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Hormonal, reproductive, and behavioural predictors of fear extinction recall in female rats
Hormones and Behavior 121 (2020) 104693
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Hormonal, reproductive, and behavioural predictors of fear extinction recall in female rats
T
Samantha Tang , Bronwyn M. Graham ⁎
School of Psychology, UNSW Sydney, NSW 2052, Australia
ARTICLE INFO
ABSTRACT
Keywords: Anxiety disorders Fear extinction Within-session extinction Estrous cycle Motherhood
The prevalence, severity and chronicity of anxiety disorders is significantly higher in women compared to men. Exposure therapy, the gold-standard treatment for anxiety disorders, can be modelled in the laboratory through Pavlovian fear extinction. Understanding the factors that influence fear extinction in females may aid in optimising the treatment of anxiety disorders in this population. The aim of the current study was therefore to explore the hormonal, reproductive and behavioural predictors of fear extinction recall in female rats by analysing data from nine published experiments that examined fear extinction in female rats. A hierarchical multiple regression analysis revealed that estrous cycle effects on extinction recall may be modulated by reproductive status. While the estrous phase in which nulliparous (virgin) rats undergo extinction training was predictive of extinction recall, no relationship between estrous phase and extinction recall was found among primiparous (one prior reproductive experience) rats. Moreover, estrous cycle predicted the relationship between early extinction and extinction recall in nulliparous rats, but not primiparous rats. Although reproductive status did not predict extinction recall, primiparous rats exhibited poor extinction recall relative to nulliparous rats extinguished during proestrus, and better extinction recall than nulliparous rats extinguished during metestrus. A faster rate of extinction, and lower fear responses at the end of extinction training were predictive of lower levels of CSelicited fear during extinction recall in both nulliparous and primiparous female rats, while the length of extinction training was not predictive of extinction recall. The potential theoretical and clinical implications of these findings are discussed.
1. Introduction Fear extinction refers to the decline in conditioned fear that occurs when subjects are repeatedly presented with a fear conditioned stimulus (CS) in the absence of an aversive outcome (US, e.g., a noise previously paired with a shock) until their fear subsides. A subject's ability to recall extinction can be tested by measuring their fear responses to the CS at a later time point (extinction recall). Low levels of CS-elicited fear are indicative of good extinction recall, while high levels of CS-elicited fear are indicative of poor extinction recall. CS-elicited fear during extinction recall is influenced by a subject's ability to acquire, consolidate and/or retrieve a fear extinction memory. Fear extinction forms the procedural basis of exposure therapy, a highly efficacious and effective treatment for anxiety disorders (Bandelow et al., 2015; Foa and McLean, 2016). Recent evidence also indicates that fear extinction performance measured prior to exposure therapy may predict exposure therapy success (Ball et al., 2017; Craske et al., 2018; Forcadell et al., 2017; Lange et al., 2019; Waters and Pine, 2016).
⁎
Understanding the factors that influence fear extinction is therefore worthwhile to identify what factors might influence the success of exposure therapy. To date, the vast majority of animal studies examining fear extinction have exclusively used male subjects. Human studies also often fail to analyse results by sex (S. H. Li and Graham, 2017). This is problematic given that women are twice as likely to be diagnosed with an anxiety disorder compared to men. Moreover, anxiety disorders in women are associated with greater symptom severity, higher rates of comorbidity and a higher burden of disease (Bandelow and Michaelis, 2015; Bekker and van Mens-Verhulst, 2007; McLean et al., 2011). Understanding the factors that influence fear extinction in female subjects may better allow us to optimise exposure therapy outcomes in women. Naturally cycling female rats and women who undergo fear extinction during periods of high endogenous estradiol (the major form of estrogen) show good extinction recall relative to female rats and women that undergo fear extinction during periods of low endogenous estradiol (Glover et al., 2012; Graham and Daher, 2016; Graham and Scott,
Corresponding author at: School of Psychology, UNSW Sydney, Australia. E-mail address: [email protected] (S. Tang).
https://doi.org/10.1016/j.yhbeh.2020.104693 Received 27 October 2019; Received in revised form 13 January 2020; Accepted 18 January 2020 0018-506X/ © 2020 Elsevier Inc. All rights reserved.
Hormones and Behavior 121 (2020) 104693
S. Tang and B.M. Graham
2018a; Gruene et al., 2015; Li and Graham, 2016; Milad et al., 2009; Milad et al., 2010; Milligan-Saville and Graham, 2016; Pineles et al., 2016; Rey et al., 2014; Wegerer et al., 2014; White and Graham, 2016; Zeidan et al., 2011). Poor extinction recall is also observed in female rats that experience chronically low levels of estradiol as a result of ovariectomy (Chang et al., 2009; Parrish et al., 2019) or the administration of hormonal contraceptives (Graham and Milad, 2013; Parrish et al., 2019; White and Graham, 2016). Systemic administration of estradiol or selective estrogen receptor agonists pre- or post-extinction training improve extinction recall in rats extinguished during periods of low endogenous estradiol (Chang et al., 2009; Graham and Milad, 2013; Graham and Scott, 2018a, 2018b; Maeng et al., 2017; Milad et al., 2009; Zeidan et al., 2011), while systemic administration of an estrogen receptor antagonist pre-extinction training impairs extinction recall (Milad et al., 2009), suggesting that estradiol not only enhances, but is also necessary for fear extinction in female rats. Notably, however, we have recently shown that the involvement of estradiol in fear extinction may be altered by reproductive experience. In contrast to nulliparous (virgin) rats, primiparous (one prior reproductive experience) rats extinguished during proestrus (i.e., high estradiol phase of estrous cycle) showed comparable levels of extinction recall as those extinguished during metestrus (i.e., low estradiol phase of estrous cycle) (Graham, 2018; Milligan-Saville and Graham, 2016). Notably, these effects persisted for up to three months post-weaning, suggesting that changes in the features of fear extinction following reproductive experience may be enduring. Primiparous rats also appear to differ from nulliparous rats in that they show extinction that appears to be resistant to relapse (Milligan-Saville and Graham, 2016), independent of the activation of N-methyl-D-aspartate (NMDA) receptors (Tang and Graham, 2019b), and resistant to the augmenting effects of pharmacological adjuncts, including d-cycloserine (DCS) and estradiol (Tang and Graham, 2019a). We have also recently demonstrated that female reproductive experience appears to influence the quality of extinction recall, with some (but not all) studies indicating that primiparous (one prior reproductive experience) rats show worse extinction recall relative to nulliparous (virgin) rats. For instance, in one study, we showed that an increased number of extinction trials was required for primiparous rats to exhibit levels of extinction recall that were comparable to that of nulliparous rats (Tang and Graham, 2019b). In another recent study, we found that reproductive experience may alter the relationship between within-session extinction and extinction recall. In this study, CS-elicited freezing during extinction recall was higher among nulliparous rats that showed high levels of freezing at the end of extinction training relative to those that showed low levels of freezing at the end of extinction training. No such difference in extinction recall was found among primiparous rats, suggesting a lack of a relationship between freezing at the end of extinction training and freezing during extinction recall in primiparous rats (Tang and Graham, 2019a). To our knowledge, no studies have examined the relationship between within-session extinction (e.g., length of training, fear exhibited at the beginning of training, extent of fear reduction achieved) and extinction recall in females, despite this being previously examined reasonably extensively in males. For example, Graham and Richardson (2019) recently examined the relationship between early extinction (i.e., CS-elicited fear exhibited at the beginning of extinction training), late extinction (i.e., CS-elicited fear exhibited at the end of extinction training), and extinction recall in male rats administered either vehicle or fibroblast growth factor-2 (FGF2, a pharmacological enhancer of fear extinction) prior to extinction training. Using a hierarchical multiple regression analysis (MRA), they found that neither early extinction, nor late extinction predicted freezing during extinction recall among rats administered vehicle. However, a positive relationship between late extinction and extinction recall emerged for FGF2-treated rats. Moreover, a number of studies have demonstrated that CS-elicited freezing during extinction recall is lower among adult male rats that exhibit a faster rate of within-session extinction relative to rats that exhibit a
slower rate of within-session extinction (Bush et al., 2007; King et al., 2017). It is important to understand the relationship between withinsession extinction and extinction recall in females as it may point to potential ways in which we can enhance extinction among females (e.g., by increasing the efficiency or effectiveness of within-session extinction). As we have now conducted multiple experiments on fear extinction in female rats in our laboratory, in this study we compiled our previously published results from 245 rats in order to achieve strong statistical power to analyse the various factors that predict fear extinction recall in female rats using a hierarchical MRA. Several key variables were included in this analysis: extinction length, reproductive status, estrous phase, early extinction (as defined above), late extinction (as defined above) and extinction rate (i.e., the first extinction trial in which the subject shows 0% CS-elicited freezing). Early extinction was included as a measure of the strength of fear conditioning, late extinction was included as a measure of within-session extinction (i.e., the degree to which CS-elicited fear decreases over the course of extinction training), and extinction rate was included in light of research showing that this variable may be predictive of extinction recall in adult male rats (Bush et al., 2007; King et al., 2017). 2. Method 2.1. Subjects Data from 245 Sprague-Dawley females rats that had undergone handling and context pre-exposure, conditioning, extinction training and extinction recall in nine previously published experiments (Graham, 2018; Milligan-Saville and Graham, 2016; Tang and Graham, 2019a, 2019b) were analysed in this study (see Table 1). Rats were obtained from the Animal Resources Centre, Perth, WA, Australia. Upon arrival, rats were housed in groups of 8–10 in plastic boxes (67 cm long × 30 cm wide × 22 cm high) filled with sawdust bedding, and covered with a wire lid. The boxes were kept inside a colony room that was maintained on a 12-h light-dark cycle (lights on at 6:30 am). All behavioural testing occurred during the light phase, in line with previous studies examining fear extinction in naturally cycling female rats (Graham and Daher, 2016; Graham and Scott, 2018a; Milligan-Saville and Graham, 2016; Rey et al., 2014; Tang and Graham, 2019a, 2019b; Woods and Bouton, 2006). Food and water were available ad libitum, and cages were cleaned on a weekly basis by an animal attendant. Rats continued to be housed under these conditions for an acclimatisation period of approximately two weeks prior to the commencement of any procedures. All rats were treated according to The Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (8th edition, 2013), and all procedures were approved by the Animal Care and Ethics Committee at The University of New South Wales. Only rats that received no drug administration or rats that were assigned to a vehicle drug group were included in this analysis, to eliminate any confounding effects of drug manipulation. 2.2. Breeding The rats were randomly assigned to remain as virgins (nulliparous, n = 110) or to be mated (primiparous, n = 135). Breeding procedures were as previously described (Milligan-Saville and Graham, 2016). Nulliparous and primiparous rats were age-matched in each of the experiments. In seven experiments, rats underwent fear conditioning at approximately 5 months of age, while in two experiments, rats underwent conditioning at approximately 8 months of age. 2.3. Apparatus Two pairs of Med Associates experimental chambers (24 cm long × 30 cm wide × 21 cm high) were used in this study. Chambers 2
Hormones and Behavior 121 (2020) 104693
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Table 1 Summary of experiments included in hierarchical MRA.
Experiment 1
Reproductive status
Phase of extinction
Sample size
Extinction trials
Reference
Nulliparous
Proestrus Metestrus Proestrus Metestrus
n n n n
= = = =
10 10 12 10
30 30 30 30
Milligan-Saville and Graham (2016)
Proestrus Metestrus Proestrus Metestrus
n n n n
= = = =
11 11 9 8
30 30 30 30
Milligan-Saville and Graham (2016)
Proestrus Metestrus Proestrus Metestrus
n n n n
= = = =
8 8 10 9
30 30 30 30
Milligan-Saville and Graham (2016)
Primiparous Experiment 2
Nulliparous Primiparous
Experiment 3
Nulliparous Primiparous
Experiment 4
Primiparous
Proestrus Metestrus
n=7 n=7
30 30
Graham (2018)
Experiment 5
Nulliparous Primiparous
Proestrus Proestrus
n = 12 n = 10
30 30
Tang and Graham (2019b)
Experiment 6
Nulliparous Primiparous
Proestrus Proestrus
n = 12 n = 15
45 45
Tang and Graham (2019b)
Experiment 7
Nulliparous Primiparous
Metestrus Metestrus
n = 16 n = 17
30 45
Tang and Graham (2019a)
Experiment 8
Nulliparous Primiparous
Metestrus Metestrus
n = 12 n = 10
30 45
Tang and Graham (2019a)
Experiment 9
Primiparous
Metestrus
n = 11
45
Tang and Graham (2019a)
Note that rats from Experiments 3a and 3b of Tang and Graham (2019b) received one, rather than two CS-US trials during fear conditioning, and were therefore not included in this analysis.
2.5. Procedure
were housed in separate wooden cabinets to minimise external auditory and visual stimulation; ventilation fans provided low, constant background noise (~58 dB). An infrared video camera mounted on the rear wall of the cabinets recorded the behaviour of each rat inside the chamber. Each pair of chambers was characterised by distinct visual features. One pair of chambers (Context A) consisted of stainless steel side walls, and a clear Perspex rear wall, ceiling and hinged front door. The flooring comprised of stainless steel rods (4 mm in diameter, 16 mm apart) that could be used to deliver an electric shock. A stainless steel tray containing corn cob bedding was placed underneath the rods. Lighting was provided by a yellow ceiling light in the experimental room. The second pair of chambers (Context B) differed from Context A in that the rod flooring was overlaid with an opaque Perspex sheet, and a piece of paper with black and white vertical stripes (2.5 cm wide) was used to cover the clear Perspex door. White light from a table lamp attached to the roof of the chamber provided illumination. The CS was a 62 dB white noise delivered via high frequency speakers embedded to the right wall of each chamber, and the US was a 0.4 mA, 1.0 s footshock. A computer running Med Associates Med-PC IV controlled presentations of both the CS and the US.
2.5.1. Handling and context pre-exposure Rats were handled for 5 min a day, for three consecutive days prior to the commencement of the experiment. Rats were also individually pre-exposed to Context A for 10 min on the last two days on handling. 2.5.2. Conditioning Rats were placed in Context A on Day 1. Following an adaptation period of 2 min, rats were exposed to two 10-second CS presentations that co-terminated with a footshock. The inter-trial interval (ITI) was 135 s. 2.5.3. Extinction training Rats underwent extinction training 24 h after conditioning (Day 2) in Context B. Following an adaptation period of 2 min, rats received 10s presentations of the CS, with an ITI of 10 s. The majority of included experiments involved 30 extinction trials (see Table 1). However, in one experiment (Tang and Graham, 2019b), it was found that 30 extinction trials produced significantly higher levels of freezing during extinction recall in primiparous rats compared to nulliparous rats. As such, subsequent experiments involved the administration of 45 extinction trials in an attempt to equate freezing during extinction recall between nulliparous and primiparous rats. Equating the strength of extinction allowed for more meaningful conclusions to be drawn about the effect of various drug manipulations on fear extinction in each group.
2.4. Vaginal cytology Vaginal smears were conducted daily to determine estrous cycle phase as previously described (Graham and Daher, 2016). Rats that did not exhibit a regular 4–5 day estrous cycle were excluded from the study. For this reason, primiparous rats were not fear conditioned until at least two weeks after weaning, when lactation had ceased and estrous cycling had recommenced (Leuner and Shors, 2006). In two of the included experiments, primiparous rats were fear conditioned approximately three months after weaning. The estrous phase in which rats underwent extinction training varied between experiments. In four experiments, half of the rats were extinguished during proestrus, while the other half were extinguished during metestrus. In two experiments, all rats were extinguished during proestrus, while in three experiments, all rats were extinguished during metestrus.
2.5.4. Extinction recall Rats were returned to Context B 24 h after extinction training (Day 3). Following an adaptation period of 1 min, rats were presented with a single 2-minute presentation of the CS. 2.6. Scoring Freezing, defined as the absence of all movement aside from that which is required for respiration (Fanselow, 1980), was used to 3
Hormones and Behavior 121 (2020) 104693
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measure conditioned fear. Rats were hand scored as freezing or not freezing every 3 s during the adaptation periods prior to extinction training and extinction recall. These scores provided baseline levels of freezing. CS-elicited freezing was measured every 3 s throughout the CS presentations during extinction training and recall. Extinction trials were collapsed into blocks consisting of the average of five trials. A percentage of observed freezing was calculated for each block of extinction training, and for extinction recall. All behaviour was scored in real time.
3. Results Results of the hierarchical MRA are presented in Table 2. The first model was significant (p = .04), with estrous phase emerging as a significant predictor of extinction recall. Undergoing extinction during metestrus resulted in higher levels of CS-elicited freezing during extinction recall relative to undergoing extinction training during proestrus. The second model was also significant (p < .01), with late extinction, and extinction rate both emerging as significant predictors of extinction recall. There was a positive association between late extinction and extinction recall, and a positive association between extinction rate and extinction recall. The third model was also significant (p < .01), with the interaction between estrous phase and reproductive experience, the interaction between estrous phase and early extinction, and the interaction between estrous phase, reproductive experience and early extinction emerging as significant. Given that both reproductive experience and estrous phase are binary variables, follow-up independent samples t-tests were conducted to determine the source of the interaction between these variables. These tests revealed that nulliparous females extinguished during metestrus showed significantly higher levels of CS-elicited freezing during extinction recall compared to nulliparous females extinguished during proestrus (see Fig. 1, t(108) = 5.04, p < .01). Primiparous females showed similar levels of CS-elicited freezing at extinction recall, regardless of the phase in which they underwent extinction training (t(133) = 0.52, p = .60). To determine how extinction recall in primiparous rats (collapsed across estrous phase) compared to extinction recall in nulliparous rats extinguished during proestrus, and nulliparous rats extinguished during metestrus, a post-hoc contrast analysis using the statistical program, PSY, was utilised. This analysis revealed that levels of CS-elicited freezing during extinction recall among primiparous rats was significantly lower than that of nulliparous rats extinguished during metestrus (F(1, 241) = 8.417, p = .004), and significantly higher than that of nulliparous rats extinguished during proestrus (F(1, 241) = 7.94, p = .005). The hierarchical MRA revealed a significant two-way interaction
2.7. Data analysis A hierarchical MRA was conducted, with CS-elicited freezing during extinction recall entered as the dependent variable. Extinction length, reproductive experience and estrous phase were entered in the first step, while the centred variables, early extinction, late extinction, and extinction rate were entered in the second step. Early extinction was defined as CS-elicited freezing during the first five-trial block of extinction training, while late extinction was defined as CS-elicited freezing during the final five-trial block of extinction training. Extinction rate was defined as the first extinction trial in which the subject showed 0% CS-elicited freezing. Rats that did not reach 0% freezing on any extinction trial were assigned the total number of extinction trials received during extinction training (i.e., 30 or 45, depending on the experiment). Estrous phase interaction terms (estrous phase × early extinction, estrous phase × late extinction, estrous phase × extinction rate, estrous phase × reproductive experience), reproductive experience interaction terms (reproductive experience × early extinction, reproductive experience × late extinction, reproductive experience × extinction rate), and estrous phase × reproductive experience interaction terms (estrous phase × reproductive experience × early extinction, estrous phase × reproductive experience × late extinction, estrous phase × reproductive experience × extinction rate) were entered in the third step (see Table 2). Follow-up analyses were conducted when appropriate, details of which will be provided below. Table 2 Results of hierarchical MRA. Step
Variable
R2
ΔR2
1
Extinction length Reproductive experience Estrous phase
0.035
0.035
2
Extinction length Reproductive experience Estrous phase Early extinction Late extinction Extinction rate
0.488
3
Extinction length Reproductive experience Estrous phase Early extinction Late extinction Extinction rate Estrous phase × Early extinction Estrous phase × Late extinction Estrous phase × Extinction rate Estrous phase × Reproductive experience Reproductive experience × Early extinction Reproductive experience × Late extinction Reproductive experience × Extinction rate Estrous phase × Reproductive experience × Early extinction Estrous phase × Reproductive experience × Late extinction Estrous phase × Reproductive experience × Extinction rate
0.545
N = 245. ⁎ p < .05. ⁎⁎ p < .001. 4
B
SE B
β
95% CI
5.439 −2.364 11.136
5.003 4.431 4.193
0.073 −0.036 0.168⁎
[−4.417, 15.295] [−11.092, 6.364] [2.877, 19.396]
0.453
−6.454 −3.184 4.789 0.143 0.252 1.986
3.759 3.278 3.127 0.103 0.053 0.225
−0.086 −0.048 0.072 0.072 0.252⁎⁎ 0.514⁎⁎
[−13.859, 0.952] [−9.642, 3.274] [−1.372, 10.949] [−0.060, 0.347] [0.148, 0.355] [1.542, 2.429]
0.057
−0.635 8.638 16.364 −0.331 0.265 2.518 0.955 −0.086 −0.769 −23.301 0.507 0.193 −0.934 −1.008 −0.255 1.351
4.079 4.518 4.835 0.221 0.117 0.572 0.338 0.163 0.785 6.542 0.286 0.160 0.727 0.428 0.214 0.954
−0.008 0.130 0.248⁎⁎ −0.168 0.265⁎ 0.651⁎⁎ 0.308⁎ −0.068 −0.152 −0.322⁎⁎ 0.187 0.144 −0.185 −0.257⁎ −0.153 0.219
[−8.672, 7.402] [−0.265, 17.541] [6.838, 25.890] [−0.766, 0.104] [0.034, 0.495] [1.391, 3.644] [0.289, 0.1.622] [−0.408, 0.236] [−2.315, 0.777] [−36.191, −10.410] [−0.056, 1.070] [−0.122, 0.509] [−2.367, 0.498] [−1.852, −0.165] [−0.678, 0.167] [−0.529, 3.231]
Hormones and Behavior 121 (2020) 104693
S. Tang and B.M. Graham
Conditioning
100
% freezing
80 60
Extinction recall
Extinction training
NP PRO NP MET PP PRO PP MET
*
Proestrus Metestrus
40 20 0
CS1
CS2
Early extinction
Late extinction
Nulliparous
Primiparous
Fig. 1. Mean ( ± SEM) levels of CS-elicited freezing during conditioning, extinction training, and extinction recall among nulliparous and primiparous females extinguished during either the metestrus and proestrus phase of the estrous cycle. While conditioning and extinction training data were not analysed in this study, this data has been included in the figure for completeness. *Nulliparous-Metestrus > Nulliparous-Proestrus (p < .05). ** NulliparousMetestrus > Primiparous > Nulliparous-Proestrus (p < .05). Note: NP = nulliparous, PP = primiparous, PRO = proestrus, MET = metestrus.
laboratory demonstrating that fear extinction in primiparous females is not influenced by endogenous (Graham, 2018; Milligan-Saville and Graham, 2016) or exogenous (Tang and Graham, 2019a) estradiol, the absence of a relationship between estrous phase and extinction recall in primiparous rats remains highly notable given the power of the regression analysis to detect subtle effects. The results of the current regression therefore suggest that the association between estradiol and fear extinction in females may be altered by reproductive experience – while fear extinction may be associated with estradiol levels in nulliparous rats, this association may not be present in primiparous rats. The results of the current study also revealed that the effect of estrous phase on the relationship between early extinction and extinction recall may be dependent on reproductive status. While there was a positive association between early extinction and extinction recall among nulliparous females extinguished during metestrus, there was no such association for primiparous females extinguished during metestrus, and for rats extinguished during proestrus, regardless of reproductive status. Although the implications of this finding are, as yet, unclear, this result does provide yet another example of a dissociable impact of estrous phase on fear extinction in nulliparous and primiparous females. This dissociation may be due to long-term changes in endogenous estradiol levels following reproductive experience. Specifically, primiparous rats have been found to show significantly lower levels of endogenous estradiol during proestrus compared to nulliparous rats (Bridges and Byrnes, 2006; Milligan-Saville and Graham, 2016). Reproductive experience has also been shown to alter the expression of estrogen receptors in areas of the brain such as the medial amygdala (Byrnes et al., 2009). It is possible that persistent changes in both endogenous estradiol levels and estrogen receptor expression alters the role of estradiol in fear extinction following reproductive experience. It was somewhat surprising that reproductive experience itself did not emerge as a significant predictor of extinction recall in our analysis, given that we have previously found that primiparous females show impaired extinction recall relative to nulliparous females, and that additional trials of extinction training are required to equate levels of extinction recall between nulliparous and primiparous females (Tang and Graham, 2019a, 2019b). However, it is of note that mean levels of freezing during extinction recall for primiparous females were between that of nulliparous females extinguished during proestrus and nulliparous females extinguished during metestrus, despite the fact that a large proportion of the primiparous rats included in the current regression received one and a half times more extinction trials than nulliparous females. This result lends some support to the idea that fear extinction may be impaired in primiparous rats. Impaired extinction recall in primiparous rats may occur as a result of an impaired ability to acquire, or consolidate extinction. Alternatively, it may be the case that primiparous rats show stronger fear conditioning compared to
between estrous phase and early extinction, and a significant three-way interaction between estrous phase, reproductive experience and early extinction. To determine the source of these interactions, four separate Pearson partial correlations examining the relationship between early extinction and extinction recall among nulliparous females extinguished during proestrus, nulliparous females extinguished during metestrus, primiparous females extinguished during proestrus, and primiparous females extinguished during metestrus were conducted. This analysis method was selected given that estrous phase is a binary variable, whereas early extinction is a continuous variable. In all correlations, late extinction and extinction rate were held constant. There was no significant association between early extinction and extinction recall among nulliparous females extinguished during proestrus, primiparous rats extinguished during proestrus, and primiparous rats extinguished during metestrus (largest r = −0.23, p = .10). However, there was a significant positive association between early extinction and CS-elicited freezing during extinction recall among nulliparous females extinguished during metestrus (r = 0.33, p = .02). 4. Discussion In the current study, a hierarchical MRA was performed on data collated from nine previously published experiments to compare the behavioural, hormonal and reproductive predictors of fear extinction in female rats. This analysis revealed that estrous cycle effects on fear extinction may be dependent on reproductive status. While estrous phase predicts extinction recall, and the relationship between early extinction and extinction recall in nulliparous females, it may not predict either of these variables in primiparous females. It was also revealed that nulliparous and primiparous females share two common behavioural predictors of fear extinction: late extinction and extinction rate. A faster rate of extinction and lower levels of CS-elicited freezing at the end of extinction training were associated with lower levels of CSelicited freezing during extinction recall among both groups of females. Reproductive experience and extinction length also did not emerge as significant predictors of fear extinction in female rats. However, primiparous rats were found to exhibit levels of extinction recall that were between than that of nulliparous rats extinguished during metestrus and nulliparous rats extinguished during proestrus. The hierarchical MRA revealed that estrous phase effects on fear extinction were dependent on reproductive status. Consistent with the results of previous studies (see Li and Graham, 2017), undergoing extinction training during metestrus was associated with impaired extinction recall relative to undergoing extinction training during proestrus in nulliparous females. However, no such association between estrous phase and extinction recall was found in primiparous females. While this latter result is consistent with previous findings from our 5
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nulliparous rats, and are therefore more resistant to extinction. Indeed, in one of our previous studies, we found that primiparous rats show higher levels of CS-elicited fear following conditioning compared to nulliparous rats (Tang and Graham, 2019b). Contrastingly, Rima et al. (2009) found that primiparous females exhibited lower CS-elicited freezing compared to nulliparous females one day after fear conditioning. However, rather than using a footshock as the US, Rima et al. (2009) used a predator noise, which is uncommon in rodent studies examining fear conditioning. Nonetheless, further research is required to disentangle whether impaired extinction recall in primiparous females is due to impaired extinction acquisition/consolidation, or strengthened conditioning. Somewhat surprisingly, extinction length also did not emerge as a significant predictor of extinction recall in the current regression, given that across two of our previous studies (Tang and Graham, 2019a, 2019b), primiparous rats were administered additional trials of extinction training to achieve levels of extinction recall that were comparable to that of nulliparous rats. However, it is of note that while increasing the length of extinction training from 30 trials to 45 trials led to comparable levels of extinction recall between nulliparous and primiparous rats, it did not lead to a significant reduction in levels of CSelicited freezing during extinction recall among primiparous rats. Rather, both an increase in the number of extinction trials, and the administration of a weaker conditioning protocol were required to achieve a significant reduction in freezing at extinction recall (Tang and Graham, 2019b). As such, the results of the current regression are broadly consistent with the findings of our previous study. The current regression revealed that late extinction and extinction rate predicted extinction recall in both nulliparous and primiparous rats. The former result is somewhat inconsistent with the results of our previous study (Tang and Graham, 2019a). In our previous study, nulliparous ‘non-extinguishers’ (i.e., rats that showed high levels of CSelicited freezing at late extinction) showed higher levels of freezing during extinction recall relative to nulliparous ‘extinguishers’ (i.e., rats that showed low levels of CS-elicited freezing at late extinction). Contrastingly, extinction recall was comparable between primiparous nonextinguishers and primiparous extinguishers. Inconsistences between the results of our previous and current study may be because the large sample size in the current study afforded greater power in detecting a relationship between late extinction and extinction recall among primiparous females. However, the fact that we were able to detect a relationship between late extinction and extinction recall among nulliparous, but not primiparous females, in our previous study might suggest that the size of this relationship is greater in nulliparous females compared to primiparous females. There are mixed findings as to the relationship between late extinction and extinction recall in adult male rats and humans. Consistent with the results of the current study, Reznikov et al. (2015) found that adult male rats exhibiting high levels of freezing at the end of extinction training showed significantly higher levels of freezing during extinction recall compared to rats that exhibited low levels of freezing at the end of extinction training. However, there are also studies demonstrating that CS-elicited freezing during extinction recall is unrelated to fear levels at the end of extinction training, in both adult male rats and humans (see Craske et al., 2008; Craske et al., 2014). For instance, as described earlier, Graham and Richardson (2019), who analysed a large sample of male rats using a similar statistical approach to the current study, found no relationship between late extinction and CS-elicited freezing during extinction recall among vehicle-treated rats, although a relationship was evident for rats treated with FGF2. A number of clinical studies have also demonstrated no relationship between fear levels at the end of an exposure therapy session and overall treatment outcomes (see Craske et al., 2008). For instance, Smits et al. (2013) found that treatment outcomes for placebo-treated patients receiving exposure therapy for Social Anxiety Disorder did not depend upon levels of self-reported fear exhibited at the end of each exposure session.
However, a relationship between treatment outcomes and self-reported fear at the end of each exposure session did emerge for DCS-treated patients. Together, these findings reveal that the relationship between late extinction and extinction recall may only emerge under specific circumstances. One possible circumstance that allowed a relationship between late extinction and extinction recall to emerge in the current study is that the parameters used in the experiments included in the regression analysis resulted in a greater level of variability in freezing at the end of extinction training compared to other animal studies examining the relationship between late extinction and extinction recall. Such variability may have better allowed for the detection of a relationship between late extinction and extinction recall. There is now a growing body of evidence suggesting that the hormonal, behavioural and neurobiological features of fear extinction are altered as a result of female reproductive experience. Unlike fear extinction in nulliparous rats, fear extinction in primiparous rats appears to be relapse-resistant (Milligan-Saville and Graham, 2016), estrouscycle independent (Graham, 2018; Milligan-Saville and Graham, 2016), NMDA receptor independent (Tang and Graham, 2019b), and resistant to the augmenting effects of systemic DCS and estradiol (Tang and Graham, 2019a). Observed differences in the features of fear extinction between nulliparous and primiparous rats have previously led us to suggest that the mechanisms underlying fear extinction may be altered as a consequence of reproductive experience. More specifically, we have previously suggested that prior to motherhood, fear extinction is predominantly new learning, and after motherhood, fear extinction is predominantly erasure (Tang and Graham, 2019a). This is because relapse following extinction, NMDA receptor involvement in extinction, and the ability to augment extinction using pharmacological adjuncts can be taken as evidence to suggest that fear extinction involves new learning. The current finding that estrous cycle effects on fear extinction may be dissociable between nulliparous and primiparous rats further supports the notion that the mechanisms underlying fear extinction may be altered by reproductive experience. However, the current study also revealed similarities in the features of fear extinction between nulliparous and primiparous rats – for instance, it revealed that late extinction and extinction rate predicted extinction recall in both groups of rats. These similarities suggest that fear extinction may, in fact, involve similar mechanisms in both nulliparous and primiparous rats. However, it is equally possible that the existence of a relationship between within-session extinction and extinction recall in nulliparous rats is reflective of the degree of learning that occurs during extinction training, whereas the same relationship in primiparous rats may be reflective of the degree of erasure that occurs during extinction training. To determine whether or not fear extinction involves new learning or erasure in primiparous rats, it may be helpful to investigate other neurobiological features of fear extinction in these rats. For instance, it may be useful to examine the involvement of gamma-Aminobutyric acid (GABA), particularly given that the involvement of GABA in fear extinction has been shown to be altered in other populations in which extinction may not involve new learning (i.e., developing rats, Kim and Richardson, 2007). Given that fear extinction serves as a laboratory model, and predictor, of exposure therapy, the results of the current study may have important clinical implications in the treatment of anxiety disorders in women. For instance, given that extinction recall appears to be impaired among primiparous rats compared to nulliparous rats extinguished during proestrus, one may speculate that exposure therapy is less effective in mothers compared to non-mothers undergoing exposure therapy during periods of high estradiol. It may therefore be of interest to identify ways of optimising exposure therapy outcomes among mothers. One approach that has been suggested in recent years involves timing exposure therapy with respect to a woman's menstrual cycle, given that there is extensive evidence showing that endogenous fluctuations in estradiol influence fear extinction in female rats and women (Li and Graham, 2017). That is, conducting exposure therapy 6
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sessions during periods of high endogenous estradiol (e.g., during ovulation), rather than periods of low endogenous estradiol (e.g., during menstruation, or while using hormonal contraceptives) may be associated with better treatment outcomes. Preliminary support for this approach has been found among women receiving exposure therapy for spider phobia (Graham et al., 2018). However, given that estrous phase does not appear to predict extinction recall in primiparous rats (as demonstrated in the current study, and previous studies from our laboratory; Graham, 2018; Milligan-Saville and Graham, 2016), it is possible that timing exposure therapy with respect to a woman's menstrual cycle is an ineffective means of enhancing treatment outcomes among women who are mothers. An alternative approach to enhancing exposure therapy outcomes in mothers may involve reducing levels of fear at the end of an exposure therapy session, given that the current study revealed that lower levels of fear at the end of extinction training predicted better extinction recall in both nulliparous and primiparous rats. There are several possible ways in which fear levels may be reduced at the end of an exposure session. One possibility involves increasing the duration of exposure. It has previously been suggested that doing so may enhance the learning that takes place during exposure, thereby improving treatment outcomes (Craske et al., 2014). However, given that the results of the current regression demonstrated that extinction length did not predict extinction recall in both nulliparous and primiparous rats, it is possible that extending the duration of exposure is an ineffective means of augmenting treatment outcomes in both reproductively experienced and reproductively inexperienced women. A second possible way in which fear levels can be reduced at the end of exposure in women may be through the use of a conditioned inhibitor (i.e., a cue that signals the non-occurrence of the US; Palmatier and Bevins, 2010). Rodent and human studies have consistently shown that subjects exhibit significantly lower levels of fear when co-presented with a CS and a conditioned inhibitor, compared to a CS alone (see Christianson et al., 2012). However, a number of laboratory studies have shown that the presence of conditioned inhibitors during extinction training impairs subsequent extinction recall (see Craske et al., 2008). Similar findings have been made in clinical studies examining the effect of conditioned inhibitors or ‘safety behaviours’ on exposure therapy outcomes (see Blakey and Abramowitz, 2016). Further research is therefore required to investigate alternative approaches to how fear responses can be lowered at the end of extinction training in primiparous rats, with the view of translating these approaches into clinical settings. It is of note that there are a number of limitations of the current study. Firstly, while the use of different extinction parameters allowed us to assess this variable as a predictor of extinction recall, the distribution of rats receiving 30 rather than 45 extinction trials was uneven among primiparous compared to nulliparous rats. Namely, the proportion of nulliparous rats receiving 30 extinction trials was greater than that of primiparous rats. As such, the current results, particularly those with regard to the impact of extinction length on fear extinction in nulliparous rats, need to be interpreted with caution. Moreover, all included data came from studies conducted in our laboratory. While the effect of estrous cycle on fear extinction in nulliparous females has been extensively replicated, it remains important to replicate the results of the current regression, especially those on reproductive experience, in other laboratories and rodent strains. A further limitation of the current study was that extinction recall involved a continuous two minute CS presentation, while both conditioning and extinction training involved a ten second CS presentation. It is possible that the extension of the CS during extinction recall altered rats' fear response. However, this is unlikely given that in a previous study conducted in our laboratory (Graham, 2018), the first 10 s of extinction recall data were analysed alongside the full 2 min of extinction recall data. Both analyses revealed an identical pattern of results. In summary, the results of the current study provide some insight into factors that may influence fear extinction in female rats.
Specifically, it was demonstrated that the rate and degree of withinsession extinction exhibited by female rats, and estrous phase might significantly predict extinction recall. However, the impact of estrous phase on extinction recall appears to be modulated by female reproductive status. Although reproductive status in itself did not emerge as a significant predictor of extinction recall, follow-up analyses provided some evidence to suggest that primiparous rats may exhibit poorer extinction recall compared to nulliparous extinguished during proestrus. Given that fear extinction forms the laboratory basis of exposure therapy, these findings may have important implications for the treatment of anxiety disorders in women across the reproductive lifespan. Funding and disclosure This work was supported by grants from the Australian Research Council (DE140100243 and DP180101563) to BMG, and an Australian Postgraduate Award to ST. The authors have no conflicts of interest to declare. 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Hormonal, reproductive, and behavioural predictors of fear extinction recall in female rats
T
Samantha Tang , Bronwyn M. Graham ⁎
School of Psychology, UNSW Sydney, NSW 2052, Australia
ARTICLE INFO
ABSTRACT
Keywords: Anxiety disorders Fear extinction Within-session extinction Estrous cycle Motherhood
The prevalence, severity and chronicity of anxiety disorders is significantly higher in women compared to men. Exposure therapy, the gold-standard treatment for anxiety disorders, can be modelled in the laboratory through Pavlovian fear extinction. Understanding the factors that influence fear extinction in females may aid in optimising the treatment of anxiety disorders in this population. The aim of the current study was therefore to explore the hormonal, reproductive and behavioural predictors of fear extinction recall in female rats by analysing data from nine published experiments that examined fear extinction in female rats. A hierarchical multiple regression analysis revealed that estrous cycle effects on extinction recall may be modulated by reproductive status. While the estrous phase in which nulliparous (virgin) rats undergo extinction training was predictive of extinction recall, no relationship between estrous phase and extinction recall was found among primiparous (one prior reproductive experience) rats. Moreover, estrous cycle predicted the relationship between early extinction and extinction recall in nulliparous rats, but not primiparous rats. Although reproductive status did not predict extinction recall, primiparous rats exhibited poor extinction recall relative to nulliparous rats extinguished during proestrus, and better extinction recall than nulliparous rats extinguished during metestrus. A faster rate of extinction, and lower fear responses at the end of extinction training were predictive of lower levels of CSelicited fear during extinction recall in both nulliparous and primiparous female rats, while the length of extinction training was not predictive of extinction recall. The potential theoretical and clinical implications of these findings are discussed.
1. Introduction Fear extinction refers to the decline in conditioned fear that occurs when subjects are repeatedly presented with a fear conditioned stimulus (CS) in the absence of an aversive outcome (US, e.g., a noise previously paired with a shock) until their fear subsides. A subject's ability to recall extinction can be tested by measuring their fear responses to the CS at a later time point (extinction recall). Low levels of CS-elicited fear are indicative of good extinction recall, while high levels of CS-elicited fear are indicative of poor extinction recall. CS-elicited fear during extinction recall is influenced by a subject's ability to acquire, consolidate and/or retrieve a fear extinction memory. Fear extinction forms the procedural basis of exposure therapy, a highly efficacious and effective treatment for anxiety disorders (Bandelow et al., 2015; Foa and McLean, 2016). Recent evidence also indicates that fear extinction performance measured prior to exposure therapy may predict exposure therapy success (Ball et al., 2017; Craske et al., 2018; Forcadell et al., 2017; Lange et al., 2019; Waters and Pine, 2016).
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Understanding the factors that influence fear extinction is therefore worthwhile to identify what factors might influence the success of exposure therapy. To date, the vast majority of animal studies examining fear extinction have exclusively used male subjects. Human studies also often fail to analyse results by sex (S. H. Li and Graham, 2017). This is problematic given that women are twice as likely to be diagnosed with an anxiety disorder compared to men. Moreover, anxiety disorders in women are associated with greater symptom severity, higher rates of comorbidity and a higher burden of disease (Bandelow and Michaelis, 2015; Bekker and van Mens-Verhulst, 2007; McLean et al., 2011). Understanding the factors that influence fear extinction in female subjects may better allow us to optimise exposure therapy outcomes in women. Naturally cycling female rats and women who undergo fear extinction during periods of high endogenous estradiol (the major form of estrogen) show good extinction recall relative to female rats and women that undergo fear extinction during periods of low endogenous estradiol (Glover et al., 2012; Graham and Daher, 2016; Graham and Scott,
Corresponding author at: School of Psychology, UNSW Sydney, Australia. E-mail address: [email protected] (S. Tang).
https://doi.org/10.1016/j.yhbeh.2020.104693 Received 27 October 2019; Received in revised form 13 January 2020; Accepted 18 January 2020 0018-506X/ © 2020 Elsevier Inc. All rights reserved.
Hormones and Behavior 121 (2020) 104693
S. Tang and B.M. Graham
2018a; Gruene et al., 2015; Li and Graham, 2016; Milad et al., 2009; Milad et al., 2010; Milligan-Saville and Graham, 2016; Pineles et al., 2016; Rey et al., 2014; Wegerer et al., 2014; White and Graham, 2016; Zeidan et al., 2011). Poor extinction recall is also observed in female rats that experience chronically low levels of estradiol as a result of ovariectomy (Chang et al., 2009; Parrish et al., 2019) or the administration of hormonal contraceptives (Graham and Milad, 2013; Parrish et al., 2019; White and Graham, 2016). Systemic administration of estradiol or selective estrogen receptor agonists pre- or post-extinction training improve extinction recall in rats extinguished during periods of low endogenous estradiol (Chang et al., 2009; Graham and Milad, 2013; Graham and Scott, 2018a, 2018b; Maeng et al., 2017; Milad et al., 2009; Zeidan et al., 2011), while systemic administration of an estrogen receptor antagonist pre-extinction training impairs extinction recall (Milad et al., 2009), suggesting that estradiol not only enhances, but is also necessary for fear extinction in female rats. Notably, however, we have recently shown that the involvement of estradiol in fear extinction may be altered by reproductive experience. In contrast to nulliparous (virgin) rats, primiparous (one prior reproductive experience) rats extinguished during proestrus (i.e., high estradiol phase of estrous cycle) showed comparable levels of extinction recall as those extinguished during metestrus (i.e., low estradiol phase of estrous cycle) (Graham, 2018; Milligan-Saville and Graham, 2016). Notably, these effects persisted for up to three months post-weaning, suggesting that changes in the features of fear extinction following reproductive experience may be enduring. Primiparous rats also appear to differ from nulliparous rats in that they show extinction that appears to be resistant to relapse (Milligan-Saville and Graham, 2016), independent of the activation of N-methyl-D-aspartate (NMDA) receptors (Tang and Graham, 2019b), and resistant to the augmenting effects of pharmacological adjuncts, including d-cycloserine (DCS) and estradiol (Tang and Graham, 2019a). We have also recently demonstrated that female reproductive experience appears to influence the quality of extinction recall, with some (but not all) studies indicating that primiparous (one prior reproductive experience) rats show worse extinction recall relative to nulliparous (virgin) rats. For instance, in one study, we showed that an increased number of extinction trials was required for primiparous rats to exhibit levels of extinction recall that were comparable to that of nulliparous rats (Tang and Graham, 2019b). In another recent study, we found that reproductive experience may alter the relationship between within-session extinction and extinction recall. In this study, CS-elicited freezing during extinction recall was higher among nulliparous rats that showed high levels of freezing at the end of extinction training relative to those that showed low levels of freezing at the end of extinction training. No such difference in extinction recall was found among primiparous rats, suggesting a lack of a relationship between freezing at the end of extinction training and freezing during extinction recall in primiparous rats (Tang and Graham, 2019a). To our knowledge, no studies have examined the relationship between within-session extinction (e.g., length of training, fear exhibited at the beginning of training, extent of fear reduction achieved) and extinction recall in females, despite this being previously examined reasonably extensively in males. For example, Graham and Richardson (2019) recently examined the relationship between early extinction (i.e., CS-elicited fear exhibited at the beginning of extinction training), late extinction (i.e., CS-elicited fear exhibited at the end of extinction training), and extinction recall in male rats administered either vehicle or fibroblast growth factor-2 (FGF2, a pharmacological enhancer of fear extinction) prior to extinction training. Using a hierarchical multiple regression analysis (MRA), they found that neither early extinction, nor late extinction predicted freezing during extinction recall among rats administered vehicle. However, a positive relationship between late extinction and extinction recall emerged for FGF2-treated rats. Moreover, a number of studies have demonstrated that CS-elicited freezing during extinction recall is lower among adult male rats that exhibit a faster rate of within-session extinction relative to rats that exhibit a
slower rate of within-session extinction (Bush et al., 2007; King et al., 2017). It is important to understand the relationship between withinsession extinction and extinction recall in females as it may point to potential ways in which we can enhance extinction among females (e.g., by increasing the efficiency or effectiveness of within-session extinction). As we have now conducted multiple experiments on fear extinction in female rats in our laboratory, in this study we compiled our previously published results from 245 rats in order to achieve strong statistical power to analyse the various factors that predict fear extinction recall in female rats using a hierarchical MRA. Several key variables were included in this analysis: extinction length, reproductive status, estrous phase, early extinction (as defined above), late extinction (as defined above) and extinction rate (i.e., the first extinction trial in which the subject shows 0% CS-elicited freezing). Early extinction was included as a measure of the strength of fear conditioning, late extinction was included as a measure of within-session extinction (i.e., the degree to which CS-elicited fear decreases over the course of extinction training), and extinction rate was included in light of research showing that this variable may be predictive of extinction recall in adult male rats (Bush et al., 2007; King et al., 2017). 2. Method 2.1. Subjects Data from 245 Sprague-Dawley females rats that had undergone handling and context pre-exposure, conditioning, extinction training and extinction recall in nine previously published experiments (Graham, 2018; Milligan-Saville and Graham, 2016; Tang and Graham, 2019a, 2019b) were analysed in this study (see Table 1). Rats were obtained from the Animal Resources Centre, Perth, WA, Australia. Upon arrival, rats were housed in groups of 8–10 in plastic boxes (67 cm long × 30 cm wide × 22 cm high) filled with sawdust bedding, and covered with a wire lid. The boxes were kept inside a colony room that was maintained on a 12-h light-dark cycle (lights on at 6:30 am). All behavioural testing occurred during the light phase, in line with previous studies examining fear extinction in naturally cycling female rats (Graham and Daher, 2016; Graham and Scott, 2018a; Milligan-Saville and Graham, 2016; Rey et al., 2014; Tang and Graham, 2019a, 2019b; Woods and Bouton, 2006). Food and water were available ad libitum, and cages were cleaned on a weekly basis by an animal attendant. Rats continued to be housed under these conditions for an acclimatisation period of approximately two weeks prior to the commencement of any procedures. All rats were treated according to The Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (8th edition, 2013), and all procedures were approved by the Animal Care and Ethics Committee at The University of New South Wales. Only rats that received no drug administration or rats that were assigned to a vehicle drug group were included in this analysis, to eliminate any confounding effects of drug manipulation. 2.2. Breeding The rats were randomly assigned to remain as virgins (nulliparous, n = 110) or to be mated (primiparous, n = 135). Breeding procedures were as previously described (Milligan-Saville and Graham, 2016). Nulliparous and primiparous rats were age-matched in each of the experiments. In seven experiments, rats underwent fear conditioning at approximately 5 months of age, while in two experiments, rats underwent conditioning at approximately 8 months of age. 2.3. Apparatus Two pairs of Med Associates experimental chambers (24 cm long × 30 cm wide × 21 cm high) were used in this study. Chambers 2
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Table 1 Summary of experiments included in hierarchical MRA.
Experiment 1
Reproductive status
Phase of extinction
Sample size
Extinction trials
Reference
Nulliparous
Proestrus Metestrus Proestrus Metestrus
n n n n
= = = =
10 10 12 10
30 30 30 30
Milligan-Saville and Graham (2016)
Proestrus Metestrus Proestrus Metestrus
n n n n
= = = =
11 11 9 8
30 30 30 30
Milligan-Saville and Graham (2016)
Proestrus Metestrus Proestrus Metestrus
n n n n
= = = =
8 8 10 9
30 30 30 30
Milligan-Saville and Graham (2016)
Primiparous Experiment 2
Nulliparous Primiparous
Experiment 3
Nulliparous Primiparous
Experiment 4
Primiparous
Proestrus Metestrus
n=7 n=7
30 30
Graham (2018)
Experiment 5
Nulliparous Primiparous
Proestrus Proestrus
n = 12 n = 10
30 30
Tang and Graham (2019b)
Experiment 6
Nulliparous Primiparous
Proestrus Proestrus
n = 12 n = 15
45 45
Tang and Graham (2019b)
Experiment 7
Nulliparous Primiparous
Metestrus Metestrus
n = 16 n = 17
30 45
Tang and Graham (2019a)
Experiment 8
Nulliparous Primiparous
Metestrus Metestrus
n = 12 n = 10
30 45
Tang and Graham (2019a)
Experiment 9
Primiparous
Metestrus
n = 11
45
Tang and Graham (2019a)
Note that rats from Experiments 3a and 3b of Tang and Graham (2019b) received one, rather than two CS-US trials during fear conditioning, and were therefore not included in this analysis.
2.5. Procedure
were housed in separate wooden cabinets to minimise external auditory and visual stimulation; ventilation fans provided low, constant background noise (~58 dB). An infrared video camera mounted on the rear wall of the cabinets recorded the behaviour of each rat inside the chamber. Each pair of chambers was characterised by distinct visual features. One pair of chambers (Context A) consisted of stainless steel side walls, and a clear Perspex rear wall, ceiling and hinged front door. The flooring comprised of stainless steel rods (4 mm in diameter, 16 mm apart) that could be used to deliver an electric shock. A stainless steel tray containing corn cob bedding was placed underneath the rods. Lighting was provided by a yellow ceiling light in the experimental room. The second pair of chambers (Context B) differed from Context A in that the rod flooring was overlaid with an opaque Perspex sheet, and a piece of paper with black and white vertical stripes (2.5 cm wide) was used to cover the clear Perspex door. White light from a table lamp attached to the roof of the chamber provided illumination. The CS was a 62 dB white noise delivered via high frequency speakers embedded to the right wall of each chamber, and the US was a 0.4 mA, 1.0 s footshock. A computer running Med Associates Med-PC IV controlled presentations of both the CS and the US.
2.5.1. Handling and context pre-exposure Rats were handled for 5 min a day, for three consecutive days prior to the commencement of the experiment. Rats were also individually pre-exposed to Context A for 10 min on the last two days on handling. 2.5.2. Conditioning Rats were placed in Context A on Day 1. Following an adaptation period of 2 min, rats were exposed to two 10-second CS presentations that co-terminated with a footshock. The inter-trial interval (ITI) was 135 s. 2.5.3. Extinction training Rats underwent extinction training 24 h after conditioning (Day 2) in Context B. Following an adaptation period of 2 min, rats received 10s presentations of the CS, with an ITI of 10 s. The majority of included experiments involved 30 extinction trials (see Table 1). However, in one experiment (Tang and Graham, 2019b), it was found that 30 extinction trials produced significantly higher levels of freezing during extinction recall in primiparous rats compared to nulliparous rats. As such, subsequent experiments involved the administration of 45 extinction trials in an attempt to equate freezing during extinction recall between nulliparous and primiparous rats. Equating the strength of extinction allowed for more meaningful conclusions to be drawn about the effect of various drug manipulations on fear extinction in each group.
2.4. Vaginal cytology Vaginal smears were conducted daily to determine estrous cycle phase as previously described (Graham and Daher, 2016). Rats that did not exhibit a regular 4–5 day estrous cycle were excluded from the study. For this reason, primiparous rats were not fear conditioned until at least two weeks after weaning, when lactation had ceased and estrous cycling had recommenced (Leuner and Shors, 2006). In two of the included experiments, primiparous rats were fear conditioned approximately three months after weaning. The estrous phase in which rats underwent extinction training varied between experiments. In four experiments, half of the rats were extinguished during proestrus, while the other half were extinguished during metestrus. In two experiments, all rats were extinguished during proestrus, while in three experiments, all rats were extinguished during metestrus.
2.5.4. Extinction recall Rats were returned to Context B 24 h after extinction training (Day 3). Following an adaptation period of 1 min, rats were presented with a single 2-minute presentation of the CS. 2.6. Scoring Freezing, defined as the absence of all movement aside from that which is required for respiration (Fanselow, 1980), was used to 3
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measure conditioned fear. Rats were hand scored as freezing or not freezing every 3 s during the adaptation periods prior to extinction training and extinction recall. These scores provided baseline levels of freezing. CS-elicited freezing was measured every 3 s throughout the CS presentations during extinction training and recall. Extinction trials were collapsed into blocks consisting of the average of five trials. A percentage of observed freezing was calculated for each block of extinction training, and for extinction recall. All behaviour was scored in real time.
3. Results Results of the hierarchical MRA are presented in Table 2. The first model was significant (p = .04), with estrous phase emerging as a significant predictor of extinction recall. Undergoing extinction during metestrus resulted in higher levels of CS-elicited freezing during extinction recall relative to undergoing extinction training during proestrus. The second model was also significant (p < .01), with late extinction, and extinction rate both emerging as significant predictors of extinction recall. There was a positive association between late extinction and extinction recall, and a positive association between extinction rate and extinction recall. The third model was also significant (p < .01), with the interaction between estrous phase and reproductive experience, the interaction between estrous phase and early extinction, and the interaction between estrous phase, reproductive experience and early extinction emerging as significant. Given that both reproductive experience and estrous phase are binary variables, follow-up independent samples t-tests were conducted to determine the source of the interaction between these variables. These tests revealed that nulliparous females extinguished during metestrus showed significantly higher levels of CS-elicited freezing during extinction recall compared to nulliparous females extinguished during proestrus (see Fig. 1, t(108) = 5.04, p < .01). Primiparous females showed similar levels of CS-elicited freezing at extinction recall, regardless of the phase in which they underwent extinction training (t(133) = 0.52, p = .60). To determine how extinction recall in primiparous rats (collapsed across estrous phase) compared to extinction recall in nulliparous rats extinguished during proestrus, and nulliparous rats extinguished during metestrus, a post-hoc contrast analysis using the statistical program, PSY, was utilised. This analysis revealed that levels of CS-elicited freezing during extinction recall among primiparous rats was significantly lower than that of nulliparous rats extinguished during metestrus (F(1, 241) = 8.417, p = .004), and significantly higher than that of nulliparous rats extinguished during proestrus (F(1, 241) = 7.94, p = .005). The hierarchical MRA revealed a significant two-way interaction
2.7. Data analysis A hierarchical MRA was conducted, with CS-elicited freezing during extinction recall entered as the dependent variable. Extinction length, reproductive experience and estrous phase were entered in the first step, while the centred variables, early extinction, late extinction, and extinction rate were entered in the second step. Early extinction was defined as CS-elicited freezing during the first five-trial block of extinction training, while late extinction was defined as CS-elicited freezing during the final five-trial block of extinction training. Extinction rate was defined as the first extinction trial in which the subject showed 0% CS-elicited freezing. Rats that did not reach 0% freezing on any extinction trial were assigned the total number of extinction trials received during extinction training (i.e., 30 or 45, depending on the experiment). Estrous phase interaction terms (estrous phase × early extinction, estrous phase × late extinction, estrous phase × extinction rate, estrous phase × reproductive experience), reproductive experience interaction terms (reproductive experience × early extinction, reproductive experience × late extinction, reproductive experience × extinction rate), and estrous phase × reproductive experience interaction terms (estrous phase × reproductive experience × early extinction, estrous phase × reproductive experience × late extinction, estrous phase × reproductive experience × extinction rate) were entered in the third step (see Table 2). Follow-up analyses were conducted when appropriate, details of which will be provided below. Table 2 Results of hierarchical MRA. Step
Variable
R2
ΔR2
1
Extinction length Reproductive experience Estrous phase
0.035
0.035
2
Extinction length Reproductive experience Estrous phase Early extinction Late extinction Extinction rate
0.488
3
Extinction length Reproductive experience Estrous phase Early extinction Late extinction Extinction rate Estrous phase × Early extinction Estrous phase × Late extinction Estrous phase × Extinction rate Estrous phase × Reproductive experience Reproductive experience × Early extinction Reproductive experience × Late extinction Reproductive experience × Extinction rate Estrous phase × Reproductive experience × Early extinction Estrous phase × Reproductive experience × Late extinction Estrous phase × Reproductive experience × Extinction rate
0.545
N = 245. ⁎ p < .05. ⁎⁎ p < .001. 4
B
SE B
β
95% CI
5.439 −2.364 11.136
5.003 4.431 4.193
0.073 −0.036 0.168⁎
[−4.417, 15.295] [−11.092, 6.364] [2.877, 19.396]
0.453
−6.454 −3.184 4.789 0.143 0.252 1.986
3.759 3.278 3.127 0.103 0.053 0.225
−0.086 −0.048 0.072 0.072 0.252⁎⁎ 0.514⁎⁎
[−13.859, 0.952] [−9.642, 3.274] [−1.372, 10.949] [−0.060, 0.347] [0.148, 0.355] [1.542, 2.429]
0.057
−0.635 8.638 16.364 −0.331 0.265 2.518 0.955 −0.086 −0.769 −23.301 0.507 0.193 −0.934 −1.008 −0.255 1.351
4.079 4.518 4.835 0.221 0.117 0.572 0.338 0.163 0.785 6.542 0.286 0.160 0.727 0.428 0.214 0.954
−0.008 0.130 0.248⁎⁎ −0.168 0.265⁎ 0.651⁎⁎ 0.308⁎ −0.068 −0.152 −0.322⁎⁎ 0.187 0.144 −0.185 −0.257⁎ −0.153 0.219
[−8.672, 7.402] [−0.265, 17.541] [6.838, 25.890] [−0.766, 0.104] [0.034, 0.495] [1.391, 3.644] [0.289, 0.1.622] [−0.408, 0.236] [−2.315, 0.777] [−36.191, −10.410] [−0.056, 1.070] [−0.122, 0.509] [−2.367, 0.498] [−1.852, −0.165] [−0.678, 0.167] [−0.529, 3.231]
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Conditioning
100
% freezing
80 60
Extinction recall
Extinction training
NP PRO NP MET PP PRO PP MET
*
Proestrus Metestrus
40 20 0
CS1
CS2
Early extinction
Late extinction
Nulliparous
Primiparous
Fig. 1. Mean ( ± SEM) levels of CS-elicited freezing during conditioning, extinction training, and extinction recall among nulliparous and primiparous females extinguished during either the metestrus and proestrus phase of the estrous cycle. While conditioning and extinction training data were not analysed in this study, this data has been included in the figure for completeness. *Nulliparous-Metestrus > Nulliparous-Proestrus (p < .05). ** NulliparousMetestrus > Primiparous > Nulliparous-Proestrus (p < .05). Note: NP = nulliparous, PP = primiparous, PRO = proestrus, MET = metestrus.
laboratory demonstrating that fear extinction in primiparous females is not influenced by endogenous (Graham, 2018; Milligan-Saville and Graham, 2016) or exogenous (Tang and Graham, 2019a) estradiol, the absence of a relationship between estrous phase and extinction recall in primiparous rats remains highly notable given the power of the regression analysis to detect subtle effects. The results of the current regression therefore suggest that the association between estradiol and fear extinction in females may be altered by reproductive experience – while fear extinction may be associated with estradiol levels in nulliparous rats, this association may not be present in primiparous rats. The results of the current study also revealed that the effect of estrous phase on the relationship between early extinction and extinction recall may be dependent on reproductive status. While there was a positive association between early extinction and extinction recall among nulliparous females extinguished during metestrus, there was no such association for primiparous females extinguished during metestrus, and for rats extinguished during proestrus, regardless of reproductive status. Although the implications of this finding are, as yet, unclear, this result does provide yet another example of a dissociable impact of estrous phase on fear extinction in nulliparous and primiparous females. This dissociation may be due to long-term changes in endogenous estradiol levels following reproductive experience. Specifically, primiparous rats have been found to show significantly lower levels of endogenous estradiol during proestrus compared to nulliparous rats (Bridges and Byrnes, 2006; Milligan-Saville and Graham, 2016). Reproductive experience has also been shown to alter the expression of estrogen receptors in areas of the brain such as the medial amygdala (Byrnes et al., 2009). It is possible that persistent changes in both endogenous estradiol levels and estrogen receptor expression alters the role of estradiol in fear extinction following reproductive experience. It was somewhat surprising that reproductive experience itself did not emerge as a significant predictor of extinction recall in our analysis, given that we have previously found that primiparous females show impaired extinction recall relative to nulliparous females, and that additional trials of extinction training are required to equate levels of extinction recall between nulliparous and primiparous females (Tang and Graham, 2019a, 2019b). However, it is of note that mean levels of freezing during extinction recall for primiparous females were between that of nulliparous females extinguished during proestrus and nulliparous females extinguished during metestrus, despite the fact that a large proportion of the primiparous rats included in the current regression received one and a half times more extinction trials than nulliparous females. This result lends some support to the idea that fear extinction may be impaired in primiparous rats. Impaired extinction recall in primiparous rats may occur as a result of an impaired ability to acquire, or consolidate extinction. Alternatively, it may be the case that primiparous rats show stronger fear conditioning compared to
between estrous phase and early extinction, and a significant three-way interaction between estrous phase, reproductive experience and early extinction. To determine the source of these interactions, four separate Pearson partial correlations examining the relationship between early extinction and extinction recall among nulliparous females extinguished during proestrus, nulliparous females extinguished during metestrus, primiparous females extinguished during proestrus, and primiparous females extinguished during metestrus were conducted. This analysis method was selected given that estrous phase is a binary variable, whereas early extinction is a continuous variable. In all correlations, late extinction and extinction rate were held constant. There was no significant association between early extinction and extinction recall among nulliparous females extinguished during proestrus, primiparous rats extinguished during proestrus, and primiparous rats extinguished during metestrus (largest r = −0.23, p = .10). However, there was a significant positive association between early extinction and CS-elicited freezing during extinction recall among nulliparous females extinguished during metestrus (r = 0.33, p = .02). 4. Discussion In the current study, a hierarchical MRA was performed on data collated from nine previously published experiments to compare the behavioural, hormonal and reproductive predictors of fear extinction in female rats. This analysis revealed that estrous cycle effects on fear extinction may be dependent on reproductive status. While estrous phase predicts extinction recall, and the relationship between early extinction and extinction recall in nulliparous females, it may not predict either of these variables in primiparous females. It was also revealed that nulliparous and primiparous females share two common behavioural predictors of fear extinction: late extinction and extinction rate. A faster rate of extinction and lower levels of CS-elicited freezing at the end of extinction training were associated with lower levels of CSelicited freezing during extinction recall among both groups of females. Reproductive experience and extinction length also did not emerge as significant predictors of fear extinction in female rats. However, primiparous rats were found to exhibit levels of extinction recall that were between than that of nulliparous rats extinguished during metestrus and nulliparous rats extinguished during proestrus. The hierarchical MRA revealed that estrous phase effects on fear extinction were dependent on reproductive status. Consistent with the results of previous studies (see Li and Graham, 2017), undergoing extinction training during metestrus was associated with impaired extinction recall relative to undergoing extinction training during proestrus in nulliparous females. However, no such association between estrous phase and extinction recall was found in primiparous females. While this latter result is consistent with previous findings from our 5
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nulliparous rats, and are therefore more resistant to extinction. Indeed, in one of our previous studies, we found that primiparous rats show higher levels of CS-elicited fear following conditioning compared to nulliparous rats (Tang and Graham, 2019b). Contrastingly, Rima et al. (2009) found that primiparous females exhibited lower CS-elicited freezing compared to nulliparous females one day after fear conditioning. However, rather than using a footshock as the US, Rima et al. (2009) used a predator noise, which is uncommon in rodent studies examining fear conditioning. Nonetheless, further research is required to disentangle whether impaired extinction recall in primiparous females is due to impaired extinction acquisition/consolidation, or strengthened conditioning. Somewhat surprisingly, extinction length also did not emerge as a significant predictor of extinction recall in the current regression, given that across two of our previous studies (Tang and Graham, 2019a, 2019b), primiparous rats were administered additional trials of extinction training to achieve levels of extinction recall that were comparable to that of nulliparous rats. However, it is of note that while increasing the length of extinction training from 30 trials to 45 trials led to comparable levels of extinction recall between nulliparous and primiparous rats, it did not lead to a significant reduction in levels of CSelicited freezing during extinction recall among primiparous rats. Rather, both an increase in the number of extinction trials, and the administration of a weaker conditioning protocol were required to achieve a significant reduction in freezing at extinction recall (Tang and Graham, 2019b). As such, the results of the current regression are broadly consistent with the findings of our previous study. The current regression revealed that late extinction and extinction rate predicted extinction recall in both nulliparous and primiparous rats. The former result is somewhat inconsistent with the results of our previous study (Tang and Graham, 2019a). In our previous study, nulliparous ‘non-extinguishers’ (i.e., rats that showed high levels of CSelicited freezing at late extinction) showed higher levels of freezing during extinction recall relative to nulliparous ‘extinguishers’ (i.e., rats that showed low levels of CS-elicited freezing at late extinction). Contrastingly, extinction recall was comparable between primiparous nonextinguishers and primiparous extinguishers. Inconsistences between the results of our previous and current study may be because the large sample size in the current study afforded greater power in detecting a relationship between late extinction and extinction recall among primiparous females. However, the fact that we were able to detect a relationship between late extinction and extinction recall among nulliparous, but not primiparous females, in our previous study might suggest that the size of this relationship is greater in nulliparous females compared to primiparous females. There are mixed findings as to the relationship between late extinction and extinction recall in adult male rats and humans. Consistent with the results of the current study, Reznikov et al. (2015) found that adult male rats exhibiting high levels of freezing at the end of extinction training showed significantly higher levels of freezing during extinction recall compared to rats that exhibited low levels of freezing at the end of extinction training. However, there are also studies demonstrating that CS-elicited freezing during extinction recall is unrelated to fear levels at the end of extinction training, in both adult male rats and humans (see Craske et al., 2008; Craske et al., 2014). For instance, as described earlier, Graham and Richardson (2019), who analysed a large sample of male rats using a similar statistical approach to the current study, found no relationship between late extinction and CS-elicited freezing during extinction recall among vehicle-treated rats, although a relationship was evident for rats treated with FGF2. A number of clinical studies have also demonstrated no relationship between fear levels at the end of an exposure therapy session and overall treatment outcomes (see Craske et al., 2008). For instance, Smits et al. (2013) found that treatment outcomes for placebo-treated patients receiving exposure therapy for Social Anxiety Disorder did not depend upon levels of self-reported fear exhibited at the end of each exposure session.
However, a relationship between treatment outcomes and self-reported fear at the end of each exposure session did emerge for DCS-treated patients. Together, these findings reveal that the relationship between late extinction and extinction recall may only emerge under specific circumstances. One possible circumstance that allowed a relationship between late extinction and extinction recall to emerge in the current study is that the parameters used in the experiments included in the regression analysis resulted in a greater level of variability in freezing at the end of extinction training compared to other animal studies examining the relationship between late extinction and extinction recall. Such variability may have better allowed for the detection of a relationship between late extinction and extinction recall. There is now a growing body of evidence suggesting that the hormonal, behavioural and neurobiological features of fear extinction are altered as a result of female reproductive experience. Unlike fear extinction in nulliparous rats, fear extinction in primiparous rats appears to be relapse-resistant (Milligan-Saville and Graham, 2016), estrouscycle independent (Graham, 2018; Milligan-Saville and Graham, 2016), NMDA receptor independent (Tang and Graham, 2019b), and resistant to the augmenting effects of systemic DCS and estradiol (Tang and Graham, 2019a). Observed differences in the features of fear extinction between nulliparous and primiparous rats have previously led us to suggest that the mechanisms underlying fear extinction may be altered as a consequence of reproductive experience. More specifically, we have previously suggested that prior to motherhood, fear extinction is predominantly new learning, and after motherhood, fear extinction is predominantly erasure (Tang and Graham, 2019a). This is because relapse following extinction, NMDA receptor involvement in extinction, and the ability to augment extinction using pharmacological adjuncts can be taken as evidence to suggest that fear extinction involves new learning. The current finding that estrous cycle effects on fear extinction may be dissociable between nulliparous and primiparous rats further supports the notion that the mechanisms underlying fear extinction may be altered by reproductive experience. However, the current study also revealed similarities in the features of fear extinction between nulliparous and primiparous rats – for instance, it revealed that late extinction and extinction rate predicted extinction recall in both groups of rats. These similarities suggest that fear extinction may, in fact, involve similar mechanisms in both nulliparous and primiparous rats. However, it is equally possible that the existence of a relationship between within-session extinction and extinction recall in nulliparous rats is reflective of the degree of learning that occurs during extinction training, whereas the same relationship in primiparous rats may be reflective of the degree of erasure that occurs during extinction training. To determine whether or not fear extinction involves new learning or erasure in primiparous rats, it may be helpful to investigate other neurobiological features of fear extinction in these rats. For instance, it may be useful to examine the involvement of gamma-Aminobutyric acid (GABA), particularly given that the involvement of GABA in fear extinction has been shown to be altered in other populations in which extinction may not involve new learning (i.e., developing rats, Kim and Richardson, 2007). Given that fear extinction serves as a laboratory model, and predictor, of exposure therapy, the results of the current study may have important clinical implications in the treatment of anxiety disorders in women. For instance, given that extinction recall appears to be impaired among primiparous rats compared to nulliparous rats extinguished during proestrus, one may speculate that exposure therapy is less effective in mothers compared to non-mothers undergoing exposure therapy during periods of high estradiol. It may therefore be of interest to identify ways of optimising exposure therapy outcomes among mothers. One approach that has been suggested in recent years involves timing exposure therapy with respect to a woman's menstrual cycle, given that there is extensive evidence showing that endogenous fluctuations in estradiol influence fear extinction in female rats and women (Li and Graham, 2017). That is, conducting exposure therapy 6
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sessions during periods of high endogenous estradiol (e.g., during ovulation), rather than periods of low endogenous estradiol (e.g., during menstruation, or while using hormonal contraceptives) may be associated with better treatment outcomes. Preliminary support for this approach has been found among women receiving exposure therapy for spider phobia (Graham et al., 2018). However, given that estrous phase does not appear to predict extinction recall in primiparous rats (as demonstrated in the current study, and previous studies from our laboratory; Graham, 2018; Milligan-Saville and Graham, 2016), it is possible that timing exposure therapy with respect to a woman's menstrual cycle is an ineffective means of enhancing treatment outcomes among women who are mothers. An alternative approach to enhancing exposure therapy outcomes in mothers may involve reducing levels of fear at the end of an exposure therapy session, given that the current study revealed that lower levels of fear at the end of extinction training predicted better extinction recall in both nulliparous and primiparous rats. There are several possible ways in which fear levels may be reduced at the end of an exposure session. One possibility involves increasing the duration of exposure. It has previously been suggested that doing so may enhance the learning that takes place during exposure, thereby improving treatment outcomes (Craske et al., 2014). However, given that the results of the current regression demonstrated that extinction length did not predict extinction recall in both nulliparous and primiparous rats, it is possible that extending the duration of exposure is an ineffective means of augmenting treatment outcomes in both reproductively experienced and reproductively inexperienced women. A second possible way in which fear levels can be reduced at the end of exposure in women may be through the use of a conditioned inhibitor (i.e., a cue that signals the non-occurrence of the US; Palmatier and Bevins, 2010). Rodent and human studies have consistently shown that subjects exhibit significantly lower levels of fear when co-presented with a CS and a conditioned inhibitor, compared to a CS alone (see Christianson et al., 2012). However, a number of laboratory studies have shown that the presence of conditioned inhibitors during extinction training impairs subsequent extinction recall (see Craske et al., 2008). Similar findings have been made in clinical studies examining the effect of conditioned inhibitors or ‘safety behaviours’ on exposure therapy outcomes (see Blakey and Abramowitz, 2016). Further research is therefore required to investigate alternative approaches to how fear responses can be lowered at the end of extinction training in primiparous rats, with the view of translating these approaches into clinical settings. It is of note that there are a number of limitations of the current study. Firstly, while the use of different extinction parameters allowed us to assess this variable as a predictor of extinction recall, the distribution of rats receiving 30 rather than 45 extinction trials was uneven among primiparous compared to nulliparous rats. Namely, the proportion of nulliparous rats receiving 30 extinction trials was greater than that of primiparous rats. As such, the current results, particularly those with regard to the impact of extinction length on fear extinction in nulliparous rats, need to be interpreted with caution. Moreover, all included data came from studies conducted in our laboratory. While the effect of estrous cycle on fear extinction in nulliparous females has been extensively replicated, it remains important to replicate the results of the current regression, especially those on reproductive experience, in other laboratories and rodent strains. A further limitation of the current study was that extinction recall involved a continuous two minute CS presentation, while both conditioning and extinction training involved a ten second CS presentation. It is possible that the extension of the CS during extinction recall altered rats' fear response. However, this is unlikely given that in a previous study conducted in our laboratory (Graham, 2018), the first 10 s of extinction recall data were analysed alongside the full 2 min of extinction recall data. Both analyses revealed an identical pattern of results. In summary, the results of the current study provide some insight into factors that may influence fear extinction in female rats.
Specifically, it was demonstrated that the rate and degree of withinsession extinction exhibited by female rats, and estrous phase might significantly predict extinction recall. However, the impact of estrous phase on extinction recall appears to be modulated by female reproductive status. Although reproductive status in itself did not emerge as a significant predictor of extinction recall, follow-up analyses provided some evidence to suggest that primiparous rats may exhibit poorer extinction recall compared to nulliparous extinguished during proestrus. Given that fear extinction forms the laboratory basis of exposure therapy, these findings may have important implications for the treatment of anxiety disorders in women across the reproductive lifespan. Funding and disclosure This work was supported by grants from the Australian Research Council (DE140100243 and DP180101563) to BMG, and an Australian Postgraduate Award to ST. The authors have no conflicts of interest to declare. 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