Kelimer Lebron-Milad

Estrous cycle phase and gonadal hormones influence conditioned fear extinction

Gonadal hormones modulate fear acquisition, but less is known about the influence of gonadal hormones on fear extinction. We assessed sex differences and the influence of gonadal hormone fluctuations and exogenous manipulations of estrogen and progesterone on acquisition, extinction learning and extinction recall in a 3 day auditory fear conditioning and extinction protocol. Experiments were conducted on males and naturally cycling female rats. Regarding female rats, significant differences in fear extinction were observed between subgroups of females, depending on their phase of the estrous cycle. Extinction that took place during the proestrus (high estrogen/progesterone) phase was more fully consolidated, as evidenced by low freezing during a recall test. This suggests that estrogen and/or progesterone facilitate extinction. In support of this, injection of both estrogen and progesterone prior to extinction learning in female rats during the metestrus phase of the cycle (low estrogen/progesterone) facilitated extinction consolidation, and blockade of estrogen and progesterone receptors during the proestrus phase impaired extinction consolidation. When comparing male to female rats without consideration of the estrous cycle phase, no significant sex differences were observed. When accounting for cycle phase in females, sex differences were observed only during extinction recall. Female rats that underwent extinction during the metestrus phase showed significantly higher freezing during the recall test relative to males. Collectively, these data suggest that gonadal hormones influence extinction behavior possibly by influencing the function of brain regions involved in the consolidation of fear extinction. Moreover, the elevated fear observed in female relative to male rats during extinction recall suggests that gonadal hormones may in part play a role in the higher prevalence of anxiety disorders in women.

Altered Processing of Contextual Information during Fear Extinction in PTSD: An fMRI Study

Medial prefrontal cortical areas have been hypothesized to underlie altered contextual processing in posttraumatic stress disorder (PTSD). We investigated brain signaling of contextual information in this disorder. Eighteen PTSD subjects and 16 healthy trauma‐exposed subjects underwent a two‐day fear conditioning and extinction paradigm. On day 1, within visual context A, a conditioned stimulus (CS) was followed 60% of the time by an electric shock (conditioning). The conditioned response was then extinguished (extinction learning) in context B. On day 2, recall of the extinction memory was tested in context B. Skin conductance response (SCR) and functional magnetic resonance imaging (fMRI) data were collected during context presentations. There were no SCR group differences in any context presentation. Concerning fMRI data, during late conditioning, when context A signaled danger, PTSD subjects showed dorsal anterior cingulate cortical (dACC) hyperactivation. During early extinction, when context B had not yet fully acquired signal value for safety, PTSD subjects still showed dACC hyperactivation. During late extinction, when context B had come to signal safety, they showed ventromedial prefrontal cortex (vmPFC) hypoactivation. During early extinction recall, when context B signaled safety, they showed both vmPFC hypoactivation and dACC hyperactivation. These findings suggest that PTSD subjects show alterations in the processing of contextual information related to danger and safety. This impairment is manifest even prior to a physiologically‐measured, cue‐elicited fear response, and characterized by hypoactivation in vmPFC and hyperactivation in dACC.

Sex differences, gonadal hormones and the fear extinction network: implications for anxiety disorders

Convergent data from rodents and human studies have led to the development of models describing the neural mechanisms of fear extinction. Key components of the now well-characterized fear extinction network include the amygdala, hippocampus, and medial prefrontal cortical regions. These models are fueling novel hypotheses that are currently being tested with much refined experimental tools to examine the interactions within this network. Lagging far behind, however, is the examination of sex differences in this network and how sex hormones influence the functional activity and reactivity of these brain regions in the context of fear inhibition. Indeed, there is a large body of literature suggesting that sex hormones, such as estrogen, do modulate neural plasticity within the fear extinction network, especially in the hippocampus.After a brief overview of the fear extinction network, we summarize what is currently known about sex differences in fear extinction and the influence of gonadal hormones on the fear extinction network. We then go on to propose possible mechanisms by which sex hormones, such as estrogen, may influence neural plasticity within the fear extinction network. We end with a discussion of how knowledge to be gained from developing this line of research may have significant ramifications towards the etiology, epidemiology and treatment of anxiety disorders.

Mechanisms of estradiol in fear circuitry: implications for sex differences in psychopathology.

Over the past two decades, substantial knowledge has been attained about the mechanisms underlying the acquisition and subsequent extinction of conditioned fear. Knowledge gained on the biological basis of Pavlovian conditioning has led to the general acceptance that fear extinction may be a useful model in understanding the underlying mechanisms in the pathophysiology of anxiety disorders and may also be a good model for current therapies treating these disorders. Lacking in the current knowledge is how men and women may or may not differ in the biology of fear and its extinction. It is also unclear how the neural correlates of fear extinction may mediate sex differences in the etiology, maintenance, and prevalence of psychiatric disorders. In this review, we begin by highlighting the epidemiological differences in incidence rate. We then discuss how estradiol (E2), a primary gonadal hormone, may modulate the mechanisms of fear extinction and mediate some of the sex differences observed in psychiatric disorders.

Sex differences in the neurobiology of fear conditioning and extinction: a preliminary fMRI study of shared sex differences with stress-arousal circuitry

BackgroundThe amygdala, hippocampus, medial prefrontal cortex (mPFC) and brain-stem subregions are implicated in fear conditioning and extinction, and are brain regions known to be sexually dimorphic. We used functional magnetic resonance imaging (fMRI) to investigate sex differences in brain activity in these regions during fear conditioning and extinction.MethodsSubjects were 12 healthy men comparable to 12 healthy women who underwent a 2-day experiment in a 3 T MR scanner. Fear conditioning and extinction learning occurred on day 1 and extinction recall occurred on day 2. The conditioned stimuli were visual cues and the unconditioned stimulus was a mild electric shock. Skin conductance responses (SCR) were recorded throughout the experiment as an index of the conditioned response. fMRI data (blood-oxygen-level-dependent [BOLD] signal changes) were analyzed using SPM8.ResultsFindings showed no significant sex differences in SCR during any experimental phases. However, during fear conditioning, there were significantly greater BOLD-signal changes in the right amygdala, right rostral anterior cingulate (rACC) and dorsal anterior cingulate cortex (dACC) in women compared with men. In contrast, men showed significantly greater signal changes in bilateral rACC during extinction recall.ConclusionsThese results indicate sex differences in brain activation within the fear circuitry of healthy subjects despite similar peripheral autonomic responses. Furthermore, we found that regions where sex differences were previously reported in response to stress, also exhibited sex differences during fear conditioning and extinction.

Low estradiol levels: a vulnerability factor for the development of posttraumatic stress disorder.

t a e t m l t r d s c t a P osttraumatic stress disorder (PTSD) affects millions of people who experience trauma in a given year and women are twice as likely to develop PTSD (1). Many preclinical and clinical studies have used fear conditioning and extinction paradigms to investigate the mechanisms underlying PTSD (2). This paradigm is thought to be a good model for PTSD because fear conditioning resembles the occurrence of a traumatic event and fear extinction forms the basis of exposure therapy, which is a commonly used treatment for anxiety. Elucidating the factors that lead to extinction deficits in PTSD may help to develop novel targets for treatment. Over the last decades, much progress has been made in defining the neural and molecular substrates of fear extinction (2). However, most preclinical studies have used male rodents as subjects, and most human studies have collapsed results across sex or not considered potential influences of menstrual cycle. Recent studies indicate that the natural variance of gonadal hormones across the estrous/menstrual cycle regulates fear responses during fear extinction in both female rats and healthy women (3). The aforementioned studies used healthy women to demonstrate the effect of gonadal hormones on fear extinction. In this issue of Biological Psychiatry, Glover et al. (4) took the important next step to ask: do stradiol levels influence fear extinction in women with PTSD? To answer this question, the authors exposed traumatized omen with and without PTSD to a fear conditioning and extincion paradigm. The conditioned stimuli were colored shapes preented on a monitor, the unconditioned stimulus (US) was an air last directed at the larynx, and fear potentiated startle of the eye link muscle contraction after presentation of the startle probe was sed as a measure of conditioned responding. Fear extinction trainng occurred 10 minutes after conditioning during which the conitioned stimuli were presented but no air blast US was delivered. lood samples were collected before startle testing to assess serum evels of estradiol. Women with PTSD were classified into highand ow-estradiol groups based on the median split of estradiol serum evels. The same was also conducted for the trauma-exposed omen without PTSD. Thus, there were a total of four experimental roups. The results showed that all groups acquired fear during onditioning and extinguished fear during extinction training. owever, PTSD women with low estradiol exhibited significantly levated conditioned responses during extinction training relative o all other groups. Moreover, among women with PTSD, those with ow estradiol had greater symptom severity relative to those with igh estradiol. The authors concluded that low levels of estrogen ight be a vulnerability factor for the development of PTSD in omen with trauma histories. This is an important and fundamenal study because it is the first to assess the involvement of estradiol n fear extinction in a clinically anxious population. The study by Glover et al. (4) extends recent findings demontrating a role for estradiol in fear extinction (3). For example, Chang

Test–Retest Reliability during Fear Acquisition and Fear Extinction in Humans

Aims: Classical fear conditioning and extinction has been used to understand the neurobiology of fear learning and its inhibition. The recall of an extinction memory involves the ventromedial prefrontal cortex and the amygdala, and patients with posttraumatic stress disorder (PTSD) have been shown to exhibit deficits in this process. Furthermore, extinction forms the basis of exposure therapies commonly used to treat PTSD patients. It is possible that effective pharmacological and/or psychological treatment regimens could influence the activity of these regions, and thereby increase the ability to retain an extinction memory. However, to test this, a fear conditioning and extinction paradigm must demonstrate within‐subject reproducibility over time. We, therefore, sought to test the within‐subject reliability of a previously used 2‐day, classical fear conditioning and extinction paradigm. Methods: Eighteen healthy participants participated in a 2‐day paradigm on three occasions, each separated by at least 12 weeks. Conditioning and extinction took place on Day 1, and extinction recall and fear renewal were evaluated on Day 2 on each of the three occasions. The conditioned stimulus was a visual cue and the unconditioned stimulus was a mild electric shock to the fingers. Skin conductance was recorded throughout the experiment to measure conditioned responses. Results: We found that conditioning and extinction recall were not significantly different across time and were correlated within subjects. Conclusion: These data illustrate the reliability of this paradigm and its potential usefulness in evaluating the influence of a given treatment on the fear extinction network in longitudinal within‐subject designs.

Estradiol shifts interactions between the infralimbic cortex and central amygdala to enhance fear extinction memory in female rats

There is growing evidence that estradiol (E2) enhances fear extinction memory consolidation. However, it is unclear how E2 influences the nodes of the fear extinction network to enhance extinction memory. This study begins to delineate the neural circuits underlying the influence of E2 on fear extinction acquisition and consolidation in female rats. After fear conditioning (day 1), naturally cycling female rats underwent extinction learning (day 2) in a low‐E2 state, receiving a systemic administration of either E2 or vehicle prior to extinction training. Extinction memory recall was then tested 24 hr later (day 3). We measured immediate early gene c‐fos expression within the extinction network during fear extinction learning and extinction recall. During extinction learning, E2 treatment increased centrolateral amygdala c‐fos activity and reduced lateral amygdala activity relative to vehicle. During extinction recall, E2‐treated rats exhibited reduced c‐fos expression in the centromedial amygdala. There were no group differences in c‐fos expression within the medial prefrontal cortex or dorsal hippocampus. Examining c‐fos ratios with the infralimbic cortex (IL) revealed that, despite the lack of group differences within the IL, E2 treatment induced greater IL activity relative to both prelimbic cortex and central amygdala (CeA) activity during extinction memory recall. Only the relationship between IL and CeA activity positively correlated with extinction retention. In conclusion, E2 appears to modify interactions between the IL and the CeA in females, shifting from stronger amygdalar modulation of fear during extinction learning to stronger IL control during extinction recall.

Acute gonadotropin-releasing hormone agonist treatment enhances extinction memory in male rats

Leuprolide acetate (LEU), also known as Lupron, is commonly used to treat prostate cancer in men. As a gonadotropin-releasing hormone (GnRH) receptor agonist, it initially stimulates the release of gonadal hormones, testosterone (T) and estradiol. This surge eventually suppresses these hormones, preventing the further growth and spread of cancer cells. Individuals receiving this treatment often report anxiety and cognitive changes, but LEUs effects on the neural mechanisms that are involved in anxiety during the trajectory of treatment are not well known. In this study, we examined the acute effects of LEU on fear extinction, hypothesizing that increased T levels following a single administration of LEU will facilitate extinction recall by altering neuronal activity within the fear extinction circuitry. Two groups of naïve adult male rats underwent a 3-day fear conditioning, extinction, and recall experiment. The delayed group (n=15) received a single injection of vehicle or LEU (1.2mg/kg) 3weeks before behavioral testing. The acute group (n=25) received an injection one day after fear conditioning, 30min prior to extinction training. Following recall, the brains for all animals were collected for c-fos immunohistochemistry. Blood samples were also collected and assayed for T levels. Acute administration of LEU increased serum T levels during extinction training and enhanced extinction recall 24h later. This enhanced extinction memory was correlated with increased c-fos activity within the infralimbic cortex and amygdala, which was not observed in the delayed group. These results suggest that the elevation in T induced by acute administration of LEU can influence extinction memory consolidation, perhaps through modification of neuronal activity within the infralimbic cortex and amygdala. This may be an important consideration in clinical applications of LEU and its effects on anxiety and cognition.

Protocol for studying extinction of conditioned fear in naturally cycling female rats.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.