Parvalbumin-Positive Interneurons in the Medial Prefrontal Cortex Regulate Stress-Induced Fear Extinction Impairments in Male and Female Rats.

Stress has profound effects on fear extinction, a form of learning that is essential to behavioral therapies for trauma-related and stressor-related disorders. Recent work reveals that acute footshock stress reduces medial prefrontal cortex (mPFC) activity that is critical for extinction learning. Reductions in mPFC activity may be mediated by parvalbumin (PV)-containing interneurons via feedforward inhibition imposed by amygdala afferents. To test this hypothesis, footshock stress-induced Fos expression was characterized in PV+ and PV- neurons in the prelimbic (PL) and infralimbic (IL) cortices. Footshock stress increased the proportion of PV+ cells expressing Fos in both male and female rats; this effect was more pronounced in IL compared with PL. To determine whether PV+ interneurons in the mPFC mediate stress-induced extinction impairments, we chemogenetically silenced these neurons before an immediate extinction procedure in PV-Cre rats. Clozapine-N-oxide (CNO) did not affect conditioned freezing during the extinction procedure. However, CNO exacerbated extinction retrieval in both male and female rats with relatively high PL expression of designer receptors exclusively activated by designer drugs (DREADD). In contrast, in rats with relatively high IL DREADD expression, CNO produced a modest facilitation of extinction in the earliest retrieval trials, but in male rats only. Conversely, excitation of IL PV interneurons was sufficient to impair delayed extinction in both male and female rats. Finally, chemogenetic inhibition of IL-projecting amygdala neurons reduced the immediate extinction deficit in male, but not female rats. These results reveal that PV interneurons regulate extinction learning under stress in a sex-dependent manner, and this effect is mediated by amygdaloprefrontal projections.SIGNIFICANCE STATEMENT Stress significantly impairs the memory of fear extinction, a type of learning that is central to behavioral therapies for trauma-based and anxiety-based disorders (e.g., post-traumatic stress disorder). Here we show that acute footshock stress recruits parvalbumin (PV) interneurons in the medial prefrontal cortex (mPFC) of male and female rats. Silencing mPFC PV interneurons or mPFC-projecting amygdala neurons during immediate extinction influenced extinction retrieval in a sex-dependent manner. This work highlights the role for PV-containing mPFC interneurons in stress-induced impairments in extinction learning.

Mindfulness training and exercise differentially impact fear extinction neurocircuitry.

BACKGROUND: The ability to extinguish a maladaptive conditioned fear response is crucial for healthy emotional processing and resiliency to aversive experiences. Therefore, enhancing fear extinction learning has immense potential emotional and health benefits. Mindfulness training enhances both fear conditioning and recall of extinguished fear; however, its effects on fear extinction learning are unknown. Here we investigated the impact of mindfulness training on brain mechanisms associated with fear-extinction learning, compared to an exercise-based program. METHODS: We investigated BOLD activations in response to a previously learned fear-inducing cue during an extinction paradigm, before and after an 8-week mindfulness-based stress reduction program (MBSR, n = 49) or exercise-based stress management education program (n = 27). RESULTS: The groups exhibited similar reductions in stress, but the MBSR group was uniquely associated with enhanced activation of salience network nodes and increased hippocampal engagement. CONCLUSIONS: Our results suggest that mindfulness training increases attention to anticipatory aversive stimuli, which in turn facilitates decreased aversive subjective responses and enhanced reappraisal of the memory.

Decoding neural reactivation of threat during fear learning, extinction, and recall in a randomized clinical trial of L-DOPA among women with PTSD.

BACKGROUND: Laboratory paradigms are widely used to study fear learning in posttraumatic stress disorder (PTSD). Recent basic science models demonstrate that, during fear learning, patterns of activity in large neuronal ensembles for the conditioned stimuli (CS) begin to reinstate neural activity patterns for the unconditioned stimuli (US), suggesting a direct way of quantifying fear memory strength for the CS. Here, we translate this concept to human neuroimaging and test the impact of post-learning dopaminergic neurotransmission on fear memory strength during fear acquisition, extinction, and recall among women with PTSD in a re-analysis of previously reported data. METHODS: Participants (N = 79) completed a context-dependent fear acquisition and extinction task on day 1 and extinction recall tests 24 h later. We decoded activity patterns in large-scale functional networks for the US, then applied this decoder to activity patterns toward the CS on day 1 and day 2. RESULTS: US decoder output for the CS+ increased during acquisition and decreased during extinction in networks traditionally implicated in human fear learning. The strength of US neural reactivation also predicted individuals skin conductance responses. Participants randomized to receive L-DOPA (n = 43) following extinction on day 1 demonstrated less US neural reactivation on day 2 relative to the placebo group (n = 28). CONCLUSION: These results support neural reactivation as a measure of memory strength between competing memories of threat and safety and further demonstrate the role of dopaminergic neurotransmission in the consolidation of fear extinction memories.

The effects of diazepam on fear extinction in nulliparous and primiparous female rats.

Benzodiazepines undermine the success of exposure therapy in humans with anxiety disorders, and impair the long-term memory of fear extinction (the laboratory basis of exposure therapy) in rodents. However, most rodent studies on fear extinction and benzodiazepines have been conducted in male rodents. In female rodents, the estrous cycle influences the consolidation of fear extinction memories and sensitivity to benzodiazepines. In addition, pregnancy leads to long-term changes in the neurobiological, hormonal, and behavioural features of fear extinction, as well as the responsivity to benzodiazepines. Therefore, the present experiments examined the impact of benzodiazepines on fear extinction in female rats with and without reproductive experience. Age-matched nulliparous (no reproductive experience) and primiparous (one prior reproductive experience; tested one-month post-weaning) rats received fear conditioning to a discrete cue. The next day, rats were administered the benzodiazepine diazepam (2 mg/kg, s.c), or vehicle, prior to or immediately after extinction training. Rats were then tested the next day, drug free, for extinction retention. Similar to previous findings in males, diazepam impaired extinction retention in both nulliparous and primiparous rats when administered either pre- or post-extinction training. These findings may have potential clinical implications as they suggest that benzodiazepine use in conjunction with exposure therapy may undermine long-term treatment success in women with and without reproductive experience, although this remains to be tested in human populations. Moreover, these findings are theoretically important when considered in light of previous studies showing dissociable mechanisms of fear extinction in females pre- versus post-pregnancy.

Infralimbic YTHDF1 is necessary for the beneficial effects of acute mild exercise on auditory fear extinction retention.

Exposure therapy is the most effective approach of behavioral therapy for anxiety and post-traumatic stress disorder (PTSD). But fear is easy to reappear even after successful extinction. So, identifying novel strategies for augmenting exposure therapy is rather important. It was reported that exercise had beneficial effects on cognitive and memory deficits. However, whether exercise could affect fear memory, especially for fear extinction remained elusive. Here, our results showed that exposure to acute mild exercise 1 or 2 h before extinction training can augment recent fear extinction retention and 2 h for the remote fear extinction retention. These beneficial effects could be attributed to increased YTHDF1 expression in medial prefrontal cortex (mPFC). Furthermore, by using an AAV-shRNA-based approach to silence YTHDF1 expression via stereotactic injection in prelimbic cortex (PL) or infralimbic cortex (IL), respectively, we demonstrated that silence YTHDF1 in IL, but not in PL, blunted augmentation of exposure therapy induced by acute mild exercise and accompanied with decreased NR2B and GluR1 expression. Moreover, YTHDF1 modulated dendritic spines remodeling of pyramidal neuron in IL. Collectively, our findings suggested that acute mild exercise acted as an effective strategy in augmenting exposure therapy with possible implications for understanding new treatment underlying PTSD.

A multi-modal assessment of fear conditioning in adolescent anorexia nervosa.

OBJECTIVE: Anorexia nervosa (AN) is a pernicious psychiatric disorder which is principally characterized by a fear of weight gain. Notwithstanding the centrality of fear in the psychopathology of AN, controlled assessments of negative valence systems are lacking. Herein we assess fear conditioning in adolescent females with AN. METHOD: Adolescent girls (Mage = 14.6 years, ±1.57) with DSM-5 diagnoses of AN (N = 25) and age-matched control girls (Mage = 14.8 years, ±1.46) with no DSM-5 diagnoses (N = 25) completed structured clinical interviews and participated in a classical three-phase Pavlovian fear conditioning paradigm. Participants with comorbid anxiety disorders were excluded. Skin conductance response (SCR) was measured, alongside self-reported fear, valence, and fear expectancy ratings. RESULTS: Both groups demonstrated significant differential acquisition across all four measures. Regarding group comparisons, no differences emerged for self-reported fear, valence, and fear expectancy ratings during acquisition, although for SCR, those with AN demonstrated reduced physiological arousal relative to controls. Both groups demonstrated significant differential extinction for unconditioned stimuli (US) expectancy, self-report fear, and self-report valence. No statistically significant group differences were evident during extinction to the conditioned stimuli (CS)+, on any outcome measure. However, controls reported more positive valence to the CS- than those with AN. CONCLUSIONS: Contrary to our hypotheses, our preliminary assessment did not find support for elevated fear responding among adolescent girls with AN with regards to fear acquisition or extinction. These data suggest that AN in adolescent girls may not be associated with a heightened propensity to acquire fear, but conversely, may suggest that exposure treatments for AN may be helpful, since extinction learning is intact in AN. PUBLIC SIGNIFICANCE: AN is characterized by fear-related symptoms, including food and weight-related fear, and behavioral avoidance, yet controlled studies assessing fear learning are limited. Our preliminary assessment of adolescent AN indicates no abnormalities in fear learning among adolescents with AN. These findings may inform existing mechanistic models of AN psychopathology, and the development of exposure-based treatments for AN.

Targeting Human Glucocorticoid Receptors in Fear Learning: A Multiscale Integrated Approach to Study Functional Connectivity.

Fear extinction is a phenomenon that involves a gradual reduction in conditioned fear responses through repeated exposure to fear-inducing cues. Functional brain connectivity assessments, such as functional magnetic resonance imaging (fMRI), provide valuable insights into how brain regions communicate during these processes. Stress, a ubiquitous aspect of life, influences fear learning and extinction by changing the activity of the amygdala, prefrontal cortex, and hippocampus, leading to enhanced fear responses and/or impaired extinction. Glucocorticoid receptors (GRs) are key to the stress response and show a dual function in fear regulation: while they enhance the consolidation of fear memories, they also facilitate extinction. Accordingly, GR dysregulation is associated with anxiety and mood disorders. Recent advancements in cognitive neuroscience underscore the need for a comprehensive understanding that integrates perspectives from the molecular, cellular, and systems levels. In particular, neuropharmacology provides valuable insights into neurotransmitter and receptor systems, aiding the investigation of mechanisms underlying fear regulation and potential therapeutic targets. A notable player in this context is cortisol, a key stress hormone, which significantly influences both fear memory reconsolidation and extinction processes. Gaining a thorough understanding of these intricate interactions has implications in terms of addressing psychiatric disorders related to stress. This review sheds light on the complex interactions between cognitive processes, emotions, and their neural bases. In this endeavor, our aim is to reshape the comprehension of fear, stress, and their implications for emotional well-being, ultimately aiding in the development of therapeutic interventions.

Reducing FKBP51 Expression in the Ventral Hippocampus Decreases Auditory Fear Conditioning in Male Rats.

Fear conditioning evokes a physiologic release of glucocorticoids that assists learning. As a cochaperone in the glucocorticoid receptor complex, FKBP51 modulates stress-induced glucocorticoid signaling and may influence conditioned fear responses. This study combines molecular and behavioral approaches to examine whether locally reducing FKBP51 expression in the ventral hippocampus is sufficient to affect fear-related behaviors. We hypothesized that reducing FKBP51 expression in the VH would increase glucocorticoid signaling to alter auditory fear conditioning. Adult male rats were injected with an adeno-associated virus (AAV) vector expressing short hairpin - RNAs (shRNA) targeting FKBP5 into the ventral hippocampus to reduce FKBP5 levels or a control AAV. Infusion of FKBP5-shRNA into the ventral hippocampus decreased auditory fear acquisition and recall. Although animals injected with FKBP5-shRNA showed less freezing during extinction recall, the difference was due to a reduced fear recall rather than improved extinction. Reducing ventral hippocampus FKBP51 did not affect exploratory behavior in either the open field test or the elevated zero maze test but did increase passive behavior in the forced swim test, suggesting that the reduction in auditory fear recall was not due to more active responses to acute stress. Furthermore, lower ventral hippocampus FKBP51 levels did not alter corticosterone release in response to restraint stress, suggesting that the reduced fear recall was not due to lower corticosterone release. Our findings suggest FKBP51 in the ventral hippocampus plays a selective role in modulating fear-learning processes and passive behavioral responses to acute stress rather than hypothalamic-pituitary-adrenal axis reactivity or exploratory responses.

Rewarded Extinction Increases Amygdalar Connectivity and Stabilizes Long-Term Memory Traces in the vmPFC.

Neurobiological evidence in rodents indicates that threat extinction incorporates reward neurocircuitry. Consequently, incorporating reward associations with an extinction memory may be an effective strategy to persistently attenuate threat responses. Moreover, while there is considerable research on the short-term effects of extinction strategies in humans, the long-term effects of extinction are rarely considered. In a within-subjects fMRI study with both female and male participants, we compared counterconditioning (CC; a form of rewarded-extinction) to standard extinction at recent (24 h) and remote (approximately one month) retrieval tests. Relative to standard extinction, rewarded extinction diminished 24-h relapse of arousal and threat expectancy, and reduced activity in brain regions associated with the appraisal and expression of threat (e.g., thalamus, insula, periaqueductal gray). The retrieval of reward-associated extinction memory was accompanied by functional connectivity between the amygdala and the ventral striatum, whereas the retrieval of standard-extinction memories was associated with connectivity between the amygdala and ventromedial prefrontal cortex (vmPFC). One month later, the retrieval of both standard-extinction and rewarded-extinction was associated with amygdala-vmPFC connectivity. However, only rewarded extinction created a stable memory trace in the vmPFC, identified through overlapping multivariate patterns of fMRI activity from extinction to 24-h and one-month retrieval. These findings provide new evidence that reward may generate a more stable and enduring memory trace of attenuated threat in humans.SIGNIFICANCE STATEMENT Prevalent treatments for pathologic fear and anxiety are based on the principles of Pavlovian extinction. Unfortunately, extinction forms weak memories that only temporarily inhibit the retrieval of threat associations. Thus, to increase the translational relevance of extinction research, it is critical to investigate whether extinction can be augmented to form a more enduring memory, especially after long intervals. Here, we used a multiday fMRI paradigm in humans to compare the short-term and long-term neurobehavioral effects of aversive-to-appetitive counterconditioning (CC), a form of augmented extinction. Our results provide novel evidence that including an appetitive stimulus during extinction can reduce short-term threat relapse and stabilize the memory trace of extinction in the ventromedial prefrontal cortex (vmPFC), for at least one month after learning.

Prefrontal GABAA(δ)R Promotes Fear Extinction through Enabling the Plastic Regulation of Neuronal Intrinsic Excitability.

Extinguishing the previously acquired fear is critical for the adaptation of an organism to the ever-changing environment, a process requiring the engagement of GABAA receptors (GABAARs). GABAARs consist of tens of structurally, pharmacologically, and functionally heterogeneous subtypes. However, the specific roles of these subtypes in fear extinction remain largely unexplored. Here, we observed that in the medial prefrontal cortex (mPFC), a core region for mood regulation, the extrasynaptically situated, δ-subunit-containing GABAARs [GABAA(δ)Rs], had a permissive role in tuning fear extinction in male mice, an effect sharply contrasting to the established but suppressive role by the whole GABAAR family. First, the fear extinction in individual mice was positively correlated with the level of GABAA(δ)R expression and function in their mPFC. Second, knockdown of GABAA(δ)R in mPFC, specifically in its infralimbic (IL) subregion, sufficed to impair the fear extinction in mice. Third, GABAA(δ)R-deficient mice also showed fear extinction deficits, and re-expressing GABAA(δ)Rs in the IL of these mice rescued the impaired extinction. Further mechanistic studies demonstrated that the permissive effect of GABAA(δ)R was associated with its role in enabling the extinction-evoked plastic regulation of neuronal excitability in IL projection neurons. By contrast, GABAA(δ)R had little influence on the extinction-evoked plasticity of glutamatergic transmission in these cells. Altogether, our findings revealed an unconventional and permissive role of extrasynaptic GABAA receptors in fear extinction through a route relying on nonsynaptic plasticity.SIGNIFICANCE STATEMENT The medial prefrontal cortex (mPFC) is one of the kernel brain regions engaged in fear extinction. Previous studies have repetitively shown that the GABAA receptor (GABAAR) family in this region act to suppress fear extinction. However, the roles of specific GABAAR subtypes in mPFC are largely unknown. We observed that the GABAAR-containing δ-subunit [GABAA(δ)R], a subtype of GABAARs exclusively situated in the extrasynaptic membrane and mediating the tonic neuronal inhibition, works oppositely to the whole GABAAR family and promotes (but does not suppress) fear extinction. More interestingly, in striking contrast to the synaptic GABAARs that suppress fear extinction by breaking the extinction-evoked plasticity of glutamatergic transmission, the GABAA(δ)R promotes fear extinction through enabling the plastic regulation of neuronal excitability in the infralimbic subregion of mPFC. Our findings thus reveal an unconventional role of GABAA(δ)R in promoting fear extinction through a route relying on nonsynaptic plasticity.