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

Persistent prelimbic cortex activity contributes to enhanced learned fear expression in females

Anxiety disorders, such as post-traumatic stress, are more prevalent in women and are characterized by impaired inhibition of learned fear and medial prefrontal cortex (mPFC) dysfunction. Here we examined sex differences in fear extinction and mPFC activity in rats. Females showed more learned fear expression during extinction and its recall, but not fear conditioning. They also showed more spontaneous fear recovery and more contextual fear before extinction and its recall. Moreover, enhanced learned fear expression in females was associated with sustained prelimbic (PL) cortex activity. These results suggest that sex differences in learned fear expression may involve persistent PL activation

Impaired contextual modulation of memories in PTSD: an fMRI and psychophysiological study of extinction retention and fear renewal

Post-traumatic stress disorder (PTSD) patients display pervasive fear memories, expressed indiscriminately. Proposed mechanisms include enhanced fear learning and impaired extinction or extinction recall. Documented extinction recall deficits and failure to use safety signals could result from general failure to use contextual information, a hippocampus-dependent process. This can be probed by adding a renewal phase to standard conditioning and extinction paradigms. Human subjects with PTSD and combat controls were conditioned (skin conductance response), extinguished, and tested for extinction retention and renewal in a scanner (fMRI). Fear conditioning (light paired with shock) occurred in one context, followed by extinction in another, to create danger and safety contexts. The next day, the extinguished conditioned stimulus (CS+E) was re-presented to assess extinction recall (safety context) and fear renewal (danger context). PTSD patients showed impaired extinction recall, with increased skin conductance and heightened amygdala activity to the extinguished CS+ in the safety context. However, they also showed impaired fear renewal; in the danger context, they had less skin conductance response to CS+E and lower activity in amygdala and ventral-medial prefrontal cortex compared with combat controls. Control subjects displayed appropriate contextual modulation of memory recall, with extinction (safety) memory prevailing in the safety context, and fear memory prevailing in the danger context. PTSD patients could not use safety context to sustain suppression of extinguished fear memory, but they also less effectively used danger context to enhance fear. They did not display globally enhanced fear expression, but rather showed a globally diminished capacity to use contextual information to modulate fear expression

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

Intolerance of uncertainty predicts fear extinction in amygdala-ventromedial prefrontal cortical circuitry

Background: Coordination of activity between the amygdala and ventromedial prefrontal cortex (vmPFC) is important for fear-extinction learning. Aberrant recruitment of this circuitry is associated with anxiety disorders. Here, we sought to determine if individual differences in future threat uncertainty sensitivity, a potential risk factor for anxiety disorders, underly compromised recruitment of fear extinction circuitry. Twenty-two healthy subjects completed a cued fear conditioning task with acquisition and extinction phases. During the task, pupil dilation, skin conductance response, and functional magnetic resonance imaging were acquired. We assessed the temporality of fear extinction learning by splitting the extinction phase into early and late extinction. Threat uncertainty sensitivity was measured using self-reported intolerance of uncertainty (IU). Results: During early extinction learning, we found low IU scores to be associated with larger skin conductance responses and right amygdala activity to learned threat vs. safety cues, whereas high IU scores were associated with no skin conductance discrimination and greater activity within the right amygdala to previously learned safety cues. In late extinction learning, low IU scores were associated with successful inhibition of previously learned threat, reflected in comparable skin conductance response and right amgydala activity to learned threat vs. safety cues, whilst high IU scores were associated with continued fear expression to learned threat, indexed by larger skin conductance and amygdala activity to threat vs. safety cues. In addition, high IU scores were associated with greater vmPFC activity to threat vs. safety cues in late extinction. Similar patterns of IU and extinction learning were found for pupil dilation. The results were specific for IU and did not generalize to self-reported trait anxiety. Conclusions: Overall, the neural and psychophysiological patterns observed here suggest high IU individuals to disproportionately generalize threat during times of uncertainty, which subsequently compromises fear extinction learning. More broadly, these findings highlight the potential of intolerance of uncertainty-based mechanisms to help understand pathological fear in anxiety disorders and inform potential treatment targets

Exploring the Role of Infralimbic Cortex Inhibitory Circuits in Context-Dependent Extinction and Renewal of Fear

Exposure therapy is an effective treatment for posttraumatic stress disorder (PTSD). However, many patients experienced relapse of fear after treatment. Pavlovian fear conditioning and extinction are effective models to study the return of fear. During fear conditioning, an auditory conditioned stimulus (CS) is paired with an electric footshock (i.e., the unconditioned stimulus, US); after conditioning, the CS evoked a conditional fear response. Presenting the CS numerous times without the US causes an extinction of fear to the CS; however, the loss of fear to the CS is context-specific. That is, extinguished fear returns or “renews” outside of the extinction context. The hippocampus, the medial prefrontal cortex (mPFC) and the amygdala are thought to be essential for context-dependent extinction retrieval and fear renewal. However, how the mPFC, especially the infralimbic cortex (IL), regulates extinction memory is not clear. To clarify this question, I first used retrograde tracing techniques with immediate early gene expression to examine the activity of the mPFC-projecting neurons in the ventral hippocampus (VH) during extinction retrieval and fear renewal. Secondly, I pharmacologically manipulated GABAᴀ receptors in the IL during either extinction retrieval or fear renewal. Lastly, I examined the activity of the interneurons in IL in extinction memory retrieval and examined their contribution to memory retrieval using cell- and circuit-specific DREADD methods. The results showed that VH projections to the prelimbic cortex (PL) and IL were both engaged by fear renewal. This pattern of results suggested that VH inhibits IL via feed-forward inhibition, a finding that was confirmed by pharmacological manipulation of GABAᴀ receptors in IL. Specifically, GABAᴀ receptor agonists interfered with extinction retrieval in the extinction context, whereas GABAᴀ receptor antagonists reduced fear renewal in a different context. However, GAD-Fos immunohistochemistry did not reveal preferential recruitment of IL interneurons during renewal. Finally, the inactivation of putative IL interneurons or activation IL-> BLA pathway did not alter fear renewal. Together, these results suggested the regulating role of IL inhibitory circuits in the context-dependent memory of extinction. Future studies will be done to understand how subtypes of IL local interneurons and the GABA receptors subtypes modulate memory of extinction

Muscarinic Acetylcholine Receptor M1’s Impact on Fear Extinction Learning

Post-Traumatic Stress Disorder (PTSD) is a mental health disorder that can occur following a traumatic event like combat, assault, or disaster. Individuals with PTSD are unable to extinguish fear memories which can become chronic and disabling. However, it remains unclear why some individuals exposed to a traumatic event develop PTSD while others are resilient. Acetylcholine plays a critical role in fear learning, but its role in fear extinction is less well understood. In this investigation, we used a rat model of fear extinction to determine if individual differences in extinction learning are correlated with markers of cholinergic signaling. Cholinergic markers included the M1 muscarinic acetylcholine receptor (M1 m-AChR) and the vesicular acetylcholine transporter (vAChT). These cholinergic markers are strongly expressed in brain regions, such as the amygdala and prefrontal cortex that contribute to the fear extinction circuit. The goal of the present study was to determine whether individual differences in cholinergic signaling in these brain regions could underlie differences in fear extinction. Expression levels of cholinergic markers were measured in amygdala and prefrontal cortex from male Long-Evans rats (N = 13) that had undergone a Pavlovian fear conditioning and extinction paradigm. We found that rats exhibited individual differences in extinction of freezing behavior following twenty presentations of a conditioned auditory stimulus. Six of 13 rats tested failed to extinguish cue-conditioned freezing behavior as defined by a median split in freezing during the last 10 tone presentations. When M1 m-AChR expression in these animals was assessed by Western blot analysis, a significant correlation was evident between expression level of M1 m-AChR in the amygdala and the freezing behavior during the extinction trials. Expression of M1 m-AChRs in amygdala of animals showing good extinction learning was significantly higher than that in animals resistant to extinction. In contrast, there was no significant correlation between vAChT expression and freezing in either amygdala or prefrontal cortex. These results suggest that low expression of M1 m-AChRs in the amygdala is correlated with deficits in fear extinction, and suggest that therapeutic strategies aimed at enhancing muscarinic signaling in amygdala may enhance fear extinction in animals and perhaps patients with PTSD

Immediate Early Gene Expression in Medial Prefrontal Cortex and Hippocampus as a Function of Aging

Normal aging is accompanied by cognitive decline that differs from other aging-related pathological states like Alzheimer\u27s disease. With an increasing proportion of the world population falling in an age group of 65 years and above, a preventive gerontological approach would improve the quality of life in the elderly. Especially important in this regard is the early detection of cognitive decline, so that appropriate measures can be taken to prevent development of cognitive deficits. Impairment in cognitive flexibility, the ability to modify a previously learnt behavior, is one such measure of impairment across species in aged animals. Previous work from our lab has demonstrated that a cognitive flexibility deficit, as measured by extinction of conditioned fear, first emerges in middle-aged animals. Extinction of conditioned fear requires coordinated activity of infralimbic (IL) and prelimbic (PL) subregions of prefrontal cortex, dorsal and ventral hippocampus, and various amygdala sub nuclei. Of these, prefrontal cortex- and hippocampus-dependent behaviors are impaired during aging, indicating that aging-related impairments within these structures could underlie extinction deficits during aging. One way to measure region-specific neuronal activation is through analysis of immediate early gene (IEG) expression. IEG expression at rest is not random but reflects ongoing memory consolidation. The role of IEGs as markers of neuronal plasticity and their critical role in memory consolidation make them ideal markers for investigating early cognitive decline. The current study investigated aging-related changes in the expression of IEGs, Zif-268 and Arc in the IL and PL subregions of the mPFC in addition to dorsal and ventral hippocampus. Specifically, the current study used western blotting and immunohistochemistry to investigate region-specific expression of the IEGs Zif-268 and Arc in naive adult, middle-aged and aged animals. We found that Zif-268 expression was reduced in IL, PL, CA1 and DG of dorsal hippocampus and DG of ventral hippocampus starting middle-age. In addition, Arc expression was reduced in IL but not PL in aged rodents. Within hippocampus, Arc expression was reduced within dorsal but not ventral subregion starting middle age. These data indicate that IEG expression changes are region-specific, can be evident starting middle age and may contribute to behavioral deficits during the aging process

Explicit and Implicit Processes in Human Aversive Conditioning

The ability to adapt to a changing environment is central to an organism’s success. The process of associating two stimuli (as in associative conditioning) requires very little in the way of neural machinery. In fact, organisms with only a few hundred neurons show conditioning that is specific to an associated cue. This type of learning is commonly referred to as implicit learning. The learning can be performed in the absence of the subject’s ability to describe it. One example of learning that is thought to be implicit is delay conditioning. Delay conditioning consists of a single cue (a tone, for example) that starts before, and then overlaps with, an outcome (like a pain stimulus). In addition to associating sensory cues, humans routinely link abstract concepts with an outcome. This more complex learning is often described as explicit since subjects are able to describe the link between the stimulus and outcome. An example of conditioning that requires this type of knowledge is trace conditioning. Trace conditioning includes a separation of a few seconds between the cue and outcome. Explicit learning is often proposed to involve a separate system, but the degree of separation between implicit associations and explicit learning is still debated. We describe aversive conditioning experiments in human subjects used to study the degree of interaction that takes place between explicit and implicit systems. We do this in three ways. First, if a higher order task (in this case a working memory task) is performed during conditioning, it reduces not only explicit learning but also implicit learning. Second, we describe the area of the brain involved in explicit learning during conditioning and confirm that it is active during both trace and delay conditioning. Third, using functional magnetic resonance imaging (fMRI), we describe hemodynamic activity changes in perceptual areas of the brain that occur during delay conditioning and persist after the learned association has faded. From these studies, we conclude that there is a strong interaction between explicit and implicit learning systems, with one often directly changing the function of the other.</p

Acute and long-lasting effects of oxytocin in cortico-limbic circuits: consequences for fear recall and extinction.

The extinction of conditioned fear responses entrains the formation of safe new memories to decrease those behavioral responses. The knowledge in neuronal mechanisms of extinction is fundamental in the treatment of anxiety and fear disorders. Interestingly, the use of pharmacological compounds that reduce anxiety and fear has been shown as a potent co-adjuvant in extinction therapy. However, the efficiency and mechanisms by which pharmacological compounds promote extinction of fear memories remains still largely unknown and would benefit from a validation based on functional neuronal circuits, and the neurotransmitters that modulate them. From this perspective, oxytocin receptor signaling, which has been shown in cortical and limbic areas to modulate numerous functions (Eliava et al. Neuron 89(6):1291-1304, 2016), among them fear and anxiety circuits, and to enhance the salience of social stimuli (Stoop Neuron 76(1):142-59, 2012), may offer an interesting perspective. Experiments in animals and humans suggest that oxytocin could be a promising pharmacological agent at adjusting memory consolidation to boost fear extinction. Additionally, it is possible that long-term changes in endogenous oxytocin signaling can also play a role in reducing expression of fear at different brain targets. In this review, we summarize the effects reported for oxytocin in cortico-limbic circuits and on fear behavior that are of relevance for the modulation and potential extinction of fear memories