C.
Sections were processed for AR immunohistochemistry according to a previously published
protocol (Chen et al., 2014). After three 10-minute washes in a phosphate buffered (PB)
gelatin Triton solution (PB-GT; 0.1% gelatin, 0.3% Triton X-100, in PB, pH 7.4) and a 15-
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minute incubation in 0.5% sodium borohydride in PB-GT, sections were exposed to 10%
goat serum in PB-GT for 1 hour to block non-specific binding. Then, sections were
incubated for 10 minutes in avidin blocking followed by 10 minutes in biotin blocking
solutions according to kit instructions (Vector Laboratories). Sections were then incubated
for 24 hours at 4 °C in 1% goat serum in PB-GT with rabbit monoclonal anti-AR antibody
(Abcam: ab52615, 1:1000). Tissues were then rinsed in PB-GT and incubated for 1 hour in
1% goat serum in PB-GT with biotinylated goat anti-rabbit secondary antibody (Vector
Laboratories, 1:500). Brain sections were then washed and incubated 1 hour in PB-GT with
an avidin–biotin complex (ABC Kit, Vector Laboratories) before a final PB-GT wash and
10-minute peroxidase reaction in a nickel-enhanced 3,3’-diaminobenzidine (DAB) solution
(Sigma Aldrich). Sections were then washed with distilled H2O, mounted onto slides,
dehydrated, cleared with citrisolv, and coverslipped using DPX mountant. All tissue for each
brain region was processed simultaneously.
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Sections were processed for ERα immunohistochemistry according to a previously
published protocol (Trainor et al., 2007). Sections were washed in PB solution, incubated for
10 minutes in 1% sodium borohydride in PB, followed by a 20-minute incubation in 20%
goat serum with 0.3% hydrogen peroxide in PB solution. Sections were incubated at 4°C in
rabbit anti- ERα antibody (EMD Millipore: 06-935, 1:25,000) in PB + 0.2% Triton with 1%
goat serum. Sections were then incubated for 60 minutes in biotinylated goat anti-rabbit
secondary antibody (1:200, Vector Laboratories) in PB-Triton. Sections were incubated in
avidin-biotin-complex (ABC Kit, Vector Laboratories) for 60 minutes, and the peroxidase
reaction was visualized using a 5-minute DAB and nickel incubation in PB. To validate the
primary antibodies used, we preincubated primary antibodies in either AR or ERα blocking
peptides and found an absence of immunostaining (data not shown).
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Photomicrographs were captured at 10X magnification using an Olympus BX51
microscope. The number of AR and ERα immuno-positive cells were quantified in the
MePV, MePD, and ventral lateral septum (vLS) using MCID Core image analysis software
(InterFocus Imaging). These brain regions were selected for quantification because they
showed status-dependent changes in AR immunoreactivity in a previous study (Clinard et
al., 2016). For each tissue section, we recorded background immunoreactivity in an
unstained portion of the image. We then defined immuno-positive cells as those that showed
staining 1.6X darker than the specific background immunoreactivity calculated for each
image. Cell counts were limited to the area within a defined clip region that was tailored to
the size of each brain region. The clip regions used for quantification were as follows (width
x height): 500 μm x 500 μm (vLS), and 870 μm x 660 μm (MePD and MePV). For each
brain region we quantified four to six sections per individual distributed evenly along a
rostral-caudal axis. We quantified immunoreactivity in a semi-automated fashion, and
software thresholds were calibrated to yield cell counts that were within 90% agreement of
manual quantification.
2.9.
Statistical Analysis
In Experiment 1, we performed two-way ANOVAs to investigate an interaction between
social status (3 levels) and drug treatment (2 levels) on behavior at conditioned defeat testing
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and the number of AR immunoreactive cells. We also used Pearson correlations to correlate
the number of AR immunoreactive cells with the duration of submissive and defensive
behavior at conditioned defeat testing. In Experiment 2, we performed two-way ANOVAs to
investigate an interaction between social status (2 levels) and drug treatment (2 levels) on
behavior at conditioned defeat testing. In Experiment 3, we performed two-way AVOVAs to
test for effects of social status (3 levels) and defeat (2 levels) on social avoidance measures
and the number of AR and ERα immunoreactive cells. We also performed repeated
measures two-way ANOVAs to investigate an interaction between social status (3 levels) and
time (2 levels) on hormone concentrations. For repeated measures ANOVAs, we used
Mauchly’s test of sphericity and found the assumption of sphericity was not violated. We
used t-tests for planned comparisons between two treatment groups, used Tukey post-hoc
tests as needed, set alpha levels at p ≤ .05, and presented data as mean ± SE. When
statistically significant effects were found, we reported partial eta squared (ηp2) as an
estimate of effect size. All statistical analyses were conducted using SPSS (version 26, IBM
Corporation).
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3.
3.1.
Results
Systemic Blockade of Androgen Receptors
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Because flutamide was administered prior to each dominance encounter, we tested whether
flutamide administration disrupted the formation or stability of dominance relationships. We
found that vehicle-treated dyads and flutamide-treated dyads formed a dominance
relationship with a clear winner and loser on day 2.4 (SE = 0.70) and 1.9 (SE = 0.55),
respectively (t(20) = 0.51, p = .616). This finding indicates that flutamide administration did
not delay the formation of dominance relationships. It is noteworthy that some subordinate
individuals showed aggression during the second or third encounter, although after they lost
a fight, they fail to display aggression for the remainder of encounters (Table S1). In
addition, we found that vehicle-treated and flutamide-treated dominants did not differ
significantly in the amount of aggression shown during daily encounters (F(1,15) = .85, p
= .371; Table S1). Similarly, flutamide-treated and vehicle-treated subordinates did not differ
significantly in the amount of submissive behavior shown (F(1,16) = 1.33, p = .267; Table
S1). Altogether, these findings indicated that systemic flutamide treatment did not alter
agonistic behavior during dominance encounters and did not impact the stability of
dominance relationships.
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The primary goal of Experiment 1 was to determine whether AR blockade would prevent
status-dependent differences in responses to social defeat stress (Fig. 1a). We found that
flutamide treatment increased the conditioned defeat response in dominant animals, whereas
it did not alter the conditioned defeat response in subordinates. Specifically, we found a
significant drug treatment X dominance status interaction for the duration of submissive
behavior displayed at testing (F(2,50) = 6.22, p = .004, ηp2 = .207; Fig. 1b). Vehicle-treated
dominant animals displayed less submissive and defensive behavior compared to flutamidetreated dominant counterparts (Tukey, p = .002). In a planned comparison, vehicle-treated
dominant animals also showed less submissive and defensive behavior compared to both
vehicle-treated subordinates and controls without dominance status, respectively (Tukey, p
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= .001, p = .03, respectively). Flutamide treatment did not alter the amount of aggressive
behavior, social behavior, or nonsocial behavior displayed during conditioned defeat testing
(Fig. 1c, d, e). There was a main effect of social status on the duration of aggressive
behavior, which indicates that at least some dominant animals displayed aggression during
conditioned defeat testing while subordinates and controls never did (F(2,50) = 4.12, p = .022,
ηp2 = .142). In addition, we found that flutamide treatment did not significantly alter the
duration of aggression subjects received (F(5,52) = 0.83, p = .367) or the number of attacks
subjects received during social defeat stress (F(5,52) = 2.11, p = .131). This finding indicates
that changes in the conditioned defeat response cannot be accounted for by differences in
intensity of social defeat (Table S2). However, more dominant animals (13 of 18) fought
back against the resident aggressor during the first social defeat trial than did subordinates (0
of 18) or no status animals (4 of 21) (χ2(2) = 24.28, p = .0001). Interestingly, flutamide
treatment did not alter the proportion of dominant animals who fought back against the
resident aggressor (6 of 8 vehicle-treated animals, 7 of 10 flutamide-treated dominants, χ2(1)
= 0.06, p = .81).
Flutamide treatment reduced the number of AR-positive cells in the MePD in dominant, but
not subordinate, animals (Fig. 1g). There was a significant drug treatment X social status
interaction for the number of AR-positive cells in the MePD (F(2,46) = 12.04, p = .001, ηp2
= .263). Importantly, vehicle-treated dominant animals showed more AR-positive cells in the
MePD compared to flutamide-treated dominant animals (Tukey, p = .001). Also, vehicletreated dominant animals showed more AR-positive cells in the MePD compared to vehicletreated subordinate and control animals (Tukey, p = .001, p = .001, respectively).
Author Manuscript
Flutamide treatment produced a similar change in AR expression in the MePV, although the
differences were not as pronounced as in the MePD (Fig. 1h). There was a significant main
effect of social status on the number of AR-positive cells in the MePV (F(2,45) = 10.18, p
= .001, ηp2 = .237), while the drug treatment x social status interaction was not statistically
significant (F(2,45) = 2.28, p = .114). A planned comparison indicated that vehicle-treated
dominant animals showed a greater number of AR-positive cells in the MePV compared to
flutamide-treated dominant animals (Tukey, p = .009). Additionally, vehicle-treated
dominant animals have a greater number of AR-positive cells in the MePV than do vehicletreated subordinate and control animals (Tukey, p = .001, p = .001, respectively).
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Flutamide treatment did not alter the number of AR-positive cells in the vLS of dominant or
subordinate animals (Fig. S1). There was a main effect of social status on the number of ARpositive cells in the vLS (F(2,44) = 14.63, p = .001, ηp2 = .322), but there was no significant
drug treatment X social status interaction (F(2,44) = 1.04, p = .361). Regardless of drug
treatment, dominant animals had more AR-positive cells in the vLS compared to their
subordinate counterparts. A planned comparison indicated that vehicle-treated dominant
animals have more AR-positive cells in the vLS compared to vehicle-treated subordinate
animals (Tukey, p = .01).
Because we found status-dependent changes in AR immunoreactivity in the MePD, MePV,
and vLS, we correlated the duration of submissive and defensive behavior with the number
of AR immunoreactive cells in each of these brain regions. We found that dominant animals
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exhibited a negative correlation between submissive and defensive behavior and AR
immunoreactivity in the MePD and MePV, but not in the vLS (Table S3). Interestingly, these
significant correlations occurred flutamide-treated dominants (MePD: r(8) = −.81, p = .004;
MePV: r(8) = −.66, p = .038), but not vehicle-treated dominants (MePD: r(5) = −. 11, p = .82;
MePV: r(5) = −.37, p = .42). These findings indicate that following chronic flutamide
treatment, dominant animals with the fewest AR immunoreactive cells in the MePD and
MePV had the largest conditioned defeat response. In contrast, subordinate animals showed
a negative correlation between submissive and defensive behavior and AR immunoreactivity
in the vLS, but not in the MePD or MePV (Table S3). This correlation was apparent in both
vehicle-treated and flutamide-treated subordinates (vLS: r(7) = −.84, p = .017; vLS: r(7) =
−.72, p = .045, respectively), which indicates that the relationship between AR
immunoreactivity in the vLS and the conditioned defeat response in subordinates is not
dependent on prior flutamide treatment.
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3.2.
Blockade of Androgen Receptors in the MeP
We found that male hamsters in Experiment 2 formed stable dominance relationships,
similar to Experiment 1 above. Dominance relationships were established on day 2.3 (SE
= .21). Also, dominants showed 106.7 sec (SE = 16.8) and 114.4 (SE = 17.7) of aggression
on the 2nd and 14th encounters, respectively (Table S4). Likewise, subordinates showed
125.1 sec (SE = 21.6) and 162.8 sec (SE = 23.1) of submissive behavior on the 2nd and 14th
encounters, respectively (Table S4). Overall, dominants never showed submission and
subordinates failed to show aggression after losing the fight, which is consistent with stable
dominance relationships.
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Injection of flutamide into the MeP prior to social defeat stress increased acquisition of the
conditioned defeat response in dominant male hamsters. We found a significant interaction
in the duration of submissive and defensive behavior during conditioned defeat testing,
which indicates that flutamide treatment increased submissive and defensive behavior in
dominants but not in subordinates (Fig. 2c; F(1,30) = 10.33, p = .003, ηp2 = .256). The effect
of dominance status on the duration of aggression during conditioned defeat testing did not
reach statistical significance (Fig. 2d; F(1,30) = 1.75, p = .195). In addition, there was a nonsignificant interaction for the duration of affiliative behavior (Fig. 2e; F(1,30) = 3.66, p
= .065). There were no significant effects of drug or dominance status on the duration of
nonsocial behavior (Fig. 2f; F(1,30) = 0.39, p = .535; F(1,30) = 1.41, p = .244, respectively).
Interestingly, among animals with misplaced injections, the duration of submissive and
defensive behavior at conditioned defeat testing did not significant differ between flutamidetreated dominants (40.4 sec ± 16.0, n = 6) and vehicle-treated dominants (35.7 sec ± 7.9, n =
4) (t(8) = 0.22, p = .830).
The injection of flutamide into the MeP prior to conditioned defeat testing did not alter
expression of the conditioned defeat response. We found a main effect of dominance status
on the duration of submissive and defensive behavior during conditioned defeat testing,
which reflects a reduced conditioned defeat response in dominant males (Fig. 2g; F(1,31) =
16.79, p = .0003, ηp2 = .351). However, there was no interaction between drug and
dominance status on duration of submissive and defensive behavior, which indicates
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flutamide injection did not alter the expression of conditioned defeat in dominants (F(1,31)
= .656, p = .424). The effect of dominance status on duration of aggression during
conditioned defeat testing did not reach statistical significance (F(1,31) = 2.89, p = .099).
Dominant males showed elevated affiliative behavior during conditioned defeat testing
compared to subordinates, which is consistent with their reduced conditioned defeat
response (Fig. 2i; F(1,31) = 5.21, p = .029, ηp2 = .144). There was no effect of drug or
dominance status on the expression of nonsocial behavior at conditioned defeat testing (Fig.
2j; F(1,31)= 2.38, p = .133, F(1,31) = .001, p = .982, respectively).
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The amount of aggression subjects received during social defeat stress is shown in Table S5.
A 3-way ANOVA of social status, drug treatment, and injection time indicated that treatment
groups did not significantly differ in the duration of aggression received (F(7,199) = 0.88, p
= .350) or in the number of attacks received (F(7,199) = 0.51, p = .476). While these data
indicate that dominants and subordinates did not differ in the amount of aggression received,
dominants were more likely to fight back against the resident aggressor during their first
social defeat trial than were their subordinate counterparts. When pooling animals by drug
treatment and injection time point, we found that 28 of 35 dominants fought back against the
resident aggressor compared to 1 of 34 subordinates (χ2(1) = 42.03, p = .0001).
3.3.
Effects of Social Dominance in Female Hamsters
Author Manuscript
Females in Experiment 3 formed stable dominance relationships, although there were subtle
differences compared to male hamsters. We found that all female dyads had a clear winner
and loser during their first encounter, which suggests that dominance relationships were
quickly established (day 1.0, SE = 0.0). Nevertheless, several subordinate animals showed
aggression (11.1 ± 5.5 sec) toward the dominant during the second encounter before quickly
losing the fight (Table S6). Dominant female hamsters were aggressive for 168.1 sec (SE =
13.4) during the second encounter, which was noticeably more than the amount of
aggression shown by dominant males (Table S6). In addition, in 8 of 28 female dyads
(28.6%) the subordinate animal attacked the dominant animal and won, which flipped the
dominance relationship and excluded the pair from analysis. It is exceedingly rare for male
subordinates to flip the dominance relationship, as we found subordinate males
counterattacked the dominant and flip the relationship in 1 of 72 dyads (1.4%), which is
significantly less than in female dyads (χ2(1) = 18.19, p = .001). These findings suggest that
female hamsters are more aggressive than males and quickly establish dominance
relationships, although subordinate females actively contest their low status and occasionally
flip the relationship.
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In blood samples collected prior to and following the first dominance encounter, we found
that changes in plasma levels of gonadal steroid hormones were not related to dominance
status. We found a main effect of time on plasma concentrations of estradiol such that
estradiol levels increased following the initial dominance encounter in each social status
condition (Fig. S2, F(1,31) = 13.24, p = .001, ηp2 = .299). Similarly, there was a significant
effect of time on plasma progesterone concentrations albeit in an opposite direction where
progesterone levels decreased following the initial dominance encounter (Fig. S2, F(1,36) =
38.06, p = .0003, ηp2 = .514). Plasma testosterone concentrations also showed a significant
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main effect of time as testosterone concentrations decreased in all treatment groups (Fig. S2,
F(1,36) = 11.68, p = .002, ηp2 = .245). Because no status control animals show a similar
change in plasma hormone levels compared to animals exposed to an agonistic encounter,
these findings suggest hormone changes were due to repeated blood collection and/or
anesthesia exposure and, importantly, not related to the outcome of aggression.
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Dominant female hamsters showed greater social approach and territorial behavior during
social avoidance testing compared to subordinates and no status controls. There was a main
effect of social defeat and dominance status on the amount of time spent in the interaction
zone (Fig. 3b, F(1,55) = 52.40, p = .0001, ηp2 = .517; F(2,55) = 5.15, p = .009, ηp2 = .174,
respectively). Likewise, there was a main effect of social defeat and dominance status on the
interaction ratio (Fig. 3c, F(1,55) = 42.41, p = .0002, ηp2 = .464; F(2,55) = 3.89, p = .027, ηp2
= .137, respectively). Although there was a main effect of social defeat on the amount of
time spent in the far side of the testing arena (Fig. 3d, F(1,55) = 49.77, p = .0001, ηp2 = .491),
the effect of dominance status was non-significant (Fig. 3d, F(2,55) = 2.50, p = .093).
Together, these findings indicate that dominant females show greater social approach in a
novel arena regardless of whether they received social defeat stress. Interestingly, we found a
significant defeat by dominance status interaction on the number of flank marks such that
dominant females displayed more flank marks only after they were exposed to social defeat
stress (Fig. 3e, F(2,55) = 3.27, p = .046, ηp2 = .118). This finding suggests that dominant
females were prepared to reassert territorial behavior via scent marking following a social
defeat stressor, while others were not.
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After dominance relationships were established and maintained, animals received social
defeat stress followed by a social interaction test. Dominant, subordinate, and no status
animals did not significantly differ in the duration of aggression received or in the number of
attacks received during social defeat stress (F(2,55) = 1.19, p = .443, F(2,55) = 1.58, p = .217,
respectively, Table S7). These findings indicate that differences in behavioral responses to
social defeat stress are unlikely related to systematic differences in the amount of aggression
received. Nevertheless, dominant females were more likely to fight back against resident
aggressors during the first social defeat trial compared to others. Specifically, 7 of 10
dominants fought back during the initial social defeat trial compared to 2 of 10 subordinates
and 1 of 10 no status animals (χ2(2) = 9.30, p = .010).
Following social interaction testing, we collected brains for quantification of ERα and AR in
the MePD and MePV. There was a main effect of dominance status on ERα expression
indicating that dominant females showed increased ERα immunoreactivity in the MePD
compared to subordinates and no status controls (Fig. 3g, F(2,49) = 6.51, p = .003, ηp2
= .232). There was a similar trend in the MePV, although the effect of dominance status on
ERα immunoreactivity did not reach statistical significance (Fig. 3h, F(2,49) = 2.19, p
= .125). We found low levels of AR immunoreactivity in the MePD and MePV (Fig. S3),
although there was no significant effect of dominance status on the number of AR
immunoreactive cells either the MePD or MePV (Fig. 3i, 3j, F(2,49) = .143, p = .867, F(2,49)
= .192, p = .826). Altogether, these findings indicate that achieving social dominance is
associated with the upregulation of ERα in the MePD.
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4.
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Discussion
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This project was focused on the mechanisms by which activity of gonadal steroid hormone
receptors contribute to status-dependent changes in vulnerability to social stress. We found
that systemic pharmacological blockade of AR throughout the creation and maintenance of
dominance relationships in male hamsters prevents the upregulation of AR in the MeP and
increases the conditioned defeat response in dominant animals. These findings indicate the
activation of AR during the creation and maintenance dominance relationships contributes to
resistance to the effects of social defeat stress in dominant males. Further, we found that
blockade of AR in the MeP during acute social defeat stress prevented the acquisition of a
resilient conditioned defeat response in dominant males. The maintenance of dominance
relationships also alters behavior and gonadal steroid hormone receptors in female hamsters,
although differently than in males. We previously found that dominant males exhibit
increased plasma testosterone after winning an aggressive encounter (Clinard et al., 2016),
but here we found that the outcome of aggressive encounters did not alter plasma
concentrations of gonadal steroids in female hamsters. Nevertheless, female hamsters
showed an upregulation of ERα in the MePD after achieving social dominance. Also,
dominant females show greater social approach in a social interaction test regardless of their
exposure to social defeat stress, which suggests that social dominance produces a general
change in approach/avoidance behavior which allows stress resilience when challenged with
social defeat.
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The challenge hypothesis proposes that social experience leads to a surge in plasma
testosterone, which in turn primes aggressive behavior in future encounters (Wingfield et al.,
1990). Winners of competitive interactions show a surge in plasma testosterone in a wide
variety of species, including song sparrows (Wingfield and Wada, 1989), cichlid fish
(Hirschenhauser et al., 2004), green anole lizards (Yang and Wilczynski, 2002), California
mice (Oyegbile and Marler, 2005), and Syrian hamsters (Clinard et al., 2016). The surge in
testosterone after winning can facilitate aggression in future interactions and promote a
winner effect. The neuroendocrine mechanisms underlying a winner effect have been
particularly well-delineated in California mice. In these mice, the winner effect depends on
animals winning aggressive encounters in their familiar home cage, which leads to a surge in
plasma testosterone and an upregulation of AR in the nucleus accumbens and bed nucleus of
the stria terminalis (Fuxjager et al., 2010; Fuxjager et al., 2009). These findings are
consistent with autoregulation of AR by androgens (Lu et al., 1998) and suggest that
induction of AR is necessary for the winner effect. One possible explanation for our findings
is that systemic flutamide treatment blocks AR activation during a testosterone surge,
prevents AR upregulation in the MePD and/or MePV, and impairs the development of a
reduced conditioned defeat response. However, flutamide treatment can have other effects,
including agonist-like effects, by increasing neuroprotection against apoptotic insults
(Nguyen et al., 2007) and increasing circulating concentrations of gonadotropins and
testicular androgens (Chandolia et al., 1991). However, these additional effects of chronic
flutamide treatment would also occur in subordinate and no status control animals. Because
the flutamide effects were specific to dominants, it seems likely that pharmacological
blockade of AR prevents AR upregulation in the MeP and contributes to a loss of stress
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resistance in dominants. One unanswered question from this study is whether a flutamideinduced decrease in MeP AR immunoreactivity leads to a reduced conditioned defeat
response or whether reduced conditioned defeat leads to a reduction in AR
immunoreactivity. However, because neither subordinates, no status animals, nor vehicletreated dominants show a correlation between MeP AR immunoreactivity and conditioned
defeat, it appears that individual variation in the conditioned defeat response does not
produce changes in MeP AR immunoreactivity. Another unanswered question is the duration
of flutamide treatment necessary to produce these neuroendocrine and behavioral effects. In
this study, we treated animals with flutamide throughout the two-week maintenance of
dominance relationship because previous research indicates that resistance to conditioned
defeat requires animals to maintain dominant social status for two weeks (Morrison et al.,
2014). Nevertheless, the time point when AR activity is critical for resistance to conditioned
defeat in dominants remains unknown.
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Interestingly, not all status-dependent differences in behavior were linked to AR activation.
For example, flutamide treatment did not alter aggressive or submissive behavior during the
creation and maintenance of dominance relationships. Thus, flutamide appears to alter the
consequences of winning aggressive encounters without affecting aggressive behavior itself,
which is consistent with both the challenge hypothesis and winner effect (Marler and
Trainor, 2020; Oliveira et al., 2009). Also, flutamide treatment did not alter aggressive
behavior exhibited by dominants during conditioned defeat testing. However, whether
flutamide treatment specifically alters a submissive behavior component of the conditioned
defeat response is difficult to evaluate because of a floor effect on aggression during
conditioned defeat testing. In addition, dominant animals were more likely to actively resist
the resident aggressor during the initial social defeat trial compared to subordinates or no
status animals, although flutamide-treated animals were as likely to fight back against the
resident aggressor as were vehicle-treated animals. This finding suggests that blocking AR
during the establishment of dominance relationships or during social defeat stress itself does
not alter initial responses to the resident aggressor. One possible explanation is that how
animals respond to an aggressor is regulated by neural ensembles outside the MeP, such as
those in the ventral medial prefrontal cortex (vmPFC). Latencies to submit to an aggressor
as well as the likelihood of fighting back is correlated with activation of vmPFC neurons
projecting to the dorsal raphe nucleus (Grizzell et al., 2020). While resisting an opponent
and delaying the onset of social defeat is indicative of a proactive coping response (Veenema
et al., 2004; Walker et al., 2009; Wood et al., 2010), it appears that this type of proactive
response to social defeat is not dependent on AR activation.
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Next, we tested whether AR activation during social defeat stress or behavioral testing was
crucial for the acquisition or expression of the conditioned defeat response, respectively.
While it is possible that elevated AR expression is essential for reduced conditioned defeat
in dominant males, it is also possible that AR-dependent neural plasticity changes neural
circuits so that their activation no longer depends on AR activity. Because we found that
flutamide treatment into the MeP increases the acquisition of conditioned defeat specifically
in dominant males, we suggest that AR activity within the MeP during social defeat stress
contributes to status-dependent changes in stress resilience. Interestingly, the expectation of
an aggressive encounter can lead to an anticipatory rise in plasma testosterone in both
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humans and other animals (Antunes and Oliveira, 2009; Fuxjager et al., 2017; Neave and
Wolfson, 2003). Because our behavioral testing procedure involves the routine transport of
animals at the same time each day, it is possible that dominant males have an anticipatory
rise in testosterone prior to social defeat. Then, blockade of AR in the MeP prevents the
effects of anticipatory testosterone release on MeP circuits that modulate stress-induced
changes in social behavior. Interestingly, flutamide treatment did not alter how animals
responded to social defeat, including whether dominant animals counter-attacked and fought
back against the resident aggressors. Similarly, chronic flutamide treatment did not alter
agonistic behavior during the formation of dominance relationships. Altogether, these
findings are consistent with the challenge hypothesis and suggest that AR activation does not
regulate ongoing aggression per se, but modulates neural circuits that regulate responses to
future confrontations (Ball and Balthazart, 2020).
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Dominance status has a strong influence on future behavior in a wide variety of animals
(Desjardins et al., 2012; Holekamp and Strauss, 2016; McEwen et al., 2015; Overli et al.,
2007; Schino, 2001). However, the effects of social dominance are extremely variable and
sometimes inconsistent between different species, which might reflect the nature of the
dominance hierarchy itself or how dominance relationships are measured. In mice, a tube
test is a convenient and reliable method for quantifying dominance relationships in pairs of
individuals (Wang et al., 2011). When dominance status was established with a tube test,
Larrieu et al (2017) found that dominant individuals exhibit increased social avoidance
following chronic social defeat stress. They suggest that loss of status in dominant animals
might make them more vulnerable to the effects of chronic social defeat stress. An
additional possibility, and one that is not mutually exclusive, is that when dominance status
is established without physical aggression, changes in testicular androgens and AR
expression may not occur and effects of future stress-related behavior may differ.
Interestingly, mice exhibit a winner effect in a tube test and synaptic input from the
mediodorsal thalamus to the dorsomedial prefrontal cortex is critical for encoding a history
of winning (Zhou et al., 2017). Thus, establishing social dominance generates plasticity in
multiple neural circuits and status-related differences in future behavior likely dependent on
the type of future behavior examined and the neural ensembles engaged.
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Changes in endogenous testosterone are associated with status-related behaviors in not only
men, but also women (Cashdan, 1995; Dabbs and Hargrove, 1997; Josephs et al., 2003). For
example, female rugby players experience an anticipatory rise in testosterone that is related
to performance, although their rise in testosterone is unrelated to winning or losing (Bateup
et al., 2002). While there are sex similarities and differences in the hormonal changes
associated with competition in humans, it is rare to include females in animal models of the
neuroendocrine mechanisms underlying status-dependent changes in behavior. Female
hamsters are larger and more aggressive than males (Floody and Pfaff, 1977; Payne and
Swanson, 1971) and provide a unique rodent model for investigating the behavioral and
neuronal changes associated with dominance status in females. We found no change in
plasma steroid hormones after a single dominance encounter in female hamsters, which
suggests that neither the experience of an aggressive interaction nor the outcome produce a
surge in steroid hormones. However, we collected blood samples according to a time course
appropriate for male hamsters and an appropriate time course for females is unknown.
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Surprisingly, we found a change in the plasma concentrations of steroid hormones after a
second blood collection in all treatment groups. Although these changes are puzzling,
general anesthesia with isoflurane can induce opposing vasodilatory and vasoconstrictive
effects, and the corresponding changes in blood flow may alter steroid hormone
concentrations (Constantinides et al., 2011). Nevertheless, we found an increased number of
ERα immunoreactivity cells in the MePD in dominant females, suggesting that the longterm maintenance of dominance status can increase ERα expression without acute changes
in plasma steroid hormone concentrations. These findings are consistent with studies using
CD-1 female mice which indicate that establishing dominant-subordinate relationships can
alter gene expression for ERα and ERβ in the ventral medial hypothalamus, although
subordinate animals showed greater mRNA expression than dominants (Williamson et al.,
2019b). In addition, formation of dominance relationships in female hamsters has been
shown to alter expression of arginine-vasopressin 1A, oxytocin, and serotonin 1A receptors
in several regions of the social behavior neural network (Grieb et al., 2021).
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The conditioned defeat test is not an effective measure of the effects of social defeat in
female hamsters, likely because the strong motivation for territorial aggression contaminates
measurement of social avoidance in a test with a freely moving novel intruder (Solomon et
al., 2007). However, testing female hamsters with a novel, restrained individual provides a
robust assay to measure defeat-induced approach/avoidance behavior (McCann et al., 2017;
Rosenhauer et al., 2017). Here, we found that dominant females showed increased social
approach compared to subordinates regardless of whether they received acute social defeat
stress. These findings suggest that social dominance alters subsequent approach/avoidance
behavior in female hamsters and not solely stress-induced changes in behavior. These
findings are consistent with previous research using male hamsters, which indicate that
dominance status alters patterns of aggression and submission in conditioned defeat testing
regardless of whether animals were exposed to social defeat stress (Morrison et al., 2012).
However, conditioned defeat and social avoidance tests are separate behavioral assays and
investigating sex differences in the effects of social dominance will require testing males in a
social avoidance test. In addition, our procedure for establishing dominance relationships
was modified to avoid testing females during estrus when they exhibit less territorial
aggression and future studies with male hamsters should employ this modified testing
procedure.
Author Manuscript
Historically, the winner effect has focused on the mechanisms by which winning aggressive
interactions promotes successful outcomes in future contests (Hsu et al., 2006). While the
mechanisms by which gonadal steroid hormones regulate the winner effect have been well
described (Marler and Trainor, 2020), the possibility remains that winning may also generate
AR-dependent plasticity in neural circuits that regulate non-aggressive behavior. Here we
show that AR activation during the formation of dominance relationships is necessary for
stress resistance in dominant males and that activation of AR in the MePV during social
defeat stress is essential for status-dependent changes in stress resistance. Although
dominant females also show changes in approach/avoidance behavior, their status-dependent
changes in behavior were associated with plasticity in ERα and not AR. Together, these
findings identify sex-specific neuroendocrine mechanisms through which achieving social
dominance reduces the effects of future traumatic stress in both males and females.
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Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
We thank our team of research assistants for their daily technical support, including Connor Borresen, Max
Burzinski, Thomas Clarity, Emily Graham, Matthew Jenkins, Mason Rodriguez, Neha Sagarad, Maya Scarbrough,
and Sydney Wyatt. Also, we thank Conner Whitten for helpful comments on a previous version of the manuscript.
This project was supported by the US National Institutes of Health grants R15 MH107007 and R15 MH122946 to
MAC.
References
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Antunes RA, Oliveira RF, 2009. Hormonal anticipation of territorial challenges in cichlid fish.
Proceedings of the National Academy of Sciences 106, 15985–15989.
Apfelbeck B, Stegherr J, Goymann W, 2011. Simulating winning in the wild—The behavioral and
hormonal response of black redstarts to single and repeated territorial challenges of high and low
intensity. Hormones and behavior 60, 565–571. [PubMed: 21872602]
Ball GF, Balthazart J, 2020. The neuroendocrine integration of environmental information, the
regulation and action of testosterone and the challenge hypothesis. Hormones and behavior 123,
104574. [PubMed: 31442427]
Baratta MV, Maier SF, 2019. New tools for understanding coping and resilience. Neuroscience letters
693, 54–57. [PubMed: 28963058]
Bartolomucci A, Palanza P, Gaspani L, Limiroli E, Panerai AE, Ceresini G, Poll MD, Parmigiani S,
2001. Social status in mice: behavioral, endocrine and immune changes are context dependent.
Physiology & behavior 73, 401–410. [PubMed: 11438368]
Bartolomucci A, Palanza P, Sacerdote P, Panerai AE, Sgoifo A, Dantzer R, Parmigiani S, 2005. Social
factors and individual vulnerability to chronic stress exposure. Neurosci. Biobehav. Rev 29, 67–81.
[PubMed: 15652256]
Bateup HS, Booth A, Shirtcliff EA, Granger DA, 2002. Testosterone, cortisol, and women's
competition. Evolution and Human Behavior 23, 181–192.
Cashdan E, 1995. Hormones, sex, and status in women. Hormones and behavior 29, 354–366.
[PubMed: 7490010]
Chandolia RK, Weinbauer GF, Simoni M, Behre HM, Nieschlag E, 1991. Comparative effects of
chronic administration of the non-steroidal antiandrogens flutamide and Casodex on the
reproductive system of the adult male rat. European Journal of Endocrinology 125, 547–555.
Chen CV, Brummet JL, Lonstein JS, Jordan CL, Breedlove SM, 2014. New knockout model confirms
a role for androgen receptors in regulating anxiety-like behaviors and HPA response in mice.
Hormones and behavior 65, 211–218. [PubMed: 24440052]
Christianson JP, Greenwood BN, 2014. Stress-protective neural circuits: not all roads lead through the
prefrontal cortex. Stress 17, 1–12. [PubMed: 23574145]
Clinard CT, Barnes AK, Adler SG, Cooper MA, 2016. Winning agonistic encounters increases
testosterone and androgen receptor expression in Syrian hamsters. Hormones and behavior 86, 27–
35. [PubMed: 27619945]
Clipperton-Allen AE, Almey A, Melichercik A, Allen CP, Choleris E, 2011. Effects of an estrogen
receptor alpha agonist on agonistic behaviour in intact and gonadectomized male and female mice.
Psychoneuroendocrinology 36, 981–995. [PubMed: 21247705]
Constantinides C, Mean R, Janssen BJ, 2011. Effects of isoflurane anesthesia on the cardiovascular
function of the C57BL/6 mouse. ILAR journal/National Research Council, Institute of Laboratory
Animal Resources 52, e21.
Cooper MA, Seddighi S, Barnes AK, Grizzell JA, Dulka BN, Clinard CT, 2017. Dominance status
alters restraint-induced neural activity in brain regions controlling stress vulnerability. Physiology
& behavior 179, 153–161. [PubMed: 28606772]
Psychoneuroendocrinology. Author manuscript; available in PMC 2022 July 01.
Cooper et al.
Page 20
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Dabbs J, Hargrove MF, 1997. Age, testosterone, and behavior among female prison inmates.
Psychosom. Med 59, 477–480. [PubMed: 9316179]
Davis J, Krause E, Melhorn S, Sakai R, Benoit S, 2009. Dominant rats are natural risk takers and
display increased motivation for food reward. Neuroscience 162, 23–30. [PubMed: 19393296]
Desjardins JK, Hofmann HA, Fernald RD, 2012. Social context influences aggressive and courtship
behavior in a cichlid fish. PLoS One 7, e32781. [PubMed: 22807996]
Dulka BN, Bagatelas ED, Bress KS, Grizzell JA, Cannon MK, Whitten CJ, Cooper MA, 2020.
Chemogenetic activation of an infralimbic cortex to basolateral amygdala projection promotes
resistance to acute social defeat stress. Scientific Reports 10, 1–13. [PubMed: 31913322]
Edinger KL, Frye CA, 2006. Intrahippocampal administration of an androgen receptor antagonist,
flutamide, can increase anxiety-like behavior in intact and DHT-replaced male rats. Hormones and
behavior 50, 216–222. [PubMed: 16631174]
Floody OR, Pfaff DW, 1977. Aggressive behavior in female hamsters: the hormonal basis for
fluctuations in female aggressiveness correlated with estrous state. J.Comp Physiol Psychol 91,
443–464. [PubMed: 559693]
Fuxjager MJ, Forbes-Lorman RM, Coss DJ, Auger CJ, Auger AP, Marler CA, 2010. Winning
territorial disputes selectively enhances androgen sensitivity in neural pathways related to
motivation and social aggression. Proc. Natl. Acad. Sci. U. S. A 107, 12393–12398. [PubMed:
20616093]
Fuxjager MJ, Mast G, Becker EA, Marler CA, 2009. The 'home advantage' is necessary for a full
winner effect and changes in post-encounter testosterone. Horm Behav 56, 214–219. [PubMed:
19426733]
Fuxjager MJ, Montgomery JL, Marler CA, 2011a. Species differences in the winner effect disappear in
response to post-victory testosterone manipulations. Proc Biol Sci 278, 3497–3503. [PubMed:
21490015]
Fuxjager MJ, Oyegbile TO, Marler CA, 2011b. Independent and additive contributions of postvictory
testosterone and social experience to the development of the winner effect. Endocrinology 152,
3422–3429. [PubMed: 21771886]
Fuxjager MJ, Trainor BC, Marler CA, 2017. What can animal research tell us about the link between
androgens and social competition in humans? Hormones and behavior 92, 182–189. [PubMed:
27914879]
Grieb Z, Ross A, McCann K, Lee S, Welch M, Gomez M, Norvelle A, Michopoulos V, Huhman K,
Albers H, 2021. Sex-dependent effects of social status on the regulation of arginine-vasopressin
(AVP) V1a, oxytocin (OT), and serotonin (5-HT) 1A receptor binding and aggression in Syrian
hamsters (Mesocricetus auratus). Hormones and behavior 127, 104878. [PubMed: 33148500]
Grizzell JA, Clarity TT, Graham NB, Dulka BN, Cooper MA, 2020. Activity of a vmPFC-DRN
pathway corresponds with resistance to acute social defeat stress. Frontiers in Neural Circuits 14.
Hardy MP, Sottas CM, Ge R, McKittrick CR, Tamashiro KL, McEwen BS, Haider SG, Markham CM,
Blanchard RJ, Blanchard DC, Sakai RR, 2002. Trends of reproductive hormones in male rats
during psychosocial stress: role of glucocorticoid metabolism in behavioral dominance. Biology of
Reproduction 67, 1750–1755. [PubMed: 12444049]
Hirschenhauser K, Oliveira RF, 2006. Social modulation of androgens in male vertebrates: metaanalyses of the challenge hypothesis. Animal Behaviour 71, 265–277.
Hirschenhauser K, Taborsky M, Oliveira T, Canario AV, Oliveira R, 2004. A test of the ‘challenge
hypothesis’ in cichlid fish: simulated partner and territory intruder experiments. Animal Behaviour
68, 741–750.
Holekamp KE, Strauss ED, 2016. Aggression and dominance: an interdisciplinary overview. Current
Opinion in Behavioral Sciences 12, 44–51.
Horton BM, Hudson WH, Ortlund EA, Shirk S, Thomas JW, Young ER, Zinzow-Kramer WM, Maney
DL, 2014. Estrogen receptor α polymorphism in a species with alternative behavioral phenotypes.
Proceedings of the National Academy of Sciences 111, 1443–1448.
Hsu Y, Earley RL, Wolf LL, 2006. Modulation of aggressive behaviour by fighting experience:
mechanisms and contest outcomes. Biological Reviews 81, 33–74. [PubMed: 16460581]
Psychoneuroendocrinology. Author manuscript; available in PMC 2022 July 01.
Cooper et al.
Page 21
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Huhman KL, Solomon MB, Janicki M, Harmon AC, Lin SM, Israel JE, Jasnow AM, 2003.
Conditioned defeat in male and female Syrian hamsters. Horm Behav 44, 293–299. [PubMed:
14609551]
Josephs RA, Newman ML, Brown RP, Beer JM, 2003. Status, testosterone, and human intellectual
performance: Stereotype threat as status concern. Psychological Science 14, 158–163. [PubMed:
12661678]
Karamihalev S, Brivio E, Flachskamm C, Stoffel R, Schmidt MV, Chen A, 2020. Sexually divergent
effects of social dominance on chronic stress outcomes in mice. bioRxiv.
Knight EL, Mehta PH, 2017. Hierarchy stability moderates the effect of status on stress and
performance in humans. Proceedings of the National Academy of Sciences 114, 78–83.
Kohn JN, Snyder-Mackler N, Barreiro LB, Johnson ZP, Tung J, Wilson ME, 2016. Dominance rank
causally affects personality and glucocorticoid regulation in female rhesus macaques.
Psychoneuroendocrinology 74, 179–188. [PubMed: 27639059]
Larrieu T, Cherix A, Duque A, Rodrigues J, Lei H, Gruetter R, Sandi C, 2017. Hierarchical status
predicts behavioral vulnerability and nucleus accumbens metabolic profile following chronic
social defeat stress. Current Biology 27, 2202–2210. e2204. [PubMed: 28712571]
Lu S.-f., McKenna SE, Cologer-Clifford A, Nau EA, Simon NG, 1998. Androgen receptor in mouse
brain: sex differences and similarities in autoregulation. Endocrinology 139, 1594–1601.
[PubMed: 9528939]
Markham CM, Huhman KL, 2008. Is the medial amygdala part of the neural circuit modulating
conditioned defeat in Syrian hamsters? Learn. Mem 15, 6–12. [PubMed: 18174368]
Marler CA, Trainor BC, 2020. The challenge hypothesis revisited: Focus on reproductive experience
and neural mechanisms. Hormones and behavior 123, 104645. [PubMed: 31778720]
McCann KE, Rosenhauer AM, Jones GM, Norvelle A, Choi DC, Huhman KL, 2017. Histone
deacetylase and acetyltransferase inhibitors modulate behavioral responses to social stress.
Psychoneuroendocrinology 75, 100–109. [PubMed: 27810703]
McEwen BS, McKittrick CR, Tamashiro KL, Sakai RR, 2015. The brain on stress: Insight from studies
using the Visible Burrow System. Physiology & behavior 146, 47–56. [PubMed: 26066722]
Morin LP, Wood RI, 2001. A stereotaxic atlas of the golden hamster brain. Academic Press, New York.
Morrison KE, Bader LR, Clinard CT, Gerhard DM, Gross SE, Cooper MA, 2014. Maintenance of
dominance status is necessary for resistance to social defeat stress in Syrian hamsters. Behav Brain
Res 270, 277–286. [PubMed: 24875769]
Morrison KE, Curry DW, Cooper MA, 2012. Social status alters defeat-induced neural activation in
Syrian hamsters. Neuroscience 210, 168–178. [PubMed: 22433296]
Naghdi N, Oryan S, Etemadi R, 2003. The study of spatial memory in adult male rats with injection of
testosterone enanthate and flutamide into the basolateral nucleus of the amygdala in Morris water
maze. Brain research 972, 1–8. [PubMed: 12711072]
Nagypál A, Wood RI, 2007. Region-specific mechanisms for testosterone-induced Fos in hamster
brain. Brain research 1141, 197–204. [PubMed: 17276422]
Neave N, Wolfson S, 2003. Testosterone, territoriality, and the ‘home advantage’. Physiology &
behavior 78, 269–275. [PubMed: 12576125]
Nguyen T-VV, Yao M, Pike CJ, 2007. Flutamide and cyproterone acetate exert agonist effects:
induction of androgen receptor-dependent neuroprotection. Endocrinology 148, 2936–2943.
[PubMed: 17347309]
Ogawa S, Eng V, Taylor J, Lubahn DB, Korach KS, Pfaff DW, 1998. Roles of estrogen receptor-α gene
expression in reproduction-related behaviors in female mice. Endocrinology 139, 5070–5081.
[PubMed: 9832446]
Oliveira RF, Silva A, Canario AV, 2009. Why do winners keep winning? Androgen mediation of
winner but not loser effects in cichlid fish. Proc Biol Sci 276, 2249–2256. [PubMed: 19324741]
Overli O, Sorensen C, Pulman KG, Pottinger TG, Korzan W, Summers CH, Nilsson GE, 2007.
Evolutionary background for stress-coping styles: relationships between physiological, behavioral,
and cognitive traits in non-mammalian vertebrates. Neurosci Biobehav Rev 31, 396–412.
[PubMed: 17182101]
Psychoneuroendocrinology. Author manuscript; available in PMC 2022 July 01.
Cooper et al.
Page 22
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Oyegbile TO, Marler CA, 2005. Winning fights elevates testosterone levels in California mice and
enhances future ability to win fights. Horm Behav 48, 259–267. [PubMed: 15979073]
Payne AP, Swanson HH, 1971. Hormonal control of aggressive dominance in the female hamster.
Physiol Behav. 6, 355–357. [PubMed: 5169917]
Rose RM, Bernstein IS, Gordon TP, 1975. Consequences of social conflict on plasma testosterone
levels in rhesus monkeys. Psychosom. Med
Rosenhauer AM, McCann KE, Norvelle A, Huhman KL, 2017. An acute social defeat stressor in early
puberty increases susceptibility to social defeat in adulthood. Hormones and behavior 93, 31–38.
[PubMed: 28390864]
Rosvall KA, Bergeon Burns CM, Barske J, Goodson JL, Schlinger BA, Sengelaub DR, Ketterson ED,
2012. Neural sensitivity to sex steroids predicts individual differences in aggression: implications
for behavioural evolution. Proc Biol Sci 279, 3547–3555. [PubMed: 22673360]
Sapolsky RM, 2005. The influence of social hierarchy on primate health. Science 308, 648–652.
[PubMed: 15860617]
Schino G, 2001. Grooming, competition and social rank among female primates: a meta-analysis.
Animal Behaviour 62, 265–271.
Sherman GD, Lee JJ, Cuddy AJ, Renshon J, Oveis C, Gross JJ, Lerner JS, 2012. Leadership is
associated with lower levels of stress. Proceedings of the National Academy of Sciences 109,
17903–17907.
Solomon MB, Karom MC, Huhman KL, 2007. Sex and estrous cycle differences in the display of
conditioned defeat in Syrian hamsters. Horm Behav 52, 211–219. [PubMed: 17555756]
Spiteri T, Musatov S, Ogawa S, Ribeiro A, Pfaff DW, Ågmo A, 2010. The role of the estrogen receptor
a in the medial amygdala and ventromedial nucleus of the hypothalamus in social recognition,
anxiety and aggression. Behavioural brain research 210, 211–220. [PubMed: 20184922]
Trainor BC, Rowland MR, Nelson RJ, 2007. Photoperiod affects estrogen receptor α, estrogen
receptor β and aggressive behavior. European Journal of Neuroscience 26, 207–218.
van Praag H, Kempermann G, Gage FH, 2000. Neural consequences of environmental enrichment. Nat
Rev Neurosci 1, 191–198. [PubMed: 11257907]
Veenema AH, Koolhaas JM, de Kloet ER, 2004. Basal and stress-induced differences in HPA axis, 5HT responsiveness, and hippocampal cell proliferation in two mouse lines. Ann N Y Acad Sci
1018, 255–265. [PubMed: 15240376]
Walker FR, Masters LM, Dielenberg RA, Day TA, 2009. Coping with defeat: acute glucocorticoid and
forebrain responses to social defeat vary with defeat episode behaviour. Neuroscience 162, 244–
253. [PubMed: 19393295]
Wang F, Zhu J, Zhu H, Zhang Q, Lin Z, Hu H, 2011. Bidirectional control of social hierarchy by
synaptic efficacy in medial prefrontal cortex. Science 334, 693–697. [PubMed: 21960531]
Williamson CM, Klein IS, Lee W, Curley JP, 2019a. Immediate early gene activation throughout the
brain is associated with dynamic changes in social context. Soc Neurosci 14, 253–265. [PubMed:
29781376]
Williamson CM, Lee W, DeCasien AR, Lanham A, Romeo RD, Curley JP, 2019b. Social hierarchy
position in female mice is associated with plasma corticosterone levels and hypothalamic gene
expression. Scientific Reports 9, 1–14. [PubMed: 30626917]
Williamson CM, Lee W, Romeo RD, Curley JP, 2017. Social context-dependent relationships between
mouse dominance rank and plasma hormone levels. Physiology & behavior 171, 110–119.
[PubMed: 28065723]
Wingfield JC, Hegner RE, Dufty AM Jr., Ball GF, 1990. The "Challenge Hypothesis": Theoretical
implications for patterns of testosterone secretion, mating systems, and breeding strategies. The
American Naturalist 136, 829–846.
Wingfield JC, Wada M, 1989. Changes in plasma levels of testosterone during male-male interactions
in the song sparrow, Melospiza melodia: time course and specificity of response. Journal of
Comparative Physiology A 166, 189–194.
Wise DA, 1974. Aggression in the female golden hamster: effects of reproductive state and social
isolation. Horm.Behav 5, 235–250. [PubMed: 4474137]
Psychoneuroendocrinology. Author manuscript; available in PMC 2022 July 01.
Cooper et al.
Page 23
Author Manuscript
Wood SK, Walker HE, Valentino RJ, Bhatnagar S, 2010. Individual differences in reactivity to social
stress predict susceptibility and resilience to a depressive phenotype: role of corticotropinreleasing factor. Endocrinology 151, 1795–1805. [PubMed: 20160137]
Yang EJ, Wilczynski W, 2002. Relationships between hormones and aggressive behavior in green
anole lizards: an analysis using structural equation modeling. Horm Behav 42, 192–205. [PubMed:
12367572]
Yehuda R, LeDoux J, 2007. Response variation following trauma: a translational neuroscience
approach to understanding PTSD. Neuron 56, 19–32. [PubMed: 17920012]
Zhou T, Zhu H, Fan Z, Wang F, Chen Y, Liang H, Yang Z, Zhang L, Lin L, Zhan Y, 2017. History of
winning remodels thalamo-PFC circuit to reinforce social dominance. Science 357, 162–168.
[PubMed: 28706064]
Zuo Z, Tam YK, Diakur J, Wiebe LI, 2002. Hydroxypropyl-β-cyclodextrin-flutamide inclusion
complex. II. Oral and intravenous pharmacokinetics of flutamide in the rat. J Pharm Pharm Sci 5,
292–298. [PubMed: 12553899]
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Author Manuscript
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Fig. 1.
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Effects of flutamide injections during development of dominance relationships. A) Timeline
of drug treatments, dominance encounters, social defeat stress, and conditioned defeat (CD)
testing. During CD testing we quantified the duration of B) submissive and defensive, C)
aggressive, D) affiliative, and E) nonsocial behavior. Dominant (Dom) animals treated with
flutamide (Flu) showed increased submissive and defensive behavior compared to dominants
treated with vehicle (Veh). There was also a main effect of social dominance on aggressive
behavior. No effects of flutamide treatment were found in subordinates (Sub) or no status
(NS) animals. F) Photomicrographs showing representative images of androgen receptor
(AR) immunoreactivity (IR) in posterior dorsal and posterior ventral medial amygdala
(MePD and MePV, respectively, 10x magnification, scale bar = 200 μm, OT: optic tract). The
number of AR-positive cells were quantified in the G) MePD and H) MePV. Flutamidetreated dominants showed fewer AR-positive cells compared to vehicle-treated dominants in
both the MePD and MePV. There were no effects of flutamide treatment on AR
immunoreactivity in subordinates (Sub) or no status (NS) animals. Asterisks (*) indicate a
significant difference between treatment groups (p < .05). n = 7-10 animals per group.
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Fig. 2.
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Effects of flutamide microinjection into posterior aspects of the medial amygdala (MeP). A)
Timeline of dominance encounters and flutamide (Flu) or vehicle (Veh) injections prior to
social defeat or conditioned defeat testing. B) The location of microinjections is shown using
illustrations adapted from a hamster stereotaxic atlas. Black circles indicate injection sites
within the MePD or MePV, while open circles indicate injection sites for anatomical
controls. Circles also indicate more than one microinjection. We injected flutamide into the
MeP prior to social defeat and quantified C) submissive and defensive, D) aggressive, E)
affiliative, and F) nonsocial behavior at conditioned defeat testing. Flutamide treatment
increased the duration of submissive and defensive behavior in dominants (Dom) but not
subordinates (Sub). We injected flutamide into the MeP prior to conditioned defeat testing
and quantified the duration of G) submissive and defensive, H) aggressive, I) affiliative, and
J) nonsocial behavior. Flutamide treatment did not alter the conditioned defeat response,
although there was a main effect of dominance status on submissive and defensive behavior
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and affiliative behavior. Asterisks (*) indicate a significant difference between treatment
groups (p < .05). n = 8-10 animals per group.
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Fig. 3.
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Effects of social dominance on gonadal steroid receptors and responses to social defeat
stress in female hamsters. A) Timeline for dominance encounters, blood collection, social
defeat stress, and social avoidance testing. Following social defeat stress, animals received
social avoidance testing and we quantified B) time spent in the interaction zone, C) the
interaction ratio, D) time spent in the far zone, and E) the frequency of flank marks. We
found that dominant (Dom) animals spent more time in the interaction zone compared to
subordinates (Sub) and no status (NS) animals following social defeat stress. In addition, in
animals not exposed to social defeat stress, dominants spent more time in the interaction
zone than subordinates. Further, we found a main effect of social dominance on the
interaction ratio and a main effect of social defeat on time spent in the far zone. Also, there
was an interaction of dominance status and social defeat in the number of flank marks. F)
Photomicrographs showing representative images of estrogen receptor alpha (ERα)
immunoreactivity (IR) in posterior dorsal and posterior ventral medial amygdala (MePD and
MePV, respectively, 10x magnification, scale bar = 200 μm, OT: optic tract). The number of
ERα-positive cells were quantified in the G) MePD and H) MePV. We found the dominant
animals showed more ERα-positive cells in the MePD compared to subordinates and no
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status animals. We also quantified the number of cells showing androgen receptor (AR)
immunoreactivity but found no significant difference in the I) MePD or J) MePV. Asterisks
(*) indicate a significant difference between treatment groups (p < .05). n = 8-12 animals per
group.
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