We examined the influence of natural fluctuations of gonadal hormones on the acquisition, extinction learning, and extinction recall of conditioned fear in women. Estradiol levels did not affect the acquisition or extinction of conditioned fear but facilitated extinction memory recall. These results suggest a possible role of estradiol in the consolidation of extinction memories. With regard to sex differences, when gonadal hormones were not considered, sex differences were observed during the acquisition of conditioned fear, with men showing elevated CRs relative to women, replicating our previous report (Milad et al., 2006a
). When taking the women's estradiol levels into consideration, however, sex differences were observed during the extinction retention test. Men and women with high estradiol levels exhibited significantly more extinction memory than women with low estradiol levels.
Several studies previously examined sex differences in fear conditioning in humans (van der Molen et al., 1988
; Zorawski et al., 2005
). However, the influence of estradiol and on fear extinction and extinction memory has not been addressed. Our findings here suggest that estradiol appears to significantly facilitate extinction recall in women. Such a role for estradiol in extinction retention has recently received support from the rodent literature. In an auditory fear conditioning paradigm, we recently showed that administration of estradiol to female rats significantly enhanced extinction recall whereas injection of estradiol receptor antagonists impaired it (Milad et al., 2009a
). Chang et al., (2009)
have shown that injections of estradiol into the hippocampus facilitate extinction of contextual fear. Moreover, estradiol has anxiolytic effects in rodents (Lund et al., 2005
; Walf and Frye, 2005
) and appears to facilitate memory formation in several behavioral paradigms (Leuner et al., 2004
), including extinction of passive avoidance (Rivas-Arancibia and Vazquez-Pereyra, 1994
) and conditioned-taste aversion (Yuan and Chambers, 1999
). Previously, we assessed the influence of menstrual phase on extinction recall by focusing on two phases of the menstrual cycle: early follicular, where estradiol and progesterone levels are low, and late follicular, where estradiol levels are high and progesterone relatively low (Milad et al., 2006a
). However, hormonal levels were not directly measured in that study, and therefore estimates relied solely on the reports of the participants regarding their menstrual phase in contrast to the study reported here.
Unlike estradiol, progesterone did not appear to have a significant effect on fear extinction. This is interesting given that we have recently shown that progesterone does appear to have a significant role in facilitating recall of fear extinction in female rats (Milad et al., 2009a
). Moreover, it has also been shown that progesterone administration to rodents facilitates cognitive performance in a variety of behavioral tasks such as object recognition (Frye and Walf, 2008
). Allopregnanolone, a metabolite of progesterone, has anxiolytic effects that are mediated through brain regions involved in fear extinction such as the amygdala and the vmPFC (Engin and Treit, 2007
). The substantial overlap of estradiol and progesterone in our sample did not allow us to fully examine the effects of progesterone independent of estradiol. Thus, it remains possible that progesterone may have an effect on fear extinction consolidation in women directly or perhaps by interacting with estradiol. Additional studies are needed to further examine the role of progesterone in fear extinction in women.
One study of healthy human subjects reported no sex differences in fear conditioning (Zorawski et al., 2005
), whereas another reported elevated CRs in women relative to men (Guimaraes et al., 1991
). The apparent discrepancy between the results of the above studies and our data presented herein may be due, in part, to at least two factors. First, these studies did not control for menstrual cycle phase in their sample. Second, these studies included an unidentified number of women on birth control (who by definition would not be cycling), with other women who were cycling, thus introducing potential confounds with regard to investigating sex effects. However, men showed elevated conditioned responses during the acquisition phase relative to women, which was consistent with our previous results (Milad et al., 2006a
), and with finding increased freezing in male relative to female rodents in cued and contextual fear conditioning (Aguilar et al., 2003
; Gupta et al., 2001
; Wiltgen et al., 2001
). Our findings are also consistent with a recent functional MRI study that demonstrated increased brain activity in stress response circuitry in men compared with women during ovulation (when estradiol is high relative to progesterone) in contrast to men compared with women during the early follicular period (during which estradiol and progesterone are low) (Goldstein et al., 2010
In the present study, similar to the stress response in our previous work (Goldstein et al., 2010
), men consistently displayed elevated expression of CRs during fear conditioning relative to both HE and LE groups in women (data not shown). Thus, these data indicate that sex differences during fear acquisition may be mediated by other mechanisms, including other hormonal systems that may or may not interact with estrogen and progesterone. For example, it has been previously shown that cortisol levels are associated with facilitated memory recall during acute stress in women (Andreano et al., 2008
). Pre-conditioning injections of cortisol reduced conditioned fear in men while it had the opposite effect in women (Merz et al., 2010
; Stark et al., 2006
), thus suggesting that sex differences during fear learning may be mediated via the impact of cortisol (Andreano and Cahill, 2009
). Another possibility for the observed sex differences during fear conditioning may be due to influences of gonadal hormones during early development on brain regions mediating fear conditioning (Dalla and Shors, 2009
The level of extinction memory in men was comparable to that in women with high estradiol, whereas women with low estradiol showed significantly lower extinction memory during the recall test relative to men. This finding is counterintuitive given that the levels of estradiol in men would be most comparable to those found in low-estradiol women, and therefore one would have expected comparable extinction between these two groups. We speculate that the observed differences may be due to the elevated levels of testosterone in men. It has been shown that testosterone injections into male rats facilitated learning of inhibitory avoidance, reduced anxiety levels, and facilitated extinction of conditioned fear in mice (Edinger and Frye, 2007
; Frye et al., 2008
). Testosterone may have direct effects on facilitating extinction in men and may have indirect effects through its aromatization into estrogen. The role of testosterone in men and women on extinction memory consolidation should be further examined given that there are few studies investigating this issue.
It is worth noting that we observed a significant group difference in the extroversion trait between the HE and LE groups and significant group difference in the agreeableness factor between men and women. In a previous study, we found that only extroversion was positively correlated with fear extinction (Rauch et al., 2005
). Yet, in the present sample, extroversion was higher in women with lower extinction memory (the lowe-stradiol group) and was lower in the women that showed enhanced extinction memory (the high estradiol group). Thus, these differences are unlikely to have influenced the between-group differences we observed in fear extinction. In addition, we observed a marginal difference between men and women in the ASI measure (supplemental table 1
). This difference does not appear to influence group psychophysiological differences as such differences remained significant when co-varying for the ASI values.
Findings in the study reported here may have clinical implications given that several epidemiological studies have reported elevated incidence of anxiety disorders in women relative to men (Kinrys and Wygant, 2005
; Pigott, 2003
). Other studies have demonstrated sex differences in brain activity in processing emotional stimuli (Andreano and Cahill, 2009
; Cahill, 2003
; Goldstein et al., 2010
; Protopopescu et al., 2005
). Importantly, women diagnosed with premenstrual dysphoric disorder (PMDD) show increased anxiety and depression symptoms that are recurrent during every cycle, and that have recently been associated with genetic variation of the estrogen receptor alpha (Huo et al., 2007
). Moreover, women diagnosed with PMDD exhibited increased amygdala and decreased vmPFC activation in the context of a behavioral inhibition paradigm (Protopopescu et al., 2008
). Results of the present study suggest that there may be an association between natural variation of gonadal hormones, estradiol in particular, and the brain circuitry involved in fear inhibition. Collectively, therefore, it appears that low levels of gonadal hormones may be related to impaired fear extinction and impaired processing of emotional stimuli in women with various psychiatric disorders. Future studies of the neural circuitry of fear extinction, such as those conducted with healthy humans and patients with anxiety disorders such as PTSD (Bremner et al., 2005
; Milad et al., 2009b
), should consider obtaining information regarding gonadal hormone levels and examining their associations with activity in brain regions involved in fear inhibition. Such studies could potentially lead to novel ways to enhance the efficacy of extinction-based therapies for anxiety disorders. For example, concentrating exposure therapy at a particular phase of the menstrual cycle in women and/or administration of estradiol in conjunction with exposure therapy may strengthen extinction consolidation.