Modulation of Fear Extinction by Stress, Stress Hormones and Estradiol: A Review
Experimental Psychology II and Biological Psychology, Institute of Psychology, University of OsnabrückOsnabrück, Germany
*Correspondence: Ursula Stockhorst ursula.stockhorst@uniosnabrueck.deAbstract
Fear acquisition and extinction are valid models for the etiology and treatment of anxiety, trauma- and stressor-related disorders. These disorders are assumed to involve aversive learning under acute and/or chronic stress. Importantly, fear conditioning and stress share common neuronal circuits. The stress response involves multiple changes interacting in a time-dependent manner: (a) the fast first-wave stress response [with central actions of noradrenaline, dopamine, serotonin, corticotropin-releasing hormone (CRH), plus increased sympathetic tone and peripheral catecholamine release] and (b) the second-wave stress response [with peripheral release of glucocorticoids (GCs) after activation of the hypothalamus-pituitary-adrenocortical (HPA) axis]. Control of fear during extinction is also sensitive to these stress-response mediators. In the present review, we will thus examine current animal and human data, addressing the role of stress and single stress-response mediators for successful acquisition, consolidation and recall of fear extinction. We report studies using pharmacological manipulations targeting a number of stress-related neurotransmitters and neuromodulators [monoamines, opioids, endocannabinoids (eCBs), neuropeptide Y, oxytocin, GCs] and behavioral stress induction. As anxiety, trauma- and stressor-related disorders are more common in women, recent research focuses on female sex hormones and identifies a potential role for estradiol in fear extinction. We will thus summarize animal and human data on the role of estradiol and explore possible interactions with stress or stress-response mediators in extinction. This also aims at identifying time-windows of enhanced (or reduced) sensitivity for fear extinction, and thus also for successful exposure therapy.
Introduction
Classical fear conditioning (consisting of fear acquisition and extinction) is an influential experimental model to study emotional learning and memory. In fear acquisition, an initially neutral stimulus (cue or context) is paired with an aversive unconditioned stimulus (US) capable of eliciting a fear response (e.g., Phillips and LeDoux, 1992). After several pairings in the acquisition phase, the neutral stimulus becomes a conditioned stimulus (CS), now capable of eliciting a conditioned fear response (CR). Fear extinction is induced when the CS is repeatedly presented without the aversive outcome (US), resulting in a decline of CRs. In rodents, fear responses are typically assessed via freezing behavior (e.g., Bouton and Bolles, 1980; Fanselow and Helmstetter, 1988) or the fear-potentiated startle reflex (e.g., Brown et al., 1951; Davis, 1986). In humans, learning indicators used alone or combined include: skin conductance responses (SCRs), fear-potentiated startle, functional imaging data, as well as subjective ratings of US expectancy and/or CS valence/arousal (e.g., Grillon et al., 2004; Stark et al., 2006; Blechert et al., 2007; Milad et al., 2010; Soeter and Kindt, 2012; Bentz et al., 2013). Fear conditioning is highly adaptive, as it enables the organism to effectively and rapidly learn to predict danger using context information and environmental cues. However, fear learning can become dysfunctional if the organism continues to display fear responses in the absence of danger.
Both, fear acquisition and extinction, are valid to model features of anxiety disorders as well as trauma- and stressor-related disorders (such as posttraumatic stress disorder, PTSD). Specifically, fear acquisition can serve to model features of the etiology of these disorders. Correspondingly, simple phobias, social phobia, panic disorder, and PTSD are characterized primarily by dysregulated fear responses (Ehlers and Clark, 2000; Parsons and Ressler, 2013). Moreover, these disorders are characterized by deficits in fear extinction. This inability to inhibit fear responses is assumed to largely contribute to the maintenance of anxiety disorders (Lissek et al., 2005; Delgado et al., 2006; Mineka and Zinbarg, 2006; Mineka and Oehlberg, 2008), as well as trauma- and stressor-related disorders (e.g., Ehlers and Clark, 2000; Mineka and Oehlberg, 2008; Cover et al., 2014). PTSD is assumed to be related to and even caused by a failure to consolidate and retrieve memory for fear extinction (Quirk and Mueller, 2008). Correspondingly, patients with anxiety and especially trauma-related disorders show deficits in fear extinction learning and extinction recall (Lissek et al., 2005; Blechert et al., 2007; Michael et al., 2007; Milad et al., 2008, 2009b; Jovanovic et al., 2010; Glover et al., 2011; Norrholm et al., 2011; Inslicht et al., 2013). Moreover, extinction learning also serves as a model for exposure techniques in behavioral therapy (e.g., Milad et al., 2014).
The development of anxiety disorders and especially PTSD can be conceptualized as learning under severe stress. Stress is a state of actual or potential disruption in the individual’s internal/external environment registered by the brain and caused by factors we call stressors (Joëls and Baram, 2009). Stress leads to activation of the stress response—including activation of the central and peripheral nervous system and release of neuromodulators, hormones and transmitters—the stress-response mediators—in the brain and periphery. The stress response enables the organism to deal with the challenge by increasing central arousal, mobilizing energy, increasing cardiovascular tone, inhibiting costly processes such as reproduction, feeding, and digestion, and by modifying immune responses (Sapolsky et al., 2000; Chrousos, 2009). Thus, stress and the stress response are important for survival and are adaptive in nature. However, in some circumstances stress may cause pathology, as is the case in PTSD and other trauma- and stressor-related disorders. Traumatic situations not only include specific fear-related behaviors (e.g., flight, freezing) but also a significant amount of the less specific stress response. Thus, understanding how stress, the stress response, and specific stress-response mediators contribute to pathological changes seen in PTSD—such as impaired extinction—is of special importance. Moreover, understanding what conditions allow for normal functioning despite (traumatic) stress could advance our understanding of resilience and advance prevention. Last but not least, PTSD itself is accompanied by heightened stress (Maren and Holmes, 2016) and is also associated with changes of the stress system (e.g., Lupien et al., 2009). Both facts could interfere with the success of extinction-based exposure therapy. Consequently, examining effects of stress and single stress-response mediators on fear extinction could help improve treatment efficacy or even provide new targets for pharmacological treatment.
While anxiety disorders and trauma- and stressor-related disorders have a nearly twofold life-time prevalence in women as compared to men (Kessler et al., 1995, 2005; Tolin and Foa, 2006; Kilpatrick et al., 2013; for an overview, see Cover et al., 2014), there is increasing evidence for a role of the female sex hormone 17β-estradiol (E2) in these sex differences. Interestingly, there is first evidence that the quality of fear extinction is related to estrogen levels as supported by better extinction recall under high as compared to low E2-levels.
In line with the focus of the articles assembled in this Research Topic, we will concentrate on fear extinction and the role of stress and stress-response mediators in animals and in humans thereby also referring to the role of E2.
Fear Acquisition and Fear Extinction
Fear Acquisition and the Neuronal Fear Circuitry
The fear system can be conceptualized as an adaptive behavioral system that allows the organism to avoid, escape or face environmental threats (Rudy, 2014). The amygdala and its connections play a major role in the regulation of innate fear responses and in fear learning.
Fear acquisition involves an interplay between the basolateral amygdala (BLA), consisting of the lateral nucleus (LA), the basolateral and basomedial nuclei (together also referred to as basal nuclei or basal amygdala, BA), the central nucleus (CE), and the intercalated cell-masses (ITC), located between the BLA and the CE (LeDoux, 2007; Pape and Paré, 2010).
The LA serves as the primary input zone of the amygdala, receiving input from the auditory, visual, olfactory, somatosensory, and nociceptive systems (LeDoux, 2007; Pape and Paré, 2010; Herry and Johansen, 2014). In fear acquisition, information about the CS and the US converges into the LA. The LA is also a necessary site of synaptic plasticity underlying fear learning (LeDoux, 2007; Pape and Paré, 2010; Herry and Johansen, 2014; Tovote et al., 2015) and a main storage site for the fear memory trace (Pape and Paré, 2010).
The LA projects to the basal nuclei, and to the ITC. The ITC inhibit neurons in the CE (Royer and Paré, 2002) and thus prevent the defensive fear responses. The basal nuclei contain two types of neurons: so-called “fear neurons” and “extinction neurons” (Herry et al., 2008). Fear neurons fire when fear is expressed, and they maintain excitatory projections to neurons in the CE and in the prelimbic cortex. Extinction neurons, on the other hand, are active when fear has been extinguished and they project to the ITC (Rudy, 2014).
The CE is one main output region of the amygdala with projections to subcortical and brainstem areas. It coordinates defensive (fear) responses including freezing and endogenous opioid-mediated analgesia (periaqueductal gray, PAG), and startle reflex potentiation (nucleus reticularis pontis caudalis; Davis, 1992; Sah et al., 2003; Fanselow and Poulos, 2005; Pape and Paré, 2010). The CE is also connected to monoamine systems in the brain, including locus coeruleus (LC; noradrenaline, NA), dorsal/ventral striatum (dopamine, DA), and raphe nuclei [serotonin (5-hydroxytryptamine, 5-HT)]. These neuromodulatory connections enable the amygdala to influence the excitability of large portions of the brain, including many areas lacking a direct connection with the amygdala (Sah et al., 2003; Pape and Paré, 2010; Duvarci and Paré, 2014). Finally, the CE also activates hypothalamic nuclei producing the classical peripheral stress response with increased sympathetic arousal, hypothalamus-pituitary-adrenocortical (HPA) axis activation, and increased release of glucocorticoids (GCs) and adrenaline/NA into the bloodstream.
Thus, in the amygdala, fear expression is controlled by a fine interplay of inhibitory and excitatory microcircuits involving the BLA, CE, and ITC (Wolff et al., 2014; reviewed in: Duvarci and Paré, 2014; Dejean et al., 2015). The hippocampus is important for context fear conditioning and for encoding of contextual information in the conditioning situation and is thus assumed to contribute to the context-specificity of fear responses. Accordingly, hippocampus–amygdala interactions are regarded to be important for contextual modulation of fear (e.g., Tovote et al., 2015). Importantly, fear expression is also controlled by the medial prefrontal cortex (mPFC). Neurons in the dorsal part of the mPFC [the prelimbic cortex (PL) in rodents, and dorsal anterior cingulate cortex (dACC) in humans and other primates] promote fear expression (Likhtik and Paz, 2015) and both, BLA and PL/dACC activity, are modulated by the hippocampus (Herry and Johansen, 2014).
Fear Extinction
While the fear system per se fulfills an adaptive function it can become maladaptive and produce pathologies such as anxiety, stressor- and trauma-related disorders (Rudy, 2014). Accordingly, conditions that improve the extinction of conditioned fear and hinder the return of fear are important in order to develop successful treatments of fear and anxiety disorders.
Stages of Fear Extinction Learning and Extinction Memory
In fear extinction, the CS is repeatedly presented without the US and CRs decline. As already suggested by Pavlov (1927), extinction learning does not destroy the original fear memory, but involves new learning. The organism acquires an inhibitory CS-no US association preventing the expression of fear responses (Bouton, 1994, 2002; Mueller and Cahill, 2010). This is supported by the observation that conditioned fear can easily reemerge even after successful extinction (Todd et al., 2014). This is the case when the US alone is presented after extinction (reinstatement), when the CS is presented in a context different from the extinction context (contextual renewal), or just with the passage of time (spontaneous recovery).
Extinction covers three phases (Figure 1A): acquisition, consolidation and retrieval of extinction (Quirk and Mueller, 2008; Mueller and Cahill, 2010). Following fear acquisition (Figure 1B), extinction acquisition (Figure 1C) manifests as the decline of CRs during the initial extinction training session (Quirk and Mueller, 2008). Extinction can be quantified by the amount and speed of this decline. During consolidation, lasting at least several hours (Quirk and Mueller, 2008), cellular signaling cascades progressively stabilize the initially labile extinction memory trace into a consolidated memory (long-term memory; Baldi and Bucherelli, 2015). Extinction retrieval (or recall) is evident when subsequent presentation of the CS triggers retrieval of the extinction memory trace and only low levels of conditioned responding occur. Thus, good extinction recall will manifest in low CRs to the original CS (Figure 1D). Poor extinction recall—or return of fear—is evident in high CRs despite successful extinction. This is the case with reinstatement (Figure 1E), renewal (Figure 1F), and spontaneous recovery (Figure 1G). These return-of-fear phenomena are also a challenge for anxiety therapy, such as exposure therapies. Moreover, if the CS is paired with the US after extinction, we usually see a much faster reemergence of the CR (rapid reacquisition, Figure 1H).
Of note, the timing of different phases relative to each other is also important. For example, immediate extinction (minutes to hours after fear acquisition) vs. delayed extinction (24 h or more) were shown to produce different effects on return-of-fear phenomena. While in some studies immediate extinction produced less return of fear compared to delayed extinction (Myers et al., 2006), this was not replicated in other studies (Archbold et al., 2010; Golkar and Öhman, 2012). There is even evidence that immediate extinction produces poorer long-term fear reduction and more return of fear (Maren and Chang, 2006; Norrholm et al., 2008; Huff et al., 2009), i.e., an “immediate extinction deficit” (for a review, see Maren, 2014).
Importantly, stress, stress-response mediators and sex hormones may influence each of the phases of extinction learning and memory formation differently. Therefore, we will try to systematically review the available literature for effects on acquisition, consolidation, and retrieval of extinction. Where available, we will also report effects on return-of-fear phenomena and consider timing differences.
Presenting the CS without the US provokes a retrieval of the original fear memory. If the number of CS presentations is limited (1–4) this can trigger reconsolidation of the fear memory rather than acquisition of extinction. As originally described in the late 1960’s (Misanin et al., 1968), retrieval temporarily destabilizes the memory trace and makes it more susceptible to disruption. The reactivated memory is then actively re-stabilized (Nader et al., 2000). The increased plasticity is assumed to start about 10 min after retrieval and to last no more than 1 h (Johnson and Casey, 2015). This opens a brief reconsolidation-window during which pharmacological or behavioral intervention may erase the labile memory and prevent a return of fear. Blocking β-adrenergic receptors was found to disrupt reconsolidation in animals (e.g., Debiec and LeDoux, 2004, 2006; Rodriguez-Romaguera et al., 2009). There is also good evidence that β-adrenergic blockade could present a pharmacological tool in humans as well (Kindt et al., 2009; Soeter and Kindt, 2010, 2012; Sevenster et al., 2013; for a review, see Otis et al., 2015), but see also Bos et al. (2014). As a behavioral procedure, extinction training in the reconsolidation window was proposed and repeatedly shown to prevent the return of fear in humans in some studies (Schiller et al., 2010; Agren et al., 2012a) but not in others (Golkar et al., 2012; Kindt and Soeter, 2013). In general, blocking reconsolidation or using the reconsolidation window to modify traumatic memories is a promising technique that might reduce fear expression in patients with anxiety disorders or PTSD (for recent discussions, see Lane et al., 2015; Sandrini et al., 2015). A detailed review of reconsolidation studies is beyond the scope of our review. In the following sections, we will focus on effects of stress, and stress-response mediators on fear extinction. However, we will describe selected results from reconsolidation studies that also include measures of extinction learning and/or memory.
Brain Structures and Circuits Involved in Fear Extinction
Extinction, just as fear acquisition, is distributed over a network of structures, mainly covering the BLA, the hippocampus and parts of the mPFC, including the PL and the more ventral infralimbic cortex (IL) in rodents, and the dACC and ventromedial prefrontal cortex (vmPFC) as the corresponding structures in humans. Due to the underlying memory processes (consolidation, retrieval, reconsolidation) the involved neural circuits change over time (Maren and Holmes, 2016). While the same structures are involved in fear acquisition and fear extinction, different sets of neurons are assumed to act through different molecular mechanisms during fear acquisition and extinction (Maren and Holmes, 2016).
Besides fear acquisition, the amygdala is also involved in the acquisition, consolidation and retrieval of extinction (Quirk and Mueller, 2008). The hippocampus is the relevant site to recall contextual information. This becomes relevant because extinguished responses are often renewed in new contexts, differing from the original extinction context. Moreover, hippocampus and prefrontal cortex have a principal role in the regulation of the retrieval of both, fear acquisition and extinction memories (Fitzgerald et al., 2014). Concretely, the IL (in rodents) and the vmPFC (in humans) are vital structures (Tovote et al., 2015) for extinction recall (or fear suppression, Fitzgerald et al., 2014). The IL mPFC integrates CS-information with contextual information from the hippocampus to determine extinction retrieval. Thus, in the extinction context, the IL/vmPFC inhibits amygdala output to reduce the CR. The role of the IL specifically for extinction retrieval is under recent debate. A recent mice study by Do-Monte et al. (2015) showed that optogenetic silencing of glutamatergic IL neurons during cued extinction retrieval (1 day or 1 week after extinction acquisition) did not abolish retrieval. However, silencing IL neurons already during extinction acquisition impaired extinction retrieval on the following day. This supports the conclusion that—while relevant for the formation of extinction memory—the IL is not necessary for the retrieval of cued fear extinction. The one necessary structure for retrieval of cued extinction appears to be the amygdala. Moreover, these results are one example for recent data challenging the “canonical view” that dorsal regions (PL/dACC) of mPFC regulate fear expression and ventral regions (IL/vmPFC) fear suppression (for current reviews, see Likhtik and Paz, 2015; Giustino and Maren, 2015).
To conclude, extinction is assumed to involve functional changes in the network of amygdala, mPFC and hippocampus so that extinction networks inhibit fear networks (for an elegant review, see Tovote et al., 2015). For extinction to occur it is critical whether the CS activates the fear neurons or the extinction neurons in the BLA, whether ITC are activated, and whether there is an activation of specific subsets of neurons within the mPFC. Subsequently, when reporting effects of stress and different stress-response mediators on extinction we will consider data on direct effects in these brain structures where available.
The Role of Stress and Stress-Response Mediators in Fear Acquisition and Extinction
Relevance
Acquisition of anxiety disorders and especially of PTSD can be conceptualized as learning under severe stress. Enhanced acquisition of trauma-relevant fear is assumed to precede the development of PTSD (Bowers and Ressler, 2015). Accordingly, stress-enhanced classical fear conditioning is used as an animal model of PTSD (e.g., Rau et al., 2005). Importantly, stress and fear responses share common neural circuits, and the neuronal structures involved in fear acquisition and extinction are also highly sensitive to stress effects (e.g., Lupien et al., 2009).
Studying the effects of stress or stress-response mediators on fear acquisition and extinction thus constitutes a relevant paradigm to examine conditions that enhance or attenuate conditioned fear and fear extinction. This approach also aims at mimicking: (a) the stressful nature of the situation prevailing during the acquisition of the disorder and (b) the stressful nature of being re-exposed to the CS afterwards, e.g., during exposure therapy. Exposing subjects explicitly to additional stress or stress-response mediators within a fear-conditioning paradigm seems to be especially important in studies with humans, since the stimuli used as US are often considerably less stressful than US used in animal experiments. Despite the potential relevance, human studies on the effects of acute stress on fear extinction are still rare (for a review, see Raio and Phelps, 2015).
Features and Mediators of the Stress Response
Stress is a process that involves multiple changes interacting in a time-dependent manner: with respect to their onset, these changes can be subdivided into a rapid first-wave and a (delayed) second-wave stress response (Sapolsky et al., 2000; Rodrigues et al., 2009). The neurotransmitters and brain areas involved in the stress response are at least partly identical with those involved in the fear circuitry.
The first-wave stress response (Figure 2) occurs within seconds and involves enhanced release of (a) monoamine neurotransmitters, i.e., of NA, DA, and 5-HT (Joëls and Baram, 2009), (b) corticotropin-releasing hormone (CRH) from the hypothalamus, followed by increased release of adrenocorticotropic hormone (ACTH) from the pituitary. Moreover, (c) there is a reduction in the release of gonadotropin-releasing hormone from the hypothalamus, and subsequently, a decrease in the secretion of gonadotropins, i.e., luteinizing hormone and follicle-stimulating hormone from the pituitary. Additional changes within the 1-min time frame (Sapolsky et al., 2000) involve the secretion of endogenous opiates, prolactin, glucagon, and growth hormone, as well as arginine-vasopressin and renin. In the periphery, the first-wave stress response includes a rapid increase in sympathetic tone and secretion of adrenaline and NA from the adrenal medulla (Sapolsky et al., 2000).
The second-wave stress response, starting after several minutes, involves (among others) increased peripheral secretion of GCs as a result of the activation of the HPA-axis, and reduced secretion of gonadal steroids (i.e., estrogens and gestagens in the ovaries, and testosterone in the testes; Sapolsky et al., 2000; Joëls and Baram, 2009). This inverse action on the secretion of peripheral GCs and gonadal steroids, and the interaction between HPA and hypothalamus—pituitary—gonadal axis under stress is of interest for the present review.
First- and second-wave changes not only have distinct temporal onsets but also differ in the onset and duration of their effects. In the brain, increased NA, DA, 5-HT, and CRH (acting on CRH-receptor 1) usually act within seconds but their effects subside quickly and rarely outlast the duration of the stressor; GC concentrations in the brain only reach peak levels about 20 min after stressor onset (Joëls and Baram, 2009; Hermans et al., 2014). Genomic actions of GCs (and other steroids) take even longer to manifest (Joëls and Baram, 2009). In fact, the slower genomic GC actions were proposed to actively reverse and normalize the rapid effects of various first-wave stress mediators (Hermans et al., 2014).
The brain also receives feedback from stress-induced changes in the periphery: peripheral catecholamines which cannot cross the blood-brain barrier, activate noradrenergic cells via binding to adrenergic receptors of the vagus nerve. The vagus nerve projects to the nucleus of the solitary tract. From there, projections reach the LC (Miyashita and Williams, 2002; Hassert et al., 2004). The amplified LC-activity increases NA levels throughout the brain, including the BLA (Rudy, 2014). Here, NA binds to adrenergic G-protein-coupled receptors and initiates the cAMP signal cascade (Rudy, 2014). Moreover, heightened central NA levels are related to an improvement in memory storage (Rudy, 2014). GCs readily cross the blood-brain barrier and exert negative feedback at hypothalamic and pituitary sites to reduce CRH and ACTH secretion (de Kloet et al., 2005). GCs also bind to higher-affinity mineralocorticoid receptors (MRs) and lower-affinity glucocorticoid receptors (GRs) throughout the brain, including the amygdala, hippocampus, mPFC, and septum (Joëls and Baram, 2009), where they can influence neural signaling and synaptic plasticity. Thus, they can also influence memory processes (Schwabe et al., 2012), such as those involved in fear extinction.
Effects of Stress on Learning—Timing is Relevant
Overview
Acknowledging the fact that fear extinction covers encoding, consolidation and retrieval, models that studied the impact of stress on memory in other areas—such as declarative memory in humans and avoidance learning in rodents—should also provide predictions for fear extinction: these models reveal that the timing of the stressor relative to the learning task is important. Moreover, acknowledging the complexity of the multiple stress-response mediators over time, we will differentiate between mediators of the first wave and those of the second wave of the stress response.
The Role of Timing
Models derived from studies with inhibitory avoidance learning and object recognition (in animals) and declarative memory (in humans) suggest that stress effects on memory depend on the temporal proximity between learning and stressor: encoding close to the stressor (during the first wave of the stress response) will be enhanced, but encoding and recall later in time (during the second wave) will be suppressed (e.g., Schwabe et al., 2012).
In detail. Shortly after stress synergistic actions of rapid NA and non-genomic GC (as part of the first-wave stress response) in the BLA promote the encoding of emotionally relevant information by enhancing synaptic excitability and long term potentiation (Joëls et al., 2011), involving mainly glutamatergic actions. Slower genomic GC effects (as part of the second-wave stress response) will then enhance the consolidation of the material learned under stress. If however, the stressor is placed long before learning (i.e., stressor and learning are “out of sync”), stress should suppress the encoding of new material and impair the retrieval of previously learned material via second-wave stress mediators (such as genomic GCs).
There is evidence for the validity of these predictions from animal (Roozendaal et al., 2006b; Liebmann et al., 2009; Pu et al., 2009; Karst et al., 2010) and human studies (Henckens et al., 2010; Zoladz et al., 2011).
In animal pharmacological fear conditioning studies, reviewed by Rodrigues et al. (2009), evidence suggests that the first-wave stress mediator NA plays an important role in enhancing fear acquisition, whereas GCs as second-wave stress mediators enhance the consolidation, but have no effect on the acquisition of fear. The relevance of this model for fear conditioning in humans has to be tested.
Sex, Sex Hormones and Fear Extinction Under Stress
Sex, Sex Hormones and Fear Extinction
Compared to men, the prevalence of anxiety (Kessler et al., 2005; Somers et al., 2006; Eaton et al., 2012) and trauma- and stressor-related disorders (Kessler et al., 1995; Tolin and Foa, 2006; Kilpatrick et al., 2013; Zoladz and Diamond, 2013) is up to two times higher in women.
Levels of circulating gonadal hormones (mainly estrogens) are regarded to contribute to these sex differences in psychopathology (Lebron-Milad and Milad, 2012; Cover et al., 2014).
Estrogens are a class of steroidal sex hormones that include estrone (E1), estradiol (E2), estriol (E3), and estetrol (E4). E2 is produced by the ovaries and (to a smaller amount) by the adrenal cortex, and by the testes in men by conversion from testosterone via the enzyme aromatase or by conversion of androstenedione to E1 and conversion of E1 to E2. E1 is the dominant estrogen during menopause and E3 and E4 dominate during pregnancy. E2 is the predominating estrogen in non-pregnant women in their reproductive years when E2-levels are considerably higher in women than in men. In humans, an idealized menstrual cycle lasts 28 days and includes the following phases of different concentrations of E2 and P4 (Becker et al., 2005): E2 and P4 are low during the early follicular phase (approx. cycle days 1–8). During the late follicular phase, P4 remains low, while E2 rises to reach its peak immediately before ovulation (midcycle, approx. days 13–14). Peak P4 levels are only reached during the mid-luteal phase, where there is also a second less prominent peak in E2. In rodents, the estrous cycle lasts 4–5 days and covers four phases: metestrus (low E2 and low P4), diestrus (moderate P4 and low E2), proestrus (high E2 and high P4), and estrus (low E2 and low P4; Maeng and Milad, 2015).
E2 exerts its effects via two types of steroidal estrogen receptors located in the cell nucleus or the cytoplasm: estrogen receptor alpha (ERα) and beta (ERβ) which are both widely distributed across the brain (Gillies and McArthur, 2010). ERα are broadly distributed across cortical and subcortical structures including hippocampus, amygdala, hypothalamus, and brain stem, whereas ERβ distribution is less wide-spread with medium to high ERβ density throughout the cortex including the vmPFC, high density in the hippocampus, and in some hypothalamic nuclei (Gillies and McArthur, 2010; Cover et al., 2014). Importantly, ERs are well expressed in brain regions critical for fear acquisition and extinction including amygdala subnuclei, hippocampus and vmPFC (Shughrue et al., 1997; Österlund et al., 1998, 2000; Zhang et al., 2002; Weiser et al., 2008).
Neuroimaging studies reveal that circulating levels of E2 in women affect the reactivity of brain structures involved in both, stress and fear acquisition and extinction. Specifically, women scanned during the midcycle phase (peak E2, but low P4) exhibited significantly less responses to high arousing, aversive visual stimuli (International Affective Picture System, IAPS; Lang et al., 2008) in the amygdala, ACC, orbitofrontal cortex, mPFC, hippocampus, PAG, and several hypothalamic nuclei compared to both, men and women tested during the early follicular phase (low E2 and low P4; Goldstein et al., 2005, 2010).
Estrogens were shown to be especially important in fear extinction: in an elegant series of experiments in rats, Chang et al. (2009) found faster extinction of contextual fear conditioning in female than male rats, especially when in the high E2-phase of the estrous cycle (proestrus). The authors also observed enhanced extinction after systemic and central E2-administration to ovariectomized animals and traced the effects back to hippocampal ERβ by using selective ERα/ERβ agonists.
There is accumulating evidence for the role of E2 especially for extinction recall in both laboratory animals and healthy humans: low E2-levels were shown to impair and high E2-levels to enhance extinction recall, typically tested 24 h after extinction training (Milad et al., 2006, 2009a, 2010; Zeidan et al., 2011; Graham and Milad, 2013). Studies also include data on low-level estrogen under contraceptives (Graham and Milad, 2013). Moreover, in mice both, high endogenous E2 and activation of ERβ, enhanced glutamatergic transmission and synaptic plasticity in the IL mPFC (Galvin and Ninan, 2014), a structure associated with consolidation and recall of extinction. Interestingly, inhibition of estradiol synthesis by an aromatase inhibitor (fadrozole) in male rats, significantly impaired their extinction recall (Graham and Milad, 2014). Adequate controls (such as delivering a single dose of E2 after previous aromatase inhibitor) gave first evidence that fear extinction in males may also depend on acute effects of estrogen synthesized de novo and acting presumably via non-genomic mechanisms. In contrast to the majority of studies supporting an enhancement of extinction by high E2-levels, there is recent evidence for the opposite effect, i.e., enhanced fear memory and impaired extinction memory under high E2-levels (McDermott et al., 2015). But this study did not assess female rats under natural E2 conditions but studied E2 replacement in female ovariectomized mice (vs. mice after sham surgery) instead. Chronic high-dose, not low-dose, E2 administration in pre-puberty (4 weeks) and in adult age (10 weeks), impaired extinction but only in contextual, not cued, fear conditioning. This suggests an adverse effect of chronic high E2-levels for hippocampus-mediated fear inhibition. Anyway, we have to take into account, that chronic high E2 actions after ovariectomy do not necessarily reflect “normal” E2 actions within the brain and periphery.
Addressing brain correlates during fear extinction and considering different E2-levels in humans revealed that women using oral contraceptives (having suppressed E2-levels) showed higher differential BOLD responses to the CS+ (previously paired with the US) during fear extinction compared to men and women in the luteal phase of the menstrual cycle (high P4, medium-high E2) in the amygdala, ACC, and vmPFC (Merz et al., 2012a). This is in line with impaired extinction when E2-levels are low. The study also included administering cortisol vs. placebo but this did not affect the results (see also “Second-wave mediators” Section). Accordingly, a recent study examining women during the early follicular and luteal phases of the menstrual cycle (Wegerer et al., 2014) reported that low E2 but not P4 was associated with poorer extinction and with higher intrusive memories.
Patients: with PTSD exhibit marked fear extinction deficits as compared to healthy controls (Milad et al., 2009b; Inslicht et al., 2013) or trauma-exposed and non-trauma-exposed healthy controls (Milad et al., 2009b). Interestingly, fear inhibition/extinction deficits have been linked to low levels of E2 in both female PTSD-patients (Glover et al., 2012) as well as in healthy and traumatized women (Glover et al., 2013). A recent study by Shvil et al. (2014), addressing sex differences in trauma-exposed healthy controls vs. PTSD-patients, found impaired extinction recall (higher SCRs to the extinguished CS+) and higher activity in the left rostral dACC in men as compared to women within the PTSD group, but not in trauma-exposed healthy controls. The result also suggests that interventions improving fear extinction are highly important in male patients with PTSD.
In sum: the available data suggest an important role of E2 for fear extinction with high E2-levels enhancing extinction acquisition and especially extinction recall (with most of the studies comparing different E2-levels within females). The assessment of E2-levels should be thus included in fear extinction studies. Comparing women in different cycle phases and under different natural E2-levels is only a quasi-experimental approach. Thus, experimental studies would be important with experimental variation of sex-hormone levels.
Interaction of Stress and Sex Hormones in Fear Extinction
Although trauma- and stressor-related disorders are associated with deficits in fear extinction, we still do not know much about the effects of acute stress on fear acquisition and extinction in healthy humans (Raio and Phelps, 2015). Importantly, animal data suggests that stress effects on fear conditioning are sex specific (Dalla and Shors, 2009). For example, in ovariectomized female rats an injection of E2 (45 μg/kg) was able to alleviate conditioned fear responses after SPS (Mirshekar et al., 2013), suggesting an E2 × stress interaction. In intact females (as compared to males), SPS did not impair extinction learning or recall, regardless of estrous cycle phase (Keller et al., 2015a).
In humans, Zorawski et al. (2005) found that the level of post-acquisition GCs was positively correlated with fear acquisition performance (SCR) in men, but not in women. This was later replicated (Zorawski et al., 2006) with an added stressor after acquisition to achieve higher GC-levels. Again, post-acquisition GCs were only correlated with acquisition in men, but not in women. This suggested a sex-specific relationship between GCs and fear acquisition. Similarly, Merz et al. (2013a) report a positive correlation between basal cortisol levels and amygdala activity during fear acquisition only in men and in women taking oral contraceptives, but not in women in their luteal cycle phase. In a first experimental study (Jackson et al., 2006) reported that psychosocial stress (inducing cortisol increases) enhanced differential SCRs during both acquisition and immediate extinction in men, but had no effect in women. However, subsequent neuroimaging studies report the opposite effect after a psychosocial stressor: reduced SCRs and BOLD responses in men during acquisition and enhanced BOLD responses after stress in women (Merz et al., 2013b). Similar results, with higher conditioned BOLD responses in women, and impaired responses in men were reported from the same group (Stark et al., 2006; Merz et al., 2010) for fear acquisition after administration of cortisol (oral, 30 mg hydrocortisone) vs. placebo.
Cortisol effects on BOLD responses during fear acquisition in women were shown to depend on gonadal hormone availability: women using oral contraceptives showed enhanced conditioned BOLD responses after cortisol, whereas cortisol impaired responding in men and free-cycling women in their early follicular (low E2, low P4) and mid-luteal (low to mid E2, high P4) cycle phases (Merz et al., 2012b). But in another study of that group, comparing men and women in their luteal phase and women taking contraceptives, the additional experimental factor of administering cortisol (vs. placebo) had no effect on extinction acquisition (Merz et al., 2012a). In a recent study of our group (Antov and Stockhorst, 2014), we tested the effect of a psychosocial stressor that preceded fear acquisition. Fear acquisition started in the maximum of the peripheral cortisol peak. We examined men and free-cycling women either in the early follicular phase (low E2, low P4) or in the mid-cycle phase (high E2, low P4) and tested fear acquisition, extinction acquisition (day 1) and extinction recall (day 2). We found an interaction between stress exposure and natural E2-status within women: in mid-cycle women, extinction recall was better when fear acquisition had been preceded by stress, whereas the inverse was true in early follicular women. Thus, extinction recall of conditioned fear acquired after stress depends on estrogen status in women. Consequently, we suggest that for extinction-based exposure therapy in women, cycle phase and/or E2-level should be taken into account. We assume that the mid-cycle phase (with high E2- and low P4-levels) provides an adequate phase to re-expose women to the aversive CSs during exposure therapy. In healthy volunteers, exposure to aversive stimuli during this high E2-cycle phase was also associated with less intrusive memories (Wegerer et al., 2014).
Considering the higher prevalence of anxiety, trauma- and stressor-related disorders in women, and extinction deficits under low as compared to high E2-levels (within women), one might wonder why low levels of E2 are disadvantageous for women, but (often) not for men who also have low levels of circulating E2. Most importantly, one has to keep in mind that testosterone is rapidly converted to estrogen in the brain via aromatase. Thus, men do take advantage of estradiol actions within the brain, although they have low levels of circulating E2. This is supported by data showing that the inhibition of estradiol synthesis by an aromatase inhibitor in male rats significantly impaired extinction (Graham and Milad, 2014). When it comes to the interaction between female sex hormones and stress in fear conditioning studies with humans, there is some initial evidence that there might be a higher resemblance in the response pattern of men and naturally cycling women in a high estrogen status than with women in a low estrogen status (Antov and Stockhorst, 2014), or than with women using contraceptives (Merz et al., 2012b). Here—in addition to the above argument—one might also consider differences in binding capacities of MRs and GRs for corticosterone, or cortisol, respectively. These binding capacities were shown to differ between males and females, and—within females—also between different cycle phases (Ter Horst et al., 2012). But, most importantly, much more studies are needed to assess the interaction between stress and sex, or sex hormones, especially with regard to extinction.
A Role of Male Sex Hormones and of Hormonal Transitions in Fear Extinction
While fear extinction studies measuring female sex hormone levels are already sparse, there are hardly any studies addressing the role of male sex hormones for fear extinction. Moreover, these remaining data are mainly based on studies using castration, not exogenous administration of androgens.
In an early study (Anagnostaras et al., 1998), adult male rats (age 12–17 weeks) were either castrated or sham-operated or remained intact. Groups did not differ in their extinction acquisition of contextual fear, or in hippocampal long-term potentiation. Different results were obtained by McDermott et al. (2012): castration during prepuberty (4 weeks old) as well as thereafter (10-weeks old) resulted in an impairment of extinction in a contextual conditioning paradigm, not in cued conditioning, suggesting that testosterone might act to improve fear extinction in hippocampal-dependent, not in amygdala-mediated fear memory. In a recent fear conditioning study with healthy men (Pace-Schott et al., 2013), testosterone levels were also measured. Interestingly, a higher testosterone/cortisol (T/C) ratio predicted better extinction acquisition, but only in the morning hours (i.e., when endogenous cortisol levels are high). The T/C ratio (not the single hormones) was negatively correlated with the remaining CR (SCRs). Although these data are only correlative, they encourage examining the role of testosterone on fear extinction under stressful conditions. Experimental studies are of interest where testosterone is manipulated under stressful vs. control conditions. In the meantime, it would be helpful to measure testosterone levels in fear conditioning studies.
Another approach to study the role of sex hormones for fear-related memory and extinction memory is to examine developmental phases of larger hormonal changes. Again, data are sparse. There is some evidence (recently reviewed by Baker et al., 2014) for impaired fear extinction in adolescents as compared to both, younger individuals as well as adults. One explanation refers to functional changes in the connectivity between prefrontal cortex and amygdala during this developmental stage. Moreover, adolescence is a stage of high vulnerability to stress (Lupien et al., 2009).
For women, pregnancy and perimenopausal stages are additional phases of larger hormonal changes. For example, there is a heightened risk for depression when E2 production is sharply reduced after menopause (for an overview, see Cover et al., 2014) and there is evidence, that estradiol replacement can improve depressive symptoms in women with a history of perimenopausal depression (e.g., Schmidt et al., 2015). More studies specifically addressing fear extinction during developmental stages and hormonal transitions are necessary. This might also include conditions where estrogen receptors are blocked. This is the case in the pharmacological adjunct therapy in estrogen-receptor positive breast cancer patients receiving the estrogen-receptor blocker tamoxifen.
Conclusion
Our review aimed at elucidating how single mediators of the stress response (hormones, neurotransmitters, and neuropeptides) and the entire natural stress response (covering the sequence of the first and the second wave of the stress response) contribute to fear extinction. Our stress approach is guided by the idea that classical stress-response mediators are also agents within the fear macro- and microcircuits. Thus, explicitly investigating stress or stress-response mediators in fear acquisition and fear extinction might mimic the natural situation of acquiring anxiety, trauma- and stressor-related disorders and of being re-exposed to the fear reminders during extinction-based exposure therapy.
Concerning the mediators of the first-wave stress response (monoamines, opioids, eCBs), animal data strongly suggest that rapidly acting NA enhances extinction learning and extinction memory processes; for humans, data are less conclusive and more human studies on NA effects in extinction are warranted. Dopaminergic as well as serotonergic signaling improves extinction memory in animals and humans, while a number of modifying conditions (dose, temporal spacing, targeted receptor-subtypes, chronic vs. acute administration, gene polymorphisms) have to be considered. An improvement of extinction also occurs under opioid signaling and eCBs, with first evidence also in humans. Endogenous GCs play an important part in successful fear extinction and extinction memory. In animal studies, data converge that GC administration can improve extinction when the learning task is in “sync” with the cortisol peak thus improving consolidation of extinction memory. In line with animal studies, in humans GC administration combined with exposure therapy has shown some promise to yield a better therapy outcome. Similar conclusions can be drawn from diurnal effects in humans. However, the few studies with GCs administration directly examining fear extinction in healthy volunteers cannot back up clinical trials yet.
Considering the natural stress response, animal studies suggest that stress impairs the consolidation/recall of fear extinction. The impairment requires time (>24 h) and is associated with structural changes in extinction-relevant structures including dendritic morphology, and receptor density. While results are mixed in humans, there is some evidence that extinction acquisition during the first-wave stress response (e.g., after the CPT) improves extinction recall. On the other hand, second-wave stress before testing impairs extinction recall.
While the prevalence of anxiety, trauma- and stressor-related disorders is up to two times higher in women than in men, studies addressing the level of E2 reveal better fear extinction, especially fear extinction recall, and higher vmPFC-activity, in cycle phases of a high as compared to low E2-level in women. On the other hand, in PTSD-patients, behavioral fear-extinction deficits as well as reduced vmPFC activity have been linked to low E2-levels. There is first evidence for an interaction between stress exposure and natural E2-status within women: in mid-cycle women, extinction recall was better when fear acquisition had been preceded by stress, whereas the inverse was true in early follicular women. Thus, extinction recall of conditioned fear acquired after stress depends on estrogen status in women. Consequently, we suggest that for extinction-based exposure therapy in women, cycle phase and/or E2-level should be taken into account. In case of low E2-levels, drugs increasing dopamine and serotonin levels might improve the success of extinction, or exposure therapy.
We predict that behavioral and pharmacological interventions using the mediators of the first-wave stress response applied prior to extinction acquisition should enhance extinction memory. For GC-related interventions (being related to the second wave of the stress response), we should consider that the consolidation phase of the conditioning task (i.e., extinction acquisition) is in “sync” with the GC peak in order to take advantage of extinction-improving GC effects whereas high GC levels at retrieval are expected to impair extinction memory retrieval.
Since exposure therapy can be described as a form of extinction learning, the presented data thus might also allow predicting which of the above pharmacological interventions and behavioral types of stress induction could constitute adjuncts to exposure therapy. Concretely, DA- and 5-HT-reuptake inhibitors, endorphins, eCBs, and GCs prior to extinction (in “sync” with the consolidation phase) as well as stressors strongly affecting the first-wave stress response (such as the CPT increasing the noradrenergic tone) could be successful. Moreover, data presented here also encourage taking cycle phase and/or contraceptive use into account when scheduling exposure sessions for women.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgements
We acknowledge support by Deutsche Forschungsgemeinschaft (DFG) and Open Access Publishing Fund of Osnabrück University.