Background
There are now substantial data indicating that structural, cellular and molecular differences exist between the male and female brains in regions that are important for cognition, memory and affect, such as the hippocampus, amygdala and prefrontal cortex. Some of these differences may have clinical relevance, as marked disparities in disease incidence, manifestation, prognosis and treatment have been observed between the sexes. For example, men have a higher prevalence of conditions that emerge early in development, such as autism, attention deficit hyperactivity disorder and schizophrenia. Women, on the other hand, have a higher prevalence of disorders that emerge in adolescence or adulthood, such as major depression and anxiety disorders. Surprisingly, very little is known about the neural mechanisms that underlie the expression of sex differences in psychiatric disorders. A 2001 report by the Institute of Medicine highlighted the need to conduct scientific studies at the cellular, molecular and whole organism level that take into account sex as a variable to investigate the neural mechanisms that lead to epidemiological differences in psychiatric disorders.
The need to examine sex differences in the network mediating fear learning and its extinction can be surmised from two different perspectives. From a clinical perspective, we need to understand what contributes to the significant epidemiological differences in psychiatric disorders that are characterized by exaggerated fear and anxiety, such as post traumatic stress disorder. From a basic neuroscience perspective, it is essential that we understand how male and female brains differ in processing fundamental neurobiological phenomena such as emotional learning and memory. There is now a clear indication that failure in the function of brain regions that mediate fear learning and fear inhibition may be associated with the psychopathology of anxiety disorders 12345. Sexual dimorphism in the amygdala, hippocampus and medial prefrontal cortices is well documented 67. These brain regions also contain elevated levels of estrogen receptors 8910. Thus, the structural and functional differences in these brain regions between the sexes may explain in part, or contribute to, some of the basic and clinical differences observed between men and women. In examining the literature pertinent to learning and memory, fear conditioning, and fear extinction, it is astonishing to note that of all that we have learned about these processes, less than 2% of this research has been focused on the female brain (Figure 1).
<p>Figure 1</p>Research studies using males vs females
Research studies using males vs females.
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The fear extinction network: a brief overview
Review of the fear extinction literature in general is beyond the scope of this article. We direct the reader to some recent reviews on fear extinction that focused on the translational aspects of this line of research 11, the general clinical relevance 1213141516 and the molecular machinery of fear extinction 17. Our review of fear extinction will be brief with the objective of setting the stage for the points we intend to make regarding sex differences in, and estrogen's influence on, the fear extinction network.
Components of the fear extinction network
Early studies of conditioned fear extinction showed that blockade of N-Methyl-D-aspartic acid (NMDA) receptors within the amygdala impair the extinction of fear potentiated startle 18. A number of studies subsequently showed that lesion or pharmacological manipulations of the basolateral amygdala (BLA) and the intercalated GABAergic neurons within the amygdala interfere with fear extinction learning 192021. Electrophysiological data recorded from the BLA during fear extinction indicate the existence of two neural populations: one signals fear and the other signals fear inhibition or extinction 22. Thus, in addition to its role in fear acquisition 23, the amygdala appears to also play a critical role in fear extinction 2425.
In addition to the amygdala, the infralimbic (IL) region of the rat ventromedial prefrontal cortex (vmPFC) appears critical for the consolidation and retrieval of the extinction memory after a delay. Lesions 262728, pharmacological manipulations 242930 and electrophysiological recording 31 studies implicate the IL in extinction memory consolidation and expression. Moreover, electrical stimulation of IL simulates extinction memory 3233. Subsequent studies have further supported these findings using different experimental tools, including measuring intrinsic excitability of IL neurons 303435, using metabolic mapping 36, potentiation of thalamic inputs to IL 37, and manipulations of hippocampal inputs to IL 3839.
Another key structure that plays a role in fear extinction is the hippocampus. While its specific role in fear extinction is currently being investigated, it is likely that the hippocampus learns about the context in which fear learning took place and learns about the context in which extinction learning takes place 4041. During extinction recall, the hippocampus can, depending on the context, allow the expression of either the fear memory via activation of the amygdala fear neurons, or safety memory via activation of IL neurons 42. A number of studies have now shown that the interaction between the hippocampus and IL during fear extinction is key to the success of extinction memory consolidation and expression 41434445.
Neuroimaging studies have translated these findings to the human brain using comparable fear conditioning paradigms, some of which use contextual manipulations of fear conditioning and extinction 46474849. Imaging studies began by implicating the human amygdala in fear conditioning and also during extinction learning 465051. Recent functional magnetic resonance imaging studies (fMRI) designed specific paradigms to examine extinction recall and found that, like the rat IL, the human vmPFC increased activation to the extinguished cue during extinction recall; the level of activation positively correlates with the extinction recall magnitude 464751. Thickness of the vmPFC is also correlated with magnitude of extinction recall 5253. Like the amygdala and vmPFC, activation of the hippocampus during fear extinction is reported in a number of imaging studies during contextual conditioning 48 and extinction memory recall 4751. Deactivation of the hippocampus at the time of the delivery of the unconditioned stimulus (US) has also been reported in humans 54.
The molecular machinery mediating learning not to fear
There now exists a large database regarding the molecular machinery involved in fear extinction within the amygdala, hippocampus and IL in rodents. In BLA, interfering with mitogen activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3-K), immediate early genes cfos and early growth response protein 1 (EGR-1) prevented consolidation of extinction 172441. Protein synthesis in BLA is also necessary for fear extinction 55. Extinction training leads to structural changes in BLA. For example, mRNA for the brain-derived neurotrophic factor (BDNF) is up-regulated 56. Furthermore, rats with lentiviral-induced reduction in BDNF receptors in the BLA can extinguish normally within a session, but were unable to recall extinction the following day, consistent with a role of BLA in consolidation of extinction 56. In IL, extinction memory requires NMDA receptor activation 5758, protein kinase A 17, MAPK 59, cannabinoid receptors 60, and protein synthesis 1729. In the hippocampus, extinction of context conditioning requires protein synthesis 61 cyclic adenosine monophosphate (c-AMP) 62, BDNF 43 and a number of protein kinases and their regulators 63.
Sex differences during conditioning and its extinction
A number of studies have investigated differences between females and males in learning and memory using a number of behavioral tasks. For example, females acquire eye-blink conditioning at a faster rate relative to males 64. In inhibitory avoidance tasks, females outperform males in escaping during a one-way avoidance task and a two-way avoidance task 65. Studies conducted to examine sex differences in the acquisition of cued and contextual fear conditioning showed that male rodents exhibit increased contextual and cued fear conditioning relative to females 6667686970, whereas other studies failed to show sex differences in these learning tasks 71.
Relative to fear acquisition, few studies have investigated potential sex differences during extinction learning and recall 7273. We showed that sex differences in fear extinction are influenced by the phase of the estrous cycle in female rats and the menstrual cycle in women 747576. When not taking cycle phase into consideration, differences in fear extinction recall were not noted in either female rats or in naturally cycling women. When females were divided into low and high endogenous estradiol groups, however, sex differences emerged. During extinction recall, male rats showed comparable levels of extinction retention to female rats with high estradiol; both were significantly higher than females with low estradiol 75. The same pattern of results was observed in women. That is, men's extinction retention was comparable to that of women with high estradiol; and both groups showed significantly higher levels of extinction retention compared to women with low estradiol 76. Thus, the lack of sex differences reported in previous studies or the discrepant results between studies may be the result of not taking into consideration the cycle phase of the animals being tested. These data also raise the following question: could sex hormones influence the learning, consolidation and plasticity typically associated with fear extinction in the female brain?
Sex hormones influencing fear extinction in the female rat and in women
The data reviewed above indicate that sex hormones in rodents and humans may contribute to differences in fear learning and fear extinction. In support of this, estrogen treatment in ovariectomized female rats enhanced the acquisition of fear conditioning 7077 and fear potentiated startle 78; though elevated estrogen levels have been associated with reduced contextual conditioning in another study 67. A few studies have shown that estrogen facilitated the extinction of passive avoidance 79 and conditioned taste aversion 80. To date, there are few published studies that specifically examined the role of sex hormones on fear extinction learning and its subsequent recall. One exception is Chang et al., (2009), who have recently shown that infusion of estrogen into the hippocampus facilitates extinction of context conditioning and enhances hippocampal long term potentiation (LTP) 81.
We have recently conducted a number of experiments examining how endogenous fluctuations and exogenous manipulations of sex hormones, particularly estrogen and progesterone, influence fear extinction in female rats. A diagram of the natural fluctuations of sex hormones during the estrous cycle in rats and the menstrual cycle in women is shown in Figure 2. We observed that naturally cycling females exhibited the least amount of freezing during extinction recall when they underwent extinction learning during the proestrus phase (high estrogen and progesterone) of the estrous cycle 75. These findings suggest that estrogen and/or progesterone may facilitate the consolidation of extinction learning (Figure 3B). In support of this, systemic pre- or post-extinction administration of estradiol into female rats undergoing extinction learning in the metestrus phase (low estrogen and progesterone) significantly reduced freezing during recall (Figure 4a, b) 75. Systemic blockade of estrogen receptors alpha (ERα) and estrogen receptor beta (ERβ) in female rats undergoing extinction in the proestrus phase significantly increased freezing 75. In a later study, we observed that administration of ERβ but not ERα agonists were able to facilitate extinction recall in female rats undergoing extinction training in the metestrus phase of the cycle 74. These data provide strong evidence that cycling sex hormones, estradiol in particular, in female rats do indeed influence extinction consolidation, possibly via the selective activation of the ERβ receptors.
<p>Figure 2</p>The menstrual cycle and the estrous cycle 164
The menstrual cycle and the estrous cycle 164.
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<p>Figure 3</p>Effects of endogenous estrogen on extinction recall
Effects of endogenous estrogen on extinction recall.
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<p>Figure 4</p>Effects of exogenous estrogen on extinction recall
Effects of exogenous estrogen on extinction recall.
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In women, neuroimaging studies have shown that measures of fear and arousal are associated with changes in hormonal levels throughout the menstrual cycle 678283. Interestingly, increased vmPFC activation was observed in women in the luteal phase relative to those in the early follicular phase of the menstrual cycle while performing a Go-No-Go Task 84, suggesting that estrogen may facilitate the functional activation of the vmPFC. We have conducted an initial psychophysiological study to assess the influence of the menstrual cycle phase on recall of fear extinction in healthy women, and found that natural fluctuations of gonadal hormones do modulate extinction recall 85. Women with high estrogen exhibited significantly enhanced extinction recall (that is, less fear) relative to women with low estrogen levels 76 (Figure 3A). In a more recent study, we replicated the effect of facilitated extinction recall in women with high estrogen and found that the increased estrogen levels in these women is associated with increased vmPFC, hippocampal and amygdala activation during extinction recall (Figure 5), further supporting the idea that estrogen may be playing a critical role in extinction memory consolidation 74.
<p>Figure 5</p>Effects of estrogen in vmPFC activation and gene expression during extinction recall
Effects of estrogen in vmPFC activation and gene expression during extinction recall.
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How might estrogen modulate extinction recall?
Estrogen and its receptors
In females, estrogen is primarily produced by the ovaries, while in males testosterone is produce by the testis and then aromatized into estrogen 86. Estrogen can also be synthesized in brain regions, such as the hippocampus 87. The most potent circulating estrogen is 17β-estradiol and the most characterized ERs are ERα and ERβ 88. These receptors belong to the nuclear receptor superfamily and can be localized in the nucleus as well as in the cytoplasm of the cell 88. ERα and ERβ are coded by different genes but share similar DNA-binding and ligand-binding domains. Estrogen binds to either ERα or ERβ through its estrogen response element DNA binding site. These result in receptor dimerization and subsequence gene transcription 89.
Localization of the estrogen receptors
Both receptors are located throughout the rostral-caudal extent of the brain and spinal cord, including regions of the fear circuitry 9091. Previous studies have shown that expression of ERα and ERβ overlap in many brain regions, such as the bed nucleus of the stria terminalis, medial and cortical amygdaloid nuclei, periaqueductal grey and locus coeruleus 91. However, differences in expression have also been shown where one type of receptor is either expressed alone or at higher concentrations relative to the other. For example, ERα is the predominant receptor expressed in the ventromedial nucleus of the hypothalamus while ERβ is most prevalent in the hippocampus 92. ERα and ERβ are expressed in the amygdala while ERβ is mostly expressed in PFC 939495. Activation of these two types of receptors leads to different behavioral consequences. Accumulating evidence now indicates that selective ERβ agonists typically exert potent anxiolytic activity when animals were tested in a number of behavioral paradigms 919697. In contrast, selective ERα agonists were found to be anxiogenic and correspondingly increased the hormonal stress response 91.
Sex differences in the estrogen receptors
ERα and ERβ have similar distributions in male and female brains, although subcellular distributions of ER to the nucleus, cytoplasm, dendrites and nerve terminals have been reported to be different in male and female human hypothalamus 98. Although the functional consequences of these differences remain to be determined, this could indicate differential effects of estrogen in the male versus female brains regarding cellular processes, such as neurite extension, synaptic plasticity and mitochondrial energy regulation via mitochondrial ERs. There is also evidence for sex differences in intracellular signaling, expression of co-regulatory proteins, and in the response of the brain ER/aromatase system to injury 88.
Estrogen-induced molecular and cellular changes
The influence of estrogen on molecular and cellular changes in the brain has been examined mostly in the hippocampus 99100 and in the hypothalamus 88. Hippocampal estrogen enhances synaptogenesis and long-term potentiation (LTP) 101, increases the formation of dendritic spines 102103, increases cell proliferation 104, and increases neural excitability 105106. Estrogen-induced LTP in the hippocampus is mediated via synaptic transmission of NMDA receptors 107, specifically through the increased expression of the NR2B subunit 108.
Increased estrogen is also associated with increased BDNF expression in the hippocampus 109110. It is possible that estrogen-enhanced extinction memory consolidation may be mediated via increasing BDNF expression in the hippocampus or the vmPFC. Indeed, BDNF expression is critical for successful fear extinction in both rodents and humans 111. Peters et al. (2010) showed that hippocampal infusion of BDNF before extinction training enhanced extinction memory in an NMDAr dependent process, suggesting that hippocampal BDNF modulates IL activity during the consolidation of extinction memory 112. Estrogen and BDNF are known to have similar mechanisms of actions, activate the same cascades, and have the same behavioral effects, especially within the hippocampus. For example, both estrogen and BDNF enhance hippocampal-dependent learning 109110. The direct interaction between estrogen and BDNF during fear extinction has not yet been investigated.
Few studies have examined the influence of estrogen on the function of the vmPFC in the rat. Estrogen has been shown to increase spine density in vmPFC 113, preserve the functional integrity of the IL when subjecting the rats to chronic stress 114, and to enhance working memory 115. These data indicate that estrogen influences the function of the vmPFC in the rat brain. Markers of neural activity and synaptic plasticity, such as c-Fos, Jun-b and BDNF, have been shown to be modulated by estrogen variance 74116117118. Therefore, we propose that estrogen may modulate fear extinction learning and consolidation via its interaction with a number of molecular markers of plasticity, within the fear extinction network.
We recently examined the influence of estradiol administration on c-fos and Jun-b mRNA expression within some components of the fear extinction network. Female rats undergoing fear extinction during the metestrus phase of the cycle (low estrogen) received estradiol immediately post-extinction training and were sacrificed immediately after a brief extinction recall test. The results of this study showed that administration of estradiol after extinction training enhanced c-fos and June-b mRNA expression in IL (Figure 5b) and significantly reduced the expression of both in the amygdala during recall 74. These data further support the idea that estrogen fluctuations may be a critical modulator of extinction memory consolidation in females. In order to fully understand the role of estrogen in fear extinction, it is necessary to identify the molecular cascades by which estrogen may enhance extinction memory and investigate sex differences in estrogen action within the extinction network.
What about progesterone and testosterone?
Progesterone
Several studies have shown that progesterone administration to ovariectomized female rats facilitates contextual and cued fear conditioning, and enhances cognitive performance in a variety of other behavioral tasks in mice 119. In healthy young women, administration of progesterone during the early follicular phase (when progesterone and estrogen are at their lowest level) led to increased reactivity in the amygdala while looking at threatening faces 120. Also, progesterone increased the functional coupling of the amygdala with the mPFC 121, indicating that progesterone influences interactions between the amygdala-PFC circuits. Moreover, progesterone is metabolized into allopregnanolone 122, which acts via GABAA receptors and appears to have anxiolytic effects when infused into the amygdala or vmPFC before an elevated plus-maze test and shock-probe burying test 122123. In other tasks, progesterone facilitates extinction of cocaine self-administration 124. We have shown that systemic administration of progesterone into female rats (either alone or in conjunction with estrogen) facilitates extinction consolidation 75, suggesting that progesterone appears to also influence the function of brain regions involved in extinction consolidation. Our data gathered in women, however, showed that variance in progesterone levels in two separate cohorts of women did not correlate with extinction recall 7476. While progesterone's influence on fear extinction may differ across species, it is important to note that we were not able to fully examine the effects of progesterone independent of estrogen. Thus, it remains possible that progesterone may have an effect on fear extinction consolidation in women directly or perhaps by interacting with estrogen. Additional studies are needed to further examine the role of progesterone in fear extinction in women.
Testosterone
It is established that besides the masculinization/defeminizing role of testosterone during sexual differentiation, this hormone is also critical for the modulation of behavioral and physiological responses to anger 125126. Men demonstrating higher dominance express high levels of testosterone. In male primates, dominance is associated with higher testosterone levels and a decrease in stress response, indicating that dominant males find dominance signals less stressful and are more primed to engage in a dominance challenge 127128. More interestingly, previous studies have shown that testosterone reduces cortisol response and stress axis reactivity 129130. Other studies demonstrated that endogenous as well as exogenous testosterone influence neural reactivity to threatening faces in the amygdala and orbitofrontal cortex (OFC) in males, and in the amygdala in women 128131. Exogenous testosterone also increases amygdala reactivity to threatening faces, but reduces functional coupling between the amygdala and OFC in middle-aged women, suggesting that the testosterone may regulate interactions between amygdala and OFC 128. It is important to note that effects of testosterone may be mediated via direct interactions with androgen receptors or via conversion to other steroids. Testosterone is metabolized to dihydrotestosterone (DHT), which also acts via androgen receptors, and to androstanediol that modulates GABAA receptor similar to allopregnanolone 132. Lastly, testosterone is also aromatized into 17b-estradiol, the most potent type of estradiol, and it has been suggested that most of the effects of testosterone are mediated by estrogen 133134. Clinical and basic studies are needed to assess the role of testosterone during fear extinction.
Clinical relevance
Increased expression of inappropriate fear is the hallmark of anxiety disorders 135136137138. A large body of evidence from neuroimaging studies indicates that the neural circuits subserving fear conditioning and extinction are impaired across the different anxiety disorders 12. For example, post-traumatic stress disorder (PTSD) patients exhibit decreased vmPFC and hippocampal activation along with exaggerated amygdala activation during the processing of emotional stimuli in a wide array of paradigms 139140141142. Experimental extinction is also deficient in PTSD patients 10143144145146. We have recently shown that fear extinction is deficient in PTSD patients and that such deficiency is associated with aberrant activation of the vmPFC, hippocampus and amygdala (in addition to insula and striatal regions) during fear acquisition, extinction learning and extinction recall 155474.
Epidemiological data suggest that the prevalence of anxiety disorders is higher in women relative to men. Women are more likely to develop panic disorder (8% vs. 3%), PTSD (12.5% vs. 6%) and generalized anxiety disorder (GAD) (7% vs. 4%) (Pigott, 2003; Breslau et al., 1998;Kinrys and Wygant, 2005). Aside from prevalence, women diagnosed with PTSD have longer symptom duration (48 vs. 12 months) 147, have higher symptom severity and functional impairment 148, and have worse quality of life 149. Women diagnosed with GAD are more likely to develop comorbid psychiatric disorders and have worse prognoses and impairments 150. In addition to increased prevalence of panic disorder in women, studies also suggest that panic attacks occur more frequently in women relative to men 151. These findings point to brain-based differences in the processing of emotional stimuli in women compared to men, and suggest that sex hormones, such as estrogen, play a key role in mediating these differences. Indeed, there are some clinical data suggesting that estrogen therapy improves anxiety symptoms in postnatal depression 152, in recurrent postpartum affective disorder 153, and in menopause 154155. Despite these glaring differences, it is puzzling that so few studies have considered sex differences as a critical variable of interest.