Essential Role of Ovarian Hormones by Finell Et Al TCR Paper Which is preparing a ½-1-page, single-spaced thought/reaction/comment (TRC) paper on each read

Essential Role of Ovarian Hormones by Finell Et Al TCR Paper Which is preparing a ½-1-page, single-spaced thought/reaction/comment (TRC) paper on each reading (except for the article you are presenting). The TRC should provide interesting or important topics for discussion and should include at least 2-3 questions, as well as comments about and reflections on the topic to stimulate class discussion. Biological
Psychiatry
Archival Report
Essential Role of Ovarian Hormones in
Susceptibility to the Consequences of Witnessing
Social Defeat in Female Rats
Julie E. Finnell, Brandon L. Muniz, Akhila R. Padi, Calliandra M. Lombard, Casey M. Moffitt,
Christopher S. Wood, L. Britt Wilson, Lawrence P. Reagan, Marlene A. Wilson, and
Susan K. Wood
ABSTRACT
BACKGROUND: Women are at greater risk than men of developing depression and comorbid disorders such as
cardiovascular disease. This enhanced risk begins at puberty and ends following menopause, suggesting a role for
ovarian hormones in this sensitivity. Here we used a model of psychosocial witness stress in female rats to determine
the stress-induced neurobiological adaptations that underlie stress susceptibility in an ovarian hormone–dependent
manner.
METHODS: Intact or ovariectomized (OVX) female rats were exposed to five daily 15-minute witness-stress exposures.
Witness-stress–evoked burying, behavioral despair, and anhedonia were measured. Cardiovascular telemetry was
combined with plasma measurements of inflammation, epinephrine, and corticosterone as indices of cardiovascular
dysfunction. Finally, levels of interleukin-1b and corticotropin-releasing factor were assessed in the central amygdala.
RESULTS: Witness stress produced anxiety-like burying, depressive-like anhedonia, and behavioral despair
selectively in intact female rats, which was associated with enhanced sympathetic responses during stress,
including increased blood pressure, heart rate, and arrhythmias. Moreover, intact female rats exhibited increases
in 12-hour resting systolic pressure and heart rate and reductions in heart rate variability. Notably, OVX female
rats remained resilient. Moreover, intact, but not OVX, female rats exposed to witness stress exhibited a sensitized
cytokine and epinephrine response to stress and distinct increases in levels of corticotropin-releasing factor and
interleukin-1b in the central amygdala.
CONCLUSIONS: Together these data suggest that ovarian hormones play a critical role in the behavioral, inflammatory, and cardiovascular susceptibility to social stress in female rats and reveal putative systems that are sensitized to stress in an ovarian hormone–dependent manner.
Keywords: Central amygdala, Corticotropin releasing factor, Cytokine, Heart rate variability, Hippocampus,
Psychosocial stress, Vicarious social defeat
https://doi.org/10.1016/j.biopsych.2018.01.013
Social stress, including abuse or witnessing violent or traumatic events, can precipitate the emergence of psychosocial
disorders such as depression and anxiety (1–3). Women are
more than twice as likely to experience depression and anxiety
compared with men (4,5) and are also at a greater risk of
developing inflammatory-related comorbidities such as coronary heart disease and myocardial infarction (6). This enhanced
susceptibility to the psychological and physiological consequences of stress emerges at the onset of puberty and remains
until menopause (4,5), suggesting that ovarian hormones may
play a crucial role in this enhanced stress sensitivity in women.
Although preclinical studies suggest that ovarian hormones
can confer behavioral (7) and cardiovascular (8) protection
under stress-free conditions, in the context of stress, ovarian
hormones have proven to increase stress susceptibility (9).
Therefore, identification of discrete stress-sensitive systems
that are affected by ovarian hormones under conditions of
social stress will be critical to understanding the mechanisms
that underlie this enhanced stress susceptibility.
Much like stress, ovarian hormones modulate a variety of
physiological processes including inflammation and the
expression of corticotropin-releasing factor (CRF) in the brain
(10,11). Both CRF and inflammation have been reported to be
dysregulated in depression and anxiety (12–18) and are known
to regulate cardiovascular activity (19,20). Specifically, proinflammatory cytokines and CRF increase the activation of brain
regions, including the central amygdala (CeA), that are known
to regulate cardiac, autonomic, endocrine, and behavioral responses to fear (21,22). Importantly, innate, fearful responses
such as burying, avoidance of open spaces, startle responses,
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Ovarian Hormone–Induced Sensitivity to Witness Stress
and anhedonia are driven in part by the CeA (23–25). Moreover,
behavioral responses regulated by CeA occur in parallel with
activation of the sympathetic nervous system; CeA activation
increases blood pressure (BP) and heart rate (HR) in awake rats
(26,27), and CeA lesions decrease the hemodynamic response
to noise stress (28). Because the CeA exhibits sensitivity to
ovarian hormones (24,29), it represents an important target to
study the pathogenesis of comorbid behavioral and cardiovascular dysfunction. Like the CeA, the hippocampus is a region that is sensitized to stress (30–32), CRF (33), and ovarian
hormones (34) and can regulate HR (35) and behavioral responses to fear (36). For these reasons, the hippocampus is an
ideal region to assess along with the CeA to determine whether
social stress demonstrates global or region-specific effects
(34,36).
Our current understanding of social stress susceptibility in
female populations is limited because of the difficulty associated with conducting social stress or defeat manipulations
using female animals. Several research teams have successfully utilized social defeat in female animals; however, this
approach requires the use of a different species (e.g., highly
territorial Syrian hamsters or California mice), use of a lactating
female resident, or modification of the male resident rat or
mouse via the use of DREADDs (designer receptors exclusively
activated by designer drugs) to activate the ventromedial hypothalamus (37–45). While the physical interaction of social
defeat is effective in adult female animals, some studies suggest that it may have a greater impact on male rodents than
female rodents (37,41). A recent modification to the resident–
intruder paradigm consists of a rodent bearing witness to a
social defeat episode between a male resident and a male
intruder (46–49). This model effectively combines social isolation and visual, olfactory, and auditory exposure to social
defeat. Studies utilizing this model have identified that both
physical (intruder) and psychological (witness or vicarious)
social stress results in the emergence of robust anxiety- and
depressive-like behaviors (46–50) as well as enhanced peripheral immune (50,51) and neuroendocrine (49) responses in
male animals. Moreover, important studies have recently
emerged documenting the effectiveness of witness stress in
inducing depressive- and anxiety-like behaviors in female mice
(52), yet whether ovarian hormones mediated susceptibility
was not determined. The aim of the current study was to
identify whether female rats display susceptibility to the
behavioral and cardiovascular consequences of repeated witness stress and whether these responses are dependent on
the presence of ovarian hormones. Additionally, CRF and
neuroinflammation were assessed in the CeA and hippocampus to determine a putative neuronal mechanism.
12-hour light/dark cycle in standard cages with ad libitum
access to food and water. The care and use of animals was
approved by the University of South Carolina’s Institutional
Animal Care and Use Committee, and the study was conducted in accordance with the National Research Council’s
Guide for the Care and Use of Laboratory Animals.
Study Design
Four separate studies were conducted as outlined in Figure 1.
Surgery
To minimize inflammatory consequences of surgical manipulation and ensure washout of endogenous hormones following
ovariectomy (53), all surgical procedures were conducted 10
days prior to the start of the stress or control mainpulations. At
approximately 9 weeks of age, all female rats were subject to
brief ovary manipulation (intact group) or surgical removal of
ovaries (OVX group). Additionally, female rats in studies A to C
(Figure 1) were implanted with HD-S11 radiotelemetric transmitters (Data Sciences International, St. Paul, MN) in the
modified lead II configuration as published previously (50,54).
Cycle Tracking
The estrous cycle was monitored in studies C and D using
daily lavage (Supplemental Figure S2) to determine whether
stress-induced behavioral or cardiovascular deficits were
estrous cycle dependent. Importantly, estrous cycle length
was unaffected by exposure to witness stress (Supplemental
Table S2).
Social Stress
Witness Stress. Female witnesses were briefly placed into
the cage of a novel resident behind a clear perforated acrylic
partition where they observed social defeat between the novel
male resident and male intruder for 15 minutes each day for 5
consecutive days (Supplemental Figure S1). This stress duration was chosen as we had previously determined that 5 days
of repeated witness stress was sufficient to induce behavioral,
cardiovascular, and inflammatory consequences in male rats
(50). Control animals were handled for 15 seconds each day.
Male-Paired Controls. To determine whether stress effects in intact female rats were driven merely by exposure to a
male rat independent of social defeat, study B evaluated the
behavioral and cardiovascular effects of intact female rats
placed behind the clear perforated acrylic partition in the home
cage of a male Sprague Dawley rat (15 minutes each day for 5
days) in the absence of the aggressive resident.
METHODS AND MATERIALS
Cardiovascular Telemetry
A complete discussion of methods and materials is presented
in the Supplement.
All cardiovascular data collected in studies A to C were acquired via Dataquest ART software (Data Sciences International) and analyzed in the Ponemah physiology platform (Data
Sciences International) as previously published (50).
Animals
Female Sprague Dawley rats (175–200 g, control rats, witness
rats), male Sprague Dawley rats (225–250 g, intruders), and
male Long-Evans retired breeders (650–850 g, residents)
(Charles River, Durham, NC) were individually housed under a
Resting Dark Cycle Systolic BP and HR. HR and BP
were collected for 9 consecutive days. Data were calculated as
a change from baseline using each individual rat’s 12-hour
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Ovarian Hormone–Induced Sensitivity to Witness Stress
Figure 1. Study design and timeline. Four separate experiments were used for the completion of
this study. (A) This panel depicts the main stress
study from which most of the behavioral, cardiovascular, and physiological data were collected
(intact control rats n = 5; intact witness rats n = 7;
ovariectomized [OVX] control rats n = 15; OVX witness rats n = 11). (B) To ensure that the robust
cardiovascular and behavioral consequences of
witness stress in intact female rats were not driven
solely by the presence of a male rat (in the absence
of defeat), a separate set of intact female rats were
placed behind a clear perforated acrylic partition in
the cage of a male intruder (intact female rats n = 6).
(C) To determine whether estrous cycle was affected
by witness stress and whether the stage of estrous
cycle contributed to the cardiovascular effects of
witness stress, a telemetry study was conducted in
which female rats were lavaged daily for 20
consecutive days (intact control rats n = 12; intact
witness rats n = 7). (D) As an additional measure of
depressive-like behavior induced by witness stress,
rats were subjected to the Porsolt forced swim test
(FST). Rats were also lavaged to determine whether
estrous cycle stage biased behavior during the
forced swim test (OVX control rats n = 8; OVX witness rats n = 8; intact control rats n = 10; intact
witness rats n = 8). (The x-ray transmitter illustration
is reproduced with permission from Data Sciences
International.)
dark cycle average, which was collected 48 hours prior to the
initial stress or control (Supplemental Table S1).
scored using a time-sampling technique by two experimenters
blinded to animal conditions (62–64).
Resting Dark Cycle HR Variability. Frequency domain
Context Re-exposure and Tissue Collection
analysis of HR variability (HRV) was conducted to determine the
spectral power within the low-frequency domain (LF), which is a
nonspecific index of sympathetic activity (55), and the highfrequency domain (HF), which is the parasympathetic (vagal)
activity (56), in addition to the LF:HF ratio, which is an estimate of
sympathovagal balance (57), as previously published (54,58,59).
Tissues collected from rats in study A were used for physiological
and neuroendocrine analyses. To screen for systems that may be
sensitized following witness-stress exposure, female rats in study
A were subjected to a 15-minute context re-exposure immediately before euthanasia (day 10): Witnesses were placed behind
the clear perforated acrylic partition in a soiled resident cage in
the presence of the intruder but absence of the resident; control
animals were handled and returned to their home cage. Brains
were flash frozen with ice-cold 2-methylbutane, hearts were
dissected to obtain ventricular weights, plasma was collected
following centrifugation (50,61), and uteri were weighed for verification of OVX (Supplemental Figure S5).
Cardiovascular Response to Stress or Control Exposure. On days 1 and 5 of the witness or control exposure,
continuous cardiovascular recordings (mean arterial pressure and HR) were obtained while animals rested in the
home cage (30 minutes before stress or control exposure)
and used to calculate the change from each rat’s baseline
during stress or control exposure. Premature ventricular
contractions were also quantified during baseline and stress
exposure on days 1 and 5 as previously described (50).
Behavioral Measurements
Video recordings of witness stress on days 1 and 5 were scored
for the spontaneous expression of witness stress–evoked burying
by an experimenter blinded to the animal’s treatment (studies A–
C). Anhedonia was assessed using the two-bottle-choice sucrose
preference test ([volume 1% sucrose/total volume consumed] 3
100%; studies A, B) as described previously (50,60,61). Behavioral despair was assessed using the Porsolt forced swim test
(study D) as described previously (62,63), and the water depth
was modified to 35 cm. Video recordings of test sessions were
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Measurement of Peripheral Substrates
Plasma corticosterone, cytokine, and chemokine concentrations in response to contextual re-exposure were assessed per
manufacturer protocol with enzyme-linked immunosorbent
assay (Enzo Life Sciences, Farmingdale, NY) and a Bio-Plex
multiplex assay (Bio-Rad Laboratories, Hercules, CA),
respectively. Plasma epinephrine concentration was determined using high-performance liquid chromatography as previously described (50,61).
Measurement of CeA and Hippocampal Substrates
Frozen brains were sliced coronally to the level of the CeA (65)
and hippocampus (65). Bilateral 131-mm punches were obtained and histologically verified (Supplemental Figures S3, S4).
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Ovarian Hormone–Induced Sensitivity to Witness Stress
Tissue was homogenized and assayed for protein concentration
using a Pierce bicinchoninic acid assay (Thermo Fisher Scientific,
Waltham, MA) as previously described (50,61). Western blotting
was used to quantify CRF expression in the CeA and hippocampus using rabbit anti-CRF (1:5000; Abcam, Cambridge,
United Kingdom), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (1:1000; Abcam), goat anti-rabbit IRDye 680RD
(1:15,000; LI-COR Biosciences, Lincoln, NE), and goat antimouse IRDye 800CW (1:15,000; LI-COR Biosciences) as
previously described (50). Neither stress (F1,33 = 0.7, p = .4) nor
hormonal condition (F1,33 = 0.3, p = .6) altered the normalizing
protein glyceraldehyde-3-phosphate dehydrogenase. CeA
neuroinflammation was determined using a Bio-Plex assay
(Bio-Rad Laboratories) as previously described (61).
Statistical Analysis
The modified Thompson s outlier test identified statistical outliers as previously described (50,61). Statistical analysis was
conducted using GraphPad Prism 6 (GraphPad Software, La
Jolla, CA) and SAS JMP 10 (SAS Institute, Cary, NC) software.
Multivariate analysis of variance (a = .05) with repeated measures
was conducted for the Porsolt forced swim test and cardiovascular data to determine effects of stress, hormone, and time.
Witness-stress–induced burying and hormone-induced differences in resting HRV between intact and OVX rats were analyzed
using standard two-tailed unpaired t tests (a = .05). All other data
sets were analyzed using standard two-way analyses of variance
(a = .05) to determine the effects of stress and hormone. All
analyses of variance were followed by a Fisher’s least significant
difference post hoc analysis. Statistical statements from data
represented in the figures are included in each figure legend.
RESULTS
Witness-Stress Exposure Produces Anxiety-like
Behaviors in Intact Female Rats
Intact female rats exhibited significantly greater anxiety-like
behaviors, as evidenced by shorter latencies to begin witnessstress–evoked burying (mean 6 SEM: intact witnesses, 25.7 6
7.4 seconds; OVX witnesses, 65.3 6 5.7 seconds; t9 = 4.8, p ,
.001) and greater bury durations (Figure 2A) compared with OVX
witnesses. Although control-handling sessions for study A were
not video recorded, exposure to the male-paired control
manipulation was evaluated (Supplemental Figure S11A) and
did not significantly induce burying behaviors. Interestingly,
intact witnesses exhibited habituation to the anxiogenic effects
of witness stress, exhibiting similar burying behaviors to that of
OVX witnesses by the fifth stress exposure (latency, t12 = 0.0,
p = 1.0; duration, t11 = 0.9, p = .4; data not shown). While burying
behavior exhibited by intact female rats was independent of the
estrous cycle stage (Supplemental Figure S6), preliminary data
indicate that estradiol replacement using time-release capsules
reinstated burying behavior in OVX witnesses (Supplemental
Figure S9A). These distinct group differences in witnessstress–evoked burying (as well as the cardiovascular and inflammatory findings described below) were not driven by differences in the quantity or intensity of observed attacks, as the
average number of attacks received by the intruder was the
same regardless of the hormonal status of the witness behind
the partition (Supplemental Table S3).
Intact Female Rats Selectively Exhibit StressInduced Depressive-like Behaviors
Repeated witness stress selectively induced anhedonia in
intact female rats as measured by the sucrose preference test
(Figure 2B). These anhedonic effects were not driven by
differences in total fluid intake (effect of hormone, F1,33 = 0.9,
p = .4; effect of stress, F1,33 = 0.4, p = .5) or preexisting
differences in sucrose preference (effect of hormone, F1,39 =
2.1, p = .2; effect of stress, F1,39 = 0.2, p = .7) and did not occur
following exposure to a male in the absence of social defeat
(Supplemental Figure S11B). Moreover, estradiol replacement
did not reinstate anhedonic behaviors in OVX witnesses
(Supplemental Figure S9B). In intact female rats, witness stress
also induced behavioral despair, as measured by increased
immobility (Figure 2C) and shorter climbing durations in the
Porsolt forced swim test (Figure 2D), which is indicative of
shifts in noradrenergic neurotransmission (66), compared with
those of intact con…
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