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Effects of estrus cycle stage on defensive behavior in female Long-Evans hooded rats
Accepted Manuscript Effects of estrus cycle stage on defensive behavior in female Long-Evans hooded rats
Nathan S. Pentkowski, Yoav Litvin, D. Caroline Blanchard, Robert J. Blanchard PII: DOI: Reference:
S0031-9384(18)30209-9 doi:10.1016/j.physbeh.2018.04.028 PHB 12179
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
26 November 2017 20 April 2018 20 April 2018
Please cite this article as: Nathan S. Pentkowski, Yoav Litvin, D. Caroline Blanchard, Robert J. Blanchard , Effects of estrus cycle stage on defensive behavior in female LongEvans hooded rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phb(2018), doi:10.1016/j.physbeh.2018.04.028
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ACCEPTED MANUSCRIPT Effects of estrus cycle stage on defensive behavior in female Long-Evans hooded rats. Nathan S. Pentkowskia,1* , Yoav Litvina, D. Caroline Blanchardb,c and Robert J. Blancharda,b a
Department of Psychology, University of Hawaii at Manoa, 2530 Dole Street, Sakamaki C 400, Honolulu, Hawaii USA 96822
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Pacific Biosciences Research Center, University of Hawaii at Manoa, 1993 East-West Road, Honolulu, Hawaii USA 96822 c
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John A. Burns School of Medicine, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, Hawaii USA 96813
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Corresponding author: Nathan S. Pentkowski, Ph.D. Department of Psychology University of New Mexico 1 University of New Mexico MSC03 2220 Logan Hall Albuquerque, NM USA 87131 Phone: (505) 277-1928 Email: [email protected]
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Present address: 1 Department of Psychology, University of New Mexico, 1 University of New Mexico, MSC03 2220, Logan Hall, Albuquerque, New Mexico USA 87131
ACCEPTED MANUSCRIPT Abstract This study investigated the influence of the estrus cycle in mediating cat odor-induced unconditioned and conditioned defensive behaviors in female Long-Evans hooded rats. Unconditioned defensive behaviors were assessed during predatory cue exposure;
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conditioned defensive behaviors were examined twenty-four hours after threat exposure.
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Estrus phases were determined by microscopic examination of vaginal smears within 10
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min of completing the behavioral tests. Compared to no-odor controls, female rats exposed to cat odor exhibited both unconditioned and conditioned defensive behaviors,
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including elevated levels of freezing, risk assessment and avoidance. Rats in proestrus
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and estrus exhibited reduced levels of defensive behavior during the unconditioned test trial compared to subjects in diestrus and metestrus. Specifically, estrus stages
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characterized by high levels of circulating estrogens and progesterone were associated
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with reduced immobility (i.e. freezing) and enhanced active defense (i.e. risk assessment), profiles that may enable mate seeking and subsequent reproduction in
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potentially dangerous or novel environments. These results suggest a specific role for
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ovarian hormone fluctuations in mediating unconditioned fear- and anxiety- like defensive
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behaviors during exposure to predatory odors.
Keywords
Defense, Anxiety, Fear, Estrus, Female, Estrogen, Predator, Freezing, Risk assessment
ACCEPTED MANUSCRIPT 1. Introduction Anxiety and stress disorders are the most prevalent psychiatric illnesses, with several forms such as generalized anxiety disorder, posttraumatic stress disorder and panic disorder disproportionately affecting women more than men [1-4]. Fluctuations in levels
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of estrogen and progesterone during the menstrual cycle may be related to depression and
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anxiety susceptibility [5-8]. Proestrus (P) and estrus (E) are have higher concentrations of
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circulating progesterone and estrogen, with peak levels occurring during P followed by fluctuations throughout estrus stages [9, 10]. In rats, P and E stages are characterized by
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reduced anxiety- like behavior [11]. For instance, rats in P and E exhibit increased time
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spent in the open arms of an elevated plus-maze (EPM) [12, 13] and obtain more punished reinforcers compared to rats in diestrus (D) and/or metestrus (M) [14]; the latter
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deficits in D and M are prevented by diazepam. Studies have linked anxiolysis with P,
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reporting reduced anxiety- like behaviors in the light-dark test [15], EPM [16-18], punished responding [19], social interaction test [16] and defensive burying test [16, 20].
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In stark contrast, other studies indicate that rats in P and E exhibit enhanced anxiety-like
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behavior in the EPM [21, 22], or report no effect of estrus stage on anxiety- like behaviors in the EPM [23, 24] or defensive burying test [24]. Based on these equivocal reports,
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further characterization of neuroendocrine influences on anxiety-like behavior using novel animal models is needed in order to develop more effective pharmacological treatments for anxiety disorders in women. In order to understand the neurobiological mechanisms underlying fear- and anxietyrelated pathologies, considerable efforts have been made to develop animal models for human anxiety disorders [25]. In laboratory settings, evaluation of antipredator defensive
ACCEPTED MANUSCRIPT behaviors allows for an ethological analysis of anxiety- like behavior by exposing rats to natural predatory cues, such as cat odor. In particular, some of the defensive behaviors elicited by these naturalistic threats, e.g., risk assessment and freezing, [26, 27] are responsive to traditional anxiolytics such as diazepam and 5-HT1A agonists [28-31].
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Importantly, female rats show enhanced antipredator defensive behavior (e.g., increased
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risk assessment and decreased contact) in response to potential (e.g., cat odor) as opposed
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to distinct (e.g., live cat exposure) threat sources, as well as differential reactivity in such situations to classic anxiolytics [32].
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To further clarify the influence of the estrus cycle on anxiety- like defensive behavior,
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this study examined unconditioned and conditioned behavioral responses in female rats exposed to cat odor. We hypothesized that fluctuations in levels of ovarian hormones
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typical of the various estrus stages would differentially modulate defensive behavior,
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with higher levels associated with reduced anxiety-like responsivity. Accordingly, we predicted that this decrease in defensiveness would manifest during P and E, stages that
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are characterized by comparatively higher levels of estrogen and progesterone compared
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to M and D.
2. Materials and Methods
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2.1 Subjects
Subjects were adult female Long-Evans hooded rats born and reared in the Snyder Hall breeding colony at the University of Hawaii at Manoa. Following weaning (21 days), all rats were singly housed in standard home cages (21.6 x 45.7 x 17.8 cm) under controlled temperature (21 ± 2 ◦C) and illumination (12-h light/dark cycle, lights on at 06:00 a.m.) with free access to food (rat chow) and water. Rats (n=53) were 11 to 13
ACCEPTED MANUSCRIPT weeks of age at the start of behavioral testing. All husbandry and experimentation adhered to the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and all experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at The University of Hawaii.
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2.2 Cat Odor Exposure
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Detailed procedures for the cat odor test have been previously described [33-35].
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Briefly, the test apparatus consisted of a white Plexiglas runway (100 x 12 x 50 cm) with a clear front panel to permit observation and recording. Separate cloth-wrapped solid
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plastic blocks (9 x 9 x 2 cm) served as the cat odor and control stimuli. The cat odor
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block was rubbed for 5 min against the fur of a laboratory-housed domestic male cat for 3 consecutive days prior to use. In order to control for potential novelty effects, control rats
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were exposed to an identical cloth-wrapped plastic block never exposed to cat odor. All
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rats were habituated to the apparatus for 10 min over 3 consecutive days without the presence of a block stimulus. On the following day (unconditioned behavior test), the
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appropriate block stimulus was placed at one end of the runway and a test subject was
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placed at the opposite end. Twenty-four hours later each rat was tested in the same apparatus without the cat odor stimulus (cue+context conditioning test). A cloth-wrapped
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solid plastic block never exposed to cat odor served as the cue during the conditioned test trial. All sessions were 10 min in duration and were conducted under red light in order to ensure that the threat stimulus remained as ambiguous as possible. Rats were tested in the same order across days, with testing occurring between 11:00 a.m. and 3:00 p.m. In order to insure that control rats were not exposed to cat odor, control and cat odor exposed
ACCEPTED MANUSCRIPT subjects were tested in separate rooms, using identical test chambers. Between trials each apparatus was thoroughly cleaned using a 10% ethanol solution. 2.3 Behavioral Measures Defensive behaviors analyzed in each test situation included: crouch freeze-complete
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cessation of movement other than respiration; risk assessment behaviors including stretch
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approach-forward ambulation with flat back and stretched neck, stretch attend-standing
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on all 4 paws with flat back and stretched neck orientated toward the threat source, and crouch sniff-olfactory investigation evidenced by vertical or lateral head movements
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(scoring initiated when nose visibly moved more than 1 cm); avoidance-duration of time
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spent in the far compartment relative to the threat- or control-block stimulus; and contactdirect contact with the block stimulus, measured as direct paw or head contact.
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Locomotor activity-crossing of lines marking the far, medium and near thirds of the
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apparatus was measured to examine potential nonspecific changes in ambulation. Behavioral measures represent the durations of events in seconds (or numbers of line
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crossings) during a 10 min observation period.
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2.4 Behavioral Analysis
All test trials were recorded using Pioneer DVD recorders, and were later analyzed
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using the behavioral analysis software Observer 5.0 (Noldus Information Technology, Wageningen, The Netherlands) by a highly trained observer blind to test conditions. Use of this software allowed for a detailed, frame-by-frame analysis. 2.5 Determination of Estrus Cycle Estrus cycle phases were determined by the consensus of three research assistants using bright-field microscopic examination (10x and 40x magnification) of vaginal
ACCEPTED MANUSCRIPT smears collected within 10 min of testing in both the unconditioned and conditioned cat odor tests as previously described [9, 36, 37]. Proestrus was identified by the predominance of nucleated epithelial cells, E was identified by the presence of dense sheets of cornified epithelial cells, M was identified by the presence of scattered,
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nucleated, or cornified epithelial cells and leukocytes, and D was identified by the
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presence of leukocytes [11].
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2.6 Statistical Analysis
To examine the effects of cat odor on unconditioned and conditioned defensive
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behaviors separate t-tests were performed on each dependent measure with data from
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each estrus cycle stage pooled across groups. To examine the influence of each estrus stage on cat odor-induced defensive behaviors separate one-way analysis of variance
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(ANOVA) were performed on each dependent measure. Significant ANOVAs were
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followed by post-hoc Tukey’s HSD tests to provide pairwise comparisons; α was set at 0.05 for all statistical comparisons.
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3. Results
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3.1 Estrus Determination
Of the 32 rats exposed to the predatory cat odor, 12 were tested during diestrus, 6
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during metestrus, 6 during proestrus and 8 during estrus. One-way ANOVA failed to demonstrate any significant behavioral effects between estrus stages in rats exposed to the control block. Thus, for ease of comparison control rats from each estrus stage were pooled into a single control group (n=21). 3.2 Effects of Cat Odor on Unconditioned Defensive Behavior
ACCEPTED MANUSCRIPT Figure 1 illustrates the effects of predator odor exposure on unconditioned defensive behaviors during the cat odor test with data from each estrus cycle stage pooled. Exposure to cat odor produced robust increases in defensive behavior. Compared to controls, rats exposed to cat odor exhibited increased levels of crouch freezing
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[t(51)=5.27, p<0.001] and suppressed locomotor activity [t(51)=8.10, p<0.001; control:
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55.67 ± 3.49, cat odor: 16.69 ± 3.15]. Rats demonstrated a clear aversion to the cat odor
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stimulus, exhibiting higher levels of avoidance [t(51)=7.48, p<0.001] and less contact [t(51)=11.56, p<0.001] with the cat odor block compared to controls. Cat odor exposure
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also potentiated risk assessment behaviors, including a reliable increase in stretch attend
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[t(51)=2.88, p<0.01] and a trend toward increased stretch approach [t(51)=1.92, p=.06], but no effect on crouch sniffing (p>0.05).
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3.3 Influence of Estrus stage on Cat Odor-Induced Unconditioned Defensive Behavior
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Figure 2 illustrates the influence of estrus stage on predator-induced defensive behaviors during the unconditioned cat odor test. The ANOVA indicated a significant
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effect of estrus stage on the duration of crouch freezing [F(3, 28)=6.68, p<0.005]. Post-
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hoc analyses revealed that rats in D exhibited higher durations of freezing compared to rats in P and E (Tukey’s, p<0.05 in each case). Cat odor-elicited risk assessment was also
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influenced by estrus stage. The ANOVA indicated a difference in the duration of crouch sniffing [F(3, 28)=6.15, p<0.005], with rats in P and E exhibiting higher levels compared to rats in D (Tukey’s, p<0.05 in each case). There was no effect of estrus stage on the durations of avoidance, contact, stretch approach, stretch attend or or the number of line crossings (P: 19.00 ± 8.14 , E: 21.75 ± 9.38, M: 17.83 ± 5.74, D: 11.58 ± 3.26). 3.4 Effects of Cat Odor on Conditioned Defensive Behavior
ACCEPTED MANUSCRIPT Figure 3 illustrates the effects of predator odor-induced conditioned defensive behaviors during the cat odor cue+context test with data from each estrus cycle stage pooled. Exposure to the cat odor cue+context induced a characteristic pattern of enhanced conditioned defensiveness. Compared to controls, rats previously exposed to cat odor
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exhibited increased levels of conditioned crouch freezing [t(51)=3.46, p<0.005] and
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suppressed locomotor activity [t(51)=3.05, p<0.005; control cue: 49.24 ± 3.66, cat odor
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cue: 31.03 ± 4.19]. Rats previously exposed to cat odor demonstrated a clear aversion to the cat odor cue stimulus, exhibiting higher levels of conditioned avoidance [t(51)=8.67,
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p<0.001] and less contact [t(51)=8.66, p<0.001] with the cue control block compared to
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controls never exposed to cat odor. Cat odor cue+context exposure also potentiated conditioned risk assessment behaviors, including reliable increases in stretch attend
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[t(51)=2.53, p<0.05] and stretch approach [t(51)=2.27, p<.05], but no effect on crouch
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sniffing (p>0.05).
3.5 Influence of Estrus stage on Cat Odor-Induced Conditioned Defensive Behavior
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Figure 4 illustrates the influence of estrus stage during initial threat exposure on
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conditioned defensive behaviors during the cat odor cue+context test. Estrus stage during the unconditioned test trial did not significantly alter any defensive behaviors during the
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cat odor cue+context conditioning test (p>0.05 in each case) and there was no effect on locomotor activity (P: 34.50 ± 11.71, E: 34.38 ± 9.00, M: 43.00 ± 10.41, D: 21.08 ± 5.03). Additionally, conditioned defensive behaviors did not differ between groups when the data were re-analyzed according to the estrus stage determined immediately (within 10 min) following cue+context testing (data not shown). 4. Discussion
ACCEPTED MANUSCRIPT Results from the present experiments demonstrate for the first time that fluctuations in ovarian hormones during stages of the estrus cycle modulate unconditioned defensive behaviors during exposure to an ethologically relevant predatory threat source (cat odor). Regardless of estrus stage, cat odor increased defensive responsivity in female rats,
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increasing freezing, risk assessment and avoidance, while suppressing locomotion and
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contact with the predatory threat stimulus (see Figure 1). This defensive profile is
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partially estrus stage-dependent, as rats in P and E exhibited reduced freezing and increased risk assessment (crouch-sniffing) compared to rats in D and M (see Figure 2), a
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shift in defensive responsivity similar to reducing the level of environmental threat (i.e., a
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gradual shift from immobility to cautious exploration, eventually leading to a return to non-defensive behaviors). These results extend upon previous research indicating that
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rats in E and/or P exhibit reduced anxiety- like behaviors in the EPM [12, 13, 16-18],
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punished responding conflict models [14, 19], light-dark test [15], social interaction test [16] and defensive burying test [16, 20] compared to rats in D and/or M.
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Similar to previous reports examining conditioning in 18-38-day old female pups [35]
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or female adults [38], rats in the present study demonstrated heightened conditioned defensiveness during exposure to the cat odor-paired cue+context including increased
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freezing, risk assessment and avoidance, as well as reduced locomotion and cue contact (see Figure 3). However, in contrast to the influence of estrus stage on unconditioned defensive behaviors, there were no reliable differences between estrus groups on defensive responsivity during the cat odor cue+context test. Non-significant effects were detected when estrus cycle stages were measured either immediately following unconditioned threat exposure (see Figure 4) or immediately following cue+context re-
ACCEPTED MANUSCRIPT exposure (data not shown), suggesting that estrus stage did not impact conditioned fear acquisition or expression. These null effects of estrus stage during conditioned test trials are consistent with a previous report that failed to find an effect of estrogen replacement in ovariectomized females on cat odor-induced conditioned fear [38].
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The influence of estrus stage on conditioned behavioral responsivity appears to be
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threat-source specific as estrus stage has been implicated in the expression of conditioned
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defensive behavior and fear extinction using footshock [39, 40] as the threat source [for review see: 41]. To confirm this hypothesis, future studies examining estrus stage effects
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on predator odor-induced conditioning should test for conditioned defensive behaviors
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more than 24 hours after initial threat exposure to minimize potential carryover stress effects. Additionally, although previous studies have firmly established that cat odor
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induces conditioned defensive behaviors in adult male rats by examining the effects of
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novel cues and/or contexts across several days of extinction [42], experiments utilizing adult female rats have not included these controls.
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Rodent antipredator defensive behaviors are valid indices of anxiety-related behaviors
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in humans [43, 44]. Therefore, the shift in cat odor-induced defensive responsivity demonstrated by rats in P and E suggests that estrogen and progesterone mediate
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antipredator defensive behaviors related to anxiety. In support of this notion, situations in which rats are exposed to potential or anticipated threat (cat odor, cat-paired contexts) evoke anxiety- like behaviors [45], an assertion supported by pharmacological data [29]. Indeed, classic anxiolytics such as the benzodiazepines diazepam and chlordiazepoxide [46], alcohol [47], the 5-HT1A agonists 8-OH-DPAT and Gepirone [48], and the tricyclic antidepressant imipramine [49] decrease freezing and avoidance while increasing risk
ACCEPTED MANUSCRIPT assessment during exposure to contexts associated with a cat, while the benzodiazepine midazolam decreases freezing and avoidance during cat odor exposure [50]. Similarly, in the present study we detected a shift from passive (freezing) to active (risk-assessment) defensiveness in females with higher levels of ovarian hormones (P and E). Although this
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shift in defensiveness could result from high levels of ovarian hormones producing
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behavioral disinhibition, this explanation is unlikely as the changes in the defensive
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repertoire were independent of effects on locomotor activity (line crossings). The neurobiological mechanisms mediating the estrus stage dependent expression of
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antipredator defensive behavior may involve estrogen and/or progesterone signaling in
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the accessory olfactory system. The accessory olfactory system mediates responsivity to both conspecific and predatory olfactory cues [50, 51]. Estrogens affect social
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recognition in mice via interaction with the oxytocinergic system in the medial amygdala
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[52, 53], a component of the accessory olfactory system that preferentially modulates innate defensive behaviors to cat odor [51, 54]. Importantly, oxytocin binding in the
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medial amygdala promotes prosocial behaviors by attenuating anxiety [52, 53].
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Furthermore, in ovariectomized rats estradiol administration reduces anxiety- like defensive behaviors during cat odor exposure [38], as well as testing in the open field
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[39] and EPM [55]. Lastly, rats in P displaying reduced anxiety in the EPM have higher levels of estradiol compared to D, and estradiol administration to rats in D eliminated their anxiogenic profile [18]. Although not directly tested in the present study, our results may involve progesterone conversion to allopregnanolone and subsequent anxiolysis via agonism at GABAA receptors [56-59]. Importantly, these processes occur in the amygdala [60] during both
ACCEPTED MANUSCRIPT behavioral E [58] and P [16]. An alternative view of the role of estrogen and progesterone effects on defensive behavior should also be considered. Rather than elevated estrogen and/or progesterone signaling during P and E reducing anxiety- like defensive behavior, enhanced defensiveness in D and M may result from falling levels of progesterone and
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allopregnanolone coupled with low stable levels of estrogen in late D [10]. Indeed,
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previous reports indicate that these types of fluctuations in ovarian hormones enhance
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anxiety- like behavior [61-64].
In summary, during predator odor exposure we suggest that estrogen and progesterone
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signaling, likely involving the medial amygdala, mediates a transition from passive
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immobility/freezing to active risk assessment by attenuating fear and promoting high levels of investigative defenses that enable approach, while maintaining vigilance. The
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adaptive utility of such a mechanism is evident; high levels of ovarian hormones needed
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for reproduction would also sustain mate seeking in the face of a potential predatory threat. From an evolutionary perspective, this behavioral profile could promote mate
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seeking and subsequent reproduction in natural environments that contain a persistent, yet
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ambiguous level of predatory threat. Here, reproduction serves as an evolutionary motivation, in addition to the acquisition of resources (i.e. food and shelter) only during
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times of receptivity, and therefore a reduction in fear (defensive) levels enables efficient exploratory behaviors that would otherwise be unnecessary. The precise neural mechanisms that regulate these behavioral processes, as well as the potential involvement of ovarian hormone modulation of accessory olfactory information and defense systems require further investigation. 5. Conclusions
ACCEPTED MANUSCRIPT The present results indicate that estrus stages characterized by higher levels of circulating estrogen and progesterone (P and E) are associated with reduced freezing and enhanced risk assessment, profiles that may promote mate seeking and subsequent reproduction in novel and/or potentially dangerous environments. These results implicate
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Acknowledgements
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like defensive behaviors during exposure to predatory odors.
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fluctuations in ovarian hormones in the expression of unconditioned fear- and anxiety-
The authors thank Chris Markham, Amy Vasconcellos, Joel Sabugo and Mark Ellis
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for their technical assistance.
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Funding source
This research did not receive any specific grant from funding agencies in the public,
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commercial, or not-for-profit sectors.
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Competing Interests
The authors have no conflicts of interest to report or any involvement to disclose,
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financial or otherwise, that may bias the conduct, interpretation or presentation of this
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work.
ACCEPTED MANUSCRIPT Figure captions Figure 1.Effects of cat odor exposure on unconditioned defensive behaviors with data from each estrus cycle stage pooled. Rats exposed to cat odor (filled bars) exhibited enhanced defensiveness compared to controls (clear bars). Defensive behaviors include
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freezing (A), stretch attend (B), stretch approach (C), crouch sniffing (D), and odor block
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avoidance (E) and contact (F). Asterisk (*) represents a difference compared to controls
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(t-test, p<0.05 in each case). Behavioral measures represent the durations of events in
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seconds (s) during the 10 min test period.
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Figure 2.Effects of estrus stage on cat odor-induced unconditioned defensive behaviors. Rats in proestrus (P) and estrus (E) exhibited reduced defensive behavior compared to
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rats in diestrus (D) and metestrus (M). Defensive behaviors include freezing (A), stretch
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attend (B), stretch approach (C), crouch sniffing (D), and odor block avoidance (E) and contact (F). Asterisk (*) represents a difference compared to diestrus (Tukey’s HSD test,
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p<0.05 in each case). Behavioral measures represent the durations of events in seconds
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(s) during the 10 min test period.
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Figure 3.Effects of cat odor cue+context exposure on conditioned defensive behaviors with data from each estrus cycle stage pooled. Rats previously exposed to cat odor (filled bars) exhibited enhanced conditioned defensiveness compared to controls (clear bars). Defensive behaviors include freezing (A), stretch attend (B), stretch approach (C), crouch sniffing (D), and odor block avoidance (E) and contact (F). Asterisk (*) represents a
ACCEPTED MANUSCRIPT difference compared to controls (t-test, p<0.05 in each case). Behavioral measures represent the durations of events in seconds (s) during the 10 min test period.
Figure 4.Effects of estrus stage on conditioned defensive behaviors during cat odor
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cue+context exposure. There were no significant differences between estrus groups
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[proestrus (P), estrus (E), diestrus (D), metestrus (M)]. Defensive behaviors include
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freezing (A), stretch attend (B), stretch approach (C), crouch sniffing (D), and odor block
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seconds (s) during the 10 min test period.
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avoidance (E) and contact (F). Behavioral measures represent the durations of events in
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ACCEPTED MANUSCRIPT Highlights
Cat odor exposure induced both unconditioned and conditioned defensive behaviors in female rats.
Rats in proestrus and estrus exhibited lower levels of unconditioned defensive
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Higher levels of estrogen and/or progesterone may enable mate seeking in
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potentially dangerous environments.
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behavior.
Nathan S. Pentkowski, Yoav Litvin, D. Caroline Blanchard, Robert J. Blanchard PII: DOI: Reference:
S0031-9384(18)30209-9 doi:10.1016/j.physbeh.2018.04.028 PHB 12179
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
26 November 2017 20 April 2018 20 April 2018
Please cite this article as: Nathan S. Pentkowski, Yoav Litvin, D. Caroline Blanchard, Robert J. Blanchard , Effects of estrus cycle stage on defensive behavior in female LongEvans hooded rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phb(2018), doi:10.1016/j.physbeh.2018.04.028
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ACCEPTED MANUSCRIPT Effects of estrus cycle stage on defensive behavior in female Long-Evans hooded rats. Nathan S. Pentkowskia,1* , Yoav Litvina, D. Caroline Blanchardb,c and Robert J. Blancharda,b a
Department of Psychology, University of Hawaii at Manoa, 2530 Dole Street, Sakamaki C 400, Honolulu, Hawaii USA 96822
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Pacific Biosciences Research Center, University of Hawaii at Manoa, 1993 East-West Road, Honolulu, Hawaii USA 96822 c
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John A. Burns School of Medicine, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, Hawaii USA 96813
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Corresponding author: Nathan S. Pentkowski, Ph.D. Department of Psychology University of New Mexico 1 University of New Mexico MSC03 2220 Logan Hall Albuquerque, NM USA 87131 Phone: (505) 277-1928 Email: [email protected]
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Present address: 1 Department of Psychology, University of New Mexico, 1 University of New Mexico, MSC03 2220, Logan Hall, Albuquerque, New Mexico USA 87131
ACCEPTED MANUSCRIPT Abstract This study investigated the influence of the estrus cycle in mediating cat odor-induced unconditioned and conditioned defensive behaviors in female Long-Evans hooded rats. Unconditioned defensive behaviors were assessed during predatory cue exposure;
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conditioned defensive behaviors were examined twenty-four hours after threat exposure.
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Estrus phases were determined by microscopic examination of vaginal smears within 10
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min of completing the behavioral tests. Compared to no-odor controls, female rats exposed to cat odor exhibited both unconditioned and conditioned defensive behaviors,
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including elevated levels of freezing, risk assessment and avoidance. Rats in proestrus
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and estrus exhibited reduced levels of defensive behavior during the unconditioned test trial compared to subjects in diestrus and metestrus. Specifically, estrus stages
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characterized by high levels of circulating estrogens and progesterone were associated
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with reduced immobility (i.e. freezing) and enhanced active defense (i.e. risk assessment), profiles that may enable mate seeking and subsequent reproduction in
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potentially dangerous or novel environments. These results suggest a specific role for
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ovarian hormone fluctuations in mediating unconditioned fear- and anxiety- like defensive
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behaviors during exposure to predatory odors.
Keywords
Defense, Anxiety, Fear, Estrus, Female, Estrogen, Predator, Freezing, Risk assessment
ACCEPTED MANUSCRIPT 1. Introduction Anxiety and stress disorders are the most prevalent psychiatric illnesses, with several forms such as generalized anxiety disorder, posttraumatic stress disorder and panic disorder disproportionately affecting women more than men [1-4]. Fluctuations in levels
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of estrogen and progesterone during the menstrual cycle may be related to depression and
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anxiety susceptibility [5-8]. Proestrus (P) and estrus (E) are have higher concentrations of
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circulating progesterone and estrogen, with peak levels occurring during P followed by fluctuations throughout estrus stages [9, 10]. In rats, P and E stages are characterized by
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reduced anxiety- like behavior [11]. For instance, rats in P and E exhibit increased time
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spent in the open arms of an elevated plus-maze (EPM) [12, 13] and obtain more punished reinforcers compared to rats in diestrus (D) and/or metestrus (M) [14]; the latter
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deficits in D and M are prevented by diazepam. Studies have linked anxiolysis with P,
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reporting reduced anxiety- like behaviors in the light-dark test [15], EPM [16-18], punished responding [19], social interaction test [16] and defensive burying test [16, 20].
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In stark contrast, other studies indicate that rats in P and E exhibit enhanced anxiety-like
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behavior in the EPM [21, 22], or report no effect of estrus stage on anxiety- like behaviors in the EPM [23, 24] or defensive burying test [24]. Based on these equivocal reports,
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further characterization of neuroendocrine influences on anxiety-like behavior using novel animal models is needed in order to develop more effective pharmacological treatments for anxiety disorders in women. In order to understand the neurobiological mechanisms underlying fear- and anxietyrelated pathologies, considerable efforts have been made to develop animal models for human anxiety disorders [25]. In laboratory settings, evaluation of antipredator defensive
ACCEPTED MANUSCRIPT behaviors allows for an ethological analysis of anxiety- like behavior by exposing rats to natural predatory cues, such as cat odor. In particular, some of the defensive behaviors elicited by these naturalistic threats, e.g., risk assessment and freezing, [26, 27] are responsive to traditional anxiolytics such as diazepam and 5-HT1A agonists [28-31].
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Importantly, female rats show enhanced antipredator defensive behavior (e.g., increased
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risk assessment and decreased contact) in response to potential (e.g., cat odor) as opposed
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to distinct (e.g., live cat exposure) threat sources, as well as differential reactivity in such situations to classic anxiolytics [32].
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To further clarify the influence of the estrus cycle on anxiety- like defensive behavior,
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this study examined unconditioned and conditioned behavioral responses in female rats exposed to cat odor. We hypothesized that fluctuations in levels of ovarian hormones
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typical of the various estrus stages would differentially modulate defensive behavior,
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with higher levels associated with reduced anxiety-like responsivity. Accordingly, we predicted that this decrease in defensiveness would manifest during P and E, stages that
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are characterized by comparatively higher levels of estrogen and progesterone compared
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to M and D.
2. Materials and Methods
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2.1 Subjects
Subjects were adult female Long-Evans hooded rats born and reared in the Snyder Hall breeding colony at the University of Hawaii at Manoa. Following weaning (21 days), all rats were singly housed in standard home cages (21.6 x 45.7 x 17.8 cm) under controlled temperature (21 ± 2 ◦C) and illumination (12-h light/dark cycle, lights on at 06:00 a.m.) with free access to food (rat chow) and water. Rats (n=53) were 11 to 13
ACCEPTED MANUSCRIPT weeks of age at the start of behavioral testing. All husbandry and experimentation adhered to the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and all experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at The University of Hawaii.
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2.2 Cat Odor Exposure
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Detailed procedures for the cat odor test have been previously described [33-35].
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Briefly, the test apparatus consisted of a white Plexiglas runway (100 x 12 x 50 cm) with a clear front panel to permit observation and recording. Separate cloth-wrapped solid
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plastic blocks (9 x 9 x 2 cm) served as the cat odor and control stimuli. The cat odor
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block was rubbed for 5 min against the fur of a laboratory-housed domestic male cat for 3 consecutive days prior to use. In order to control for potential novelty effects, control rats
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were exposed to an identical cloth-wrapped plastic block never exposed to cat odor. All
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rats were habituated to the apparatus for 10 min over 3 consecutive days without the presence of a block stimulus. On the following day (unconditioned behavior test), the
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appropriate block stimulus was placed at one end of the runway and a test subject was
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placed at the opposite end. Twenty-four hours later each rat was tested in the same apparatus without the cat odor stimulus (cue+context conditioning test). A cloth-wrapped
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solid plastic block never exposed to cat odor served as the cue during the conditioned test trial. All sessions were 10 min in duration and were conducted under red light in order to ensure that the threat stimulus remained as ambiguous as possible. Rats were tested in the same order across days, with testing occurring between 11:00 a.m. and 3:00 p.m. In order to insure that control rats were not exposed to cat odor, control and cat odor exposed
ACCEPTED MANUSCRIPT subjects were tested in separate rooms, using identical test chambers. Between trials each apparatus was thoroughly cleaned using a 10% ethanol solution. 2.3 Behavioral Measures Defensive behaviors analyzed in each test situation included: crouch freeze-complete
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cessation of movement other than respiration; risk assessment behaviors including stretch
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approach-forward ambulation with flat back and stretched neck, stretch attend-standing
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on all 4 paws with flat back and stretched neck orientated toward the threat source, and crouch sniff-olfactory investigation evidenced by vertical or lateral head movements
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(scoring initiated when nose visibly moved more than 1 cm); avoidance-duration of time
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spent in the far compartment relative to the threat- or control-block stimulus; and contactdirect contact with the block stimulus, measured as direct paw or head contact.
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Locomotor activity-crossing of lines marking the far, medium and near thirds of the
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apparatus was measured to examine potential nonspecific changes in ambulation. Behavioral measures represent the durations of events in seconds (or numbers of line
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crossings) during a 10 min observation period.
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2.4 Behavioral Analysis
All test trials were recorded using Pioneer DVD recorders, and were later analyzed
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using the behavioral analysis software Observer 5.0 (Noldus Information Technology, Wageningen, The Netherlands) by a highly trained observer blind to test conditions. Use of this software allowed for a detailed, frame-by-frame analysis. 2.5 Determination of Estrus Cycle Estrus cycle phases were determined by the consensus of three research assistants using bright-field microscopic examination (10x and 40x magnification) of vaginal
ACCEPTED MANUSCRIPT smears collected within 10 min of testing in both the unconditioned and conditioned cat odor tests as previously described [9, 36, 37]. Proestrus was identified by the predominance of nucleated epithelial cells, E was identified by the presence of dense sheets of cornified epithelial cells, M was identified by the presence of scattered,
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nucleated, or cornified epithelial cells and leukocytes, and D was identified by the
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presence of leukocytes [11].
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2.6 Statistical Analysis
To examine the effects of cat odor on unconditioned and conditioned defensive
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behaviors separate t-tests were performed on each dependent measure with data from
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each estrus cycle stage pooled across groups. To examine the influence of each estrus stage on cat odor-induced defensive behaviors separate one-way analysis of variance
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(ANOVA) were performed on each dependent measure. Significant ANOVAs were
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followed by post-hoc Tukey’s HSD tests to provide pairwise comparisons; α was set at 0.05 for all statistical comparisons.
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3. Results
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3.1 Estrus Determination
Of the 32 rats exposed to the predatory cat odor, 12 were tested during diestrus, 6
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during metestrus, 6 during proestrus and 8 during estrus. One-way ANOVA failed to demonstrate any significant behavioral effects between estrus stages in rats exposed to the control block. Thus, for ease of comparison control rats from each estrus stage were pooled into a single control group (n=21). 3.2 Effects of Cat Odor on Unconditioned Defensive Behavior
ACCEPTED MANUSCRIPT Figure 1 illustrates the effects of predator odor exposure on unconditioned defensive behaviors during the cat odor test with data from each estrus cycle stage pooled. Exposure to cat odor produced robust increases in defensive behavior. Compared to controls, rats exposed to cat odor exhibited increased levels of crouch freezing
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[t(51)=5.27, p<0.001] and suppressed locomotor activity [t(51)=8.10, p<0.001; control:
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55.67 ± 3.49, cat odor: 16.69 ± 3.15]. Rats demonstrated a clear aversion to the cat odor
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stimulus, exhibiting higher levels of avoidance [t(51)=7.48, p<0.001] and less contact [t(51)=11.56, p<0.001] with the cat odor block compared to controls. Cat odor exposure
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also potentiated risk assessment behaviors, including a reliable increase in stretch attend
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[t(51)=2.88, p<0.01] and a trend toward increased stretch approach [t(51)=1.92, p=.06], but no effect on crouch sniffing (p>0.05).
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3.3 Influence of Estrus stage on Cat Odor-Induced Unconditioned Defensive Behavior
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Figure 2 illustrates the influence of estrus stage on predator-induced defensive behaviors during the unconditioned cat odor test. The ANOVA indicated a significant
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effect of estrus stage on the duration of crouch freezing [F(3, 28)=6.68, p<0.005]. Post-
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hoc analyses revealed that rats in D exhibited higher durations of freezing compared to rats in P and E (Tukey’s, p<0.05 in each case). Cat odor-elicited risk assessment was also
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influenced by estrus stage. The ANOVA indicated a difference in the duration of crouch sniffing [F(3, 28)=6.15, p<0.005], with rats in P and E exhibiting higher levels compared to rats in D (Tukey’s, p<0.05 in each case). There was no effect of estrus stage on the durations of avoidance, contact, stretch approach, stretch attend or or the number of line crossings (P: 19.00 ± 8.14 , E: 21.75 ± 9.38, M: 17.83 ± 5.74, D: 11.58 ± 3.26). 3.4 Effects of Cat Odor on Conditioned Defensive Behavior
ACCEPTED MANUSCRIPT Figure 3 illustrates the effects of predator odor-induced conditioned defensive behaviors during the cat odor cue+context test with data from each estrus cycle stage pooled. Exposure to the cat odor cue+context induced a characteristic pattern of enhanced conditioned defensiveness. Compared to controls, rats previously exposed to cat odor
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exhibited increased levels of conditioned crouch freezing [t(51)=3.46, p<0.005] and
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suppressed locomotor activity [t(51)=3.05, p<0.005; control cue: 49.24 ± 3.66, cat odor
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cue: 31.03 ± 4.19]. Rats previously exposed to cat odor demonstrated a clear aversion to the cat odor cue stimulus, exhibiting higher levels of conditioned avoidance [t(51)=8.67,
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p<0.001] and less contact [t(51)=8.66, p<0.001] with the cue control block compared to
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controls never exposed to cat odor. Cat odor cue+context exposure also potentiated conditioned risk assessment behaviors, including reliable increases in stretch attend
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[t(51)=2.53, p<0.05] and stretch approach [t(51)=2.27, p<.05], but no effect on crouch
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sniffing (p>0.05).
3.5 Influence of Estrus stage on Cat Odor-Induced Conditioned Defensive Behavior
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Figure 4 illustrates the influence of estrus stage during initial threat exposure on
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conditioned defensive behaviors during the cat odor cue+context test. Estrus stage during the unconditioned test trial did not significantly alter any defensive behaviors during the
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cat odor cue+context conditioning test (p>0.05 in each case) and there was no effect on locomotor activity (P: 34.50 ± 11.71, E: 34.38 ± 9.00, M: 43.00 ± 10.41, D: 21.08 ± 5.03). Additionally, conditioned defensive behaviors did not differ between groups when the data were re-analyzed according to the estrus stage determined immediately (within 10 min) following cue+context testing (data not shown). 4. Discussion
ACCEPTED MANUSCRIPT Results from the present experiments demonstrate for the first time that fluctuations in ovarian hormones during stages of the estrus cycle modulate unconditioned defensive behaviors during exposure to an ethologically relevant predatory threat source (cat odor). Regardless of estrus stage, cat odor increased defensive responsivity in female rats,
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increasing freezing, risk assessment and avoidance, while suppressing locomotion and
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contact with the predatory threat stimulus (see Figure 1). This defensive profile is
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partially estrus stage-dependent, as rats in P and E exhibited reduced freezing and increased risk assessment (crouch-sniffing) compared to rats in D and M (see Figure 2), a
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shift in defensive responsivity similar to reducing the level of environmental threat (i.e., a
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gradual shift from immobility to cautious exploration, eventually leading to a return to non-defensive behaviors). These results extend upon previous research indicating that
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rats in E and/or P exhibit reduced anxiety- like behaviors in the EPM [12, 13, 16-18],
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punished responding conflict models [14, 19], light-dark test [15], social interaction test [16] and defensive burying test [16, 20] compared to rats in D and/or M.
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Similar to previous reports examining conditioning in 18-38-day old female pups [35]
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or female adults [38], rats in the present study demonstrated heightened conditioned defensiveness during exposure to the cat odor-paired cue+context including increased
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freezing, risk assessment and avoidance, as well as reduced locomotion and cue contact (see Figure 3). However, in contrast to the influence of estrus stage on unconditioned defensive behaviors, there were no reliable differences between estrus groups on defensive responsivity during the cat odor cue+context test. Non-significant effects were detected when estrus cycle stages were measured either immediately following unconditioned threat exposure (see Figure 4) or immediately following cue+context re-
ACCEPTED MANUSCRIPT exposure (data not shown), suggesting that estrus stage did not impact conditioned fear acquisition or expression. These null effects of estrus stage during conditioned test trials are consistent with a previous report that failed to find an effect of estrogen replacement in ovariectomized females on cat odor-induced conditioned fear [38].
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The influence of estrus stage on conditioned behavioral responsivity appears to be
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threat-source specific as estrus stage has been implicated in the expression of conditioned
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defensive behavior and fear extinction using footshock [39, 40] as the threat source [for review see: 41]. To confirm this hypothesis, future studies examining estrus stage effects
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on predator odor-induced conditioning should test for conditioned defensive behaviors
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more than 24 hours after initial threat exposure to minimize potential carryover stress effects. Additionally, although previous studies have firmly established that cat odor
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induces conditioned defensive behaviors in adult male rats by examining the effects of
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novel cues and/or contexts across several days of extinction [42], experiments utilizing adult female rats have not included these controls.
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Rodent antipredator defensive behaviors are valid indices of anxiety-related behaviors
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in humans [43, 44]. Therefore, the shift in cat odor-induced defensive responsivity demonstrated by rats in P and E suggests that estrogen and progesterone mediate
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antipredator defensive behaviors related to anxiety. In support of this notion, situations in which rats are exposed to potential or anticipated threat (cat odor, cat-paired contexts) evoke anxiety- like behaviors [45], an assertion supported by pharmacological data [29]. Indeed, classic anxiolytics such as the benzodiazepines diazepam and chlordiazepoxide [46], alcohol [47], the 5-HT1A agonists 8-OH-DPAT and Gepirone [48], and the tricyclic antidepressant imipramine [49] decrease freezing and avoidance while increasing risk
ACCEPTED MANUSCRIPT assessment during exposure to contexts associated with a cat, while the benzodiazepine midazolam decreases freezing and avoidance during cat odor exposure [50]. Similarly, in the present study we detected a shift from passive (freezing) to active (risk-assessment) defensiveness in females with higher levels of ovarian hormones (P and E). Although this
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shift in defensiveness could result from high levels of ovarian hormones producing
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behavioral disinhibition, this explanation is unlikely as the changes in the defensive
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repertoire were independent of effects on locomotor activity (line crossings). The neurobiological mechanisms mediating the estrus stage dependent expression of
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antipredator defensive behavior may involve estrogen and/or progesterone signaling in
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the accessory olfactory system. The accessory olfactory system mediates responsivity to both conspecific and predatory olfactory cues [50, 51]. Estrogens affect social
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recognition in mice via interaction with the oxytocinergic system in the medial amygdala
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[52, 53], a component of the accessory olfactory system that preferentially modulates innate defensive behaviors to cat odor [51, 54]. Importantly, oxytocin binding in the
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medial amygdala promotes prosocial behaviors by attenuating anxiety [52, 53].
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Furthermore, in ovariectomized rats estradiol administration reduces anxiety- like defensive behaviors during cat odor exposure [38], as well as testing in the open field
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[39] and EPM [55]. Lastly, rats in P displaying reduced anxiety in the EPM have higher levels of estradiol compared to D, and estradiol administration to rats in D eliminated their anxiogenic profile [18]. Although not directly tested in the present study, our results may involve progesterone conversion to allopregnanolone and subsequent anxiolysis via agonism at GABAA receptors [56-59]. Importantly, these processes occur in the amygdala [60] during both
ACCEPTED MANUSCRIPT behavioral E [58] and P [16]. An alternative view of the role of estrogen and progesterone effects on defensive behavior should also be considered. Rather than elevated estrogen and/or progesterone signaling during P and E reducing anxiety- like defensive behavior, enhanced defensiveness in D and M may result from falling levels of progesterone and
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allopregnanolone coupled with low stable levels of estrogen in late D [10]. Indeed,
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previous reports indicate that these types of fluctuations in ovarian hormones enhance
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anxiety- like behavior [61-64].
In summary, during predator odor exposure we suggest that estrogen and progesterone
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signaling, likely involving the medial amygdala, mediates a transition from passive
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immobility/freezing to active risk assessment by attenuating fear and promoting high levels of investigative defenses that enable approach, while maintaining vigilance. The
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adaptive utility of such a mechanism is evident; high levels of ovarian hormones needed
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for reproduction would also sustain mate seeking in the face of a potential predatory threat. From an evolutionary perspective, this behavioral profile could promote mate
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seeking and subsequent reproduction in natural environments that contain a persistent, yet
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ambiguous level of predatory threat. Here, reproduction serves as an evolutionary motivation, in addition to the acquisition of resources (i.e. food and shelter) only during
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times of receptivity, and therefore a reduction in fear (defensive) levels enables efficient exploratory behaviors that would otherwise be unnecessary. The precise neural mechanisms that regulate these behavioral processes, as well as the potential involvement of ovarian hormone modulation of accessory olfactory information and defense systems require further investigation. 5. Conclusions
ACCEPTED MANUSCRIPT The present results indicate that estrus stages characterized by higher levels of circulating estrogen and progesterone (P and E) are associated with reduced freezing and enhanced risk assessment, profiles that may promote mate seeking and subsequent reproduction in novel and/or potentially dangerous environments. These results implicate
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Acknowledgements
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like defensive behaviors during exposure to predatory odors.
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fluctuations in ovarian hormones in the expression of unconditioned fear- and anxiety-
The authors thank Chris Markham, Amy Vasconcellos, Joel Sabugo and Mark Ellis
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for their technical assistance.
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Funding source
This research did not receive any specific grant from funding agencies in the public,
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commercial, or not-for-profit sectors.
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Competing Interests
The authors have no conflicts of interest to report or any involvement to disclose,
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financial or otherwise, that may bias the conduct, interpretation or presentation of this
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work.
ACCEPTED MANUSCRIPT Figure captions Figure 1.Effects of cat odor exposure on unconditioned defensive behaviors with data from each estrus cycle stage pooled. Rats exposed to cat odor (filled bars) exhibited enhanced defensiveness compared to controls (clear bars). Defensive behaviors include
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freezing (A), stretch attend (B), stretch approach (C), crouch sniffing (D), and odor block
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avoidance (E) and contact (F). Asterisk (*) represents a difference compared to controls
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(t-test, p<0.05 in each case). Behavioral measures represent the durations of events in
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seconds (s) during the 10 min test period.
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Figure 2.Effects of estrus stage on cat odor-induced unconditioned defensive behaviors. Rats in proestrus (P) and estrus (E) exhibited reduced defensive behavior compared to
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rats in diestrus (D) and metestrus (M). Defensive behaviors include freezing (A), stretch
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attend (B), stretch approach (C), crouch sniffing (D), and odor block avoidance (E) and contact (F). Asterisk (*) represents a difference compared to diestrus (Tukey’s HSD test,
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p<0.05 in each case). Behavioral measures represent the durations of events in seconds
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(s) during the 10 min test period.
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Figure 3.Effects of cat odor cue+context exposure on conditioned defensive behaviors with data from each estrus cycle stage pooled. Rats previously exposed to cat odor (filled bars) exhibited enhanced conditioned defensiveness compared to controls (clear bars). Defensive behaviors include freezing (A), stretch attend (B), stretch approach (C), crouch sniffing (D), and odor block avoidance (E) and contact (F). Asterisk (*) represents a
ACCEPTED MANUSCRIPT difference compared to controls (t-test, p<0.05 in each case). Behavioral measures represent the durations of events in seconds (s) during the 10 min test period.
Figure 4.Effects of estrus stage on conditioned defensive behaviors during cat odor
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cue+context exposure. There were no significant differences between estrus groups
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[proestrus (P), estrus (E), diestrus (D), metestrus (M)]. Defensive behaviors include
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freezing (A), stretch attend (B), stretch approach (C), crouch sniffing (D), and odor block
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seconds (s) during the 10 min test period.
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avoidance (E) and contact (F). Behavioral measures represent the durations of events in
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ACCEPTED MANUSCRIPT Highlights
Cat odor exposure induced both unconditioned and conditioned defensive behaviors in female rats.
Rats in proestrus and estrus exhibited lower levels of unconditioned defensive
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Higher levels of estrogen and/or progesterone may enable mate seeking in
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potentially dangerous environments.
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behavior.