Lisa Y. Maeng

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.

The Prefrontal Cortex Communicates with the Amygdala to Impair Learning after Acute Stress in Females but Not in Males

Acute stress exposure enhances classical eyeblink conditioning in male rats, whereas exposure to the same event dramatically impairs performance in females (Wood and Shors, 1998; Wood et al., 2001). We hypothesized that stress affects learning differently in males and females because different brain regions and circuits are being activated. In the first experiment, we determined that neuronal activity within the medial prefrontal cortex (mPFC) during the stressful event is necessary to disrupt learning in females. In both males and females, the mPFC was bilaterally inactivated with GABA agonist muscimol before the stressor. Inactivation prevented only the impaired performance in females; it had no consequence for performance in males. However, in the second experiment, excitation of the mPFC alone with GABA antagonist picrotoxin was insufficient to elicit the stress effect that was prevented through the inactivation of this region in females. Therefore, we hypothesized that the mPFC communicates with the basolateral amygdala to disrupt learning in females after the stressor. To test this hypothesis, these structures were disconnected from each other with unilateral excitotoxic (NMDA) lesions on either the same or opposite sides of the brain. Females with contralateral lesions, which disrupt the connections on both sides of the brain, were able to learn after the stressful event, whereas those with ipsilateral lesions, which disrupt only one connection, did not learn after the stressor. Together, these data indicate that the mPFC is critically involved in females during stress to impair subsequent learning and does so via communication with the amygdala.

Physical Skill Training Increases the Number of Surviving New Cells in the Adult Hippocampus

The dentate gyrus is a major site of plasticity in the adult brain, giving rise to thousands of new neurons every day, through the process of adult neurogenesis. Although the majority of these cells die within two weeks of their birth, they can be rescued from death by various forms of learning. Successful acquisition of select types of associative and spatial memories increases the number of these cells that survive. Here, we investigated the possibility that an entirely different form of learning, physical skill learning, could rescue new hippocampal cells from death. To test this possibility, rats were trained with a physically-demanding and technically-difficult version of a rotarod procedure. Acquisition of the physical skill greatly increased the number of new hippocampal cells that survived. The number of surviving cells positively correlated with performance on the task. Only animals that successfully mastered the task retained the cells that would have otherwise died. Animals that failed to learn, and those that did not learn well did not retain any more cells than those that were untrained. Importantly, acute voluntary exercise in activity wheels did not increase the number of surviving cells. These data suggest that acquisition of a physical skill can increase the number of surviving hippocampal cells. Moreover, learning an easier version of the task did not increase cell survival. These results are consistent with previous reports revealing that learning only rescues new neurons from death when acquisition is sufficiently difficult to achieve. Finally, complete hippocampal lesions did not disrupt acquisition of this physical skill. Therefore, physical skill training that does not depend on the hippocampus can effectively increase the number of surviving cells in the adult hippocampus, the vast majority of which become mature neurons.

Once a Mother, Always a Mother: Maternal Experience Protects Females From the Negative Effects of Stress on Learning

Women experience profound hormonal fluctuations throughout their reproductive lives. They are especially susceptible to disturbances in mood and cognition during the transition from pregnancy into postpartum and motherhood (Brummelte & Galea, 2010). Their behavioral and hormonal responses to stressful stimuli are also altered during this time. These changes are not limited to humans but occur in many mammalian species. Virgin female rats express a severe learning deficit in associative eyeblink conditioning after a stressful life event (Wood, Beylin, & Shors, 2001; Wood & Shors, 1998), but lactating females or those that are caring for young learn well even after the stressor (Leuner & Shors, 2006). However, we do not know whether maternal experience persistently alters learning after a stressful event. Here we hypothesized that females that had been maternal at some time in their lives would learn well even after exposure to a stressful event. To test this hypothesis, females that had at least one brood of young and expressed a normal estrous cycle were exposed to an acute stressful event that reliably impairs learning in virgin females. Animals were trained 24 hr later with classical eyeblink conditioning. Exposure to the stressor suppressed learning in virgins but not in females that had been mothers at some time in their lives. These data suggest that maternal experience induces a protective mechanism in mothers, which promotes associative learning long after the offspring have left their care.

The stressed female brain: neuronal activity in the prelimbic but not infralimbic region of the medial prefrontal cortex suppresses learning after acute stress

Women are nearly twice as likely as men to suffer from anxiety and post-traumatic stress disorder (PTSD), indicating that many females are especially vulnerable to stressful life experience. A profound sex difference in the response to stress is also observed in laboratory animals. Acute exposure to an uncontrollable stressful event disrupts associative learning during classical eyeblink conditioning in female rats but enhances this same type of learning process in males. These sex differences in response to stress are dependent on neuronal activity in similar but also different brain regions. Neuronal activity in the basolateral nucleus of the amygdala (BLA) is necessary in both males and females. However, neuronal activity in the medial prefrontal cortex (mPFC) during the stressor is necessary to modify learning in females but not in males. The mPFC is often divided into its prelimbic (PL) and infralimbic (IL) subregions, which differ both in structure and function. Through its connections to the BLA, we hypothesized that neuronal activity within the PL, but not IL, during the stressor is necessary to suppress learning in females. To test this hypothesis, either the PL or IL of adult female rats was bilaterally inactivated with GABAA agonist muscimol during acute inescapable swim stress. About 24 h later, all subjects were trained with classical eyeblink conditioning. Though stressed, females without neuronal activity in the PL learned well. In contrast, females with IL inactivation during the stressor did not learn well, behaving similarly to stressed vehicle-treated females. These data suggest that exposure to a stressful event critically engages the PL, but not IL, to disrupt associative learning in females. Together with previous studies, these data indicate that the PL communicates with the BLA to suppress learning after a stressful experience in females. This circuit may be similarly engaged in women who become cognitively impaired after stressful life events.

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.

Post-Traumatic Stress Disorder: The Relationship Between the Fear Response and Chronic Stress:

Post-traumatic stress disorder (PTSD) is a disabling psychiatric condition that can develop following a physical, psychological, or sexual trauma. Despite the growing body of literature examining the psychological and biological factors involved in PTSD psychopathology, specific biomarkers that may improve diagnosis and treatment of PTSD have yet to be identified and validated. This challenge may be attributed to the diverse array of symptoms that individuals with the disorder manifest. Examining the interrelated stress and fear systems allows for a more comprehensive study of these symptoms, and through this approach, which aligns with the research domain criteria (RDoC) framework, neural and psychophysiological measures of PTSD have emerged. In this review, we discuss PTSD neurobiology and treatment within the context of fear and stress network interactions and elucidate the advantages of using an RDoC approach to better understand PTSD with fear conditioning and extinction paradigms.

Sex Differences in Anxiety Disorders: Gonadal Hormone Interactions with Pathophysiology, Neurobiology, and Treatment

Studies have documented sex differences in psychiatric illness from diagnosis to treatment. In particular, there is a profound disparity in the prevalence of stress- and fear-based disorders, such as anxiety and posttraumatic stress disorder, in which women are more vulnerable. Fear processes have been extensively studied, and their relevance to anxiety and stress-related disorders is being increasingly examined. The mechanisms for fear learning and its subsequent extinction may provide a framework for understanding how gonadal hormones might contribute to the disproportionate incidence of these psychopathologies. Here, we discuss the influence of gonadal hormones in fear regulation processes and their contributions to the sex differences observed in the pathophysiology, neurobiology, and treatment of anxiety disorders. A better understanding of these interactions may contribute to the development of more effective, personalized treatments that take hormonal status into account.