The effects of a dual orexin receptor antagonist on fear extinction memory and sleep in mice: Implications for exposure therapy.

Extinction of conditioned fear is considered a fundamental process in the recovery from posttraumatic stress disorder and anxiety disorders. Sleep, especially rapid-eye-movement (REM) sleep, has been implicated in promoting extinction memory. The orexin system contributes to the regulation of sleep and wakefulness and emotional behaviors. In rodents, administrations of an orexin receptor antagonist following fear extinction training enhanced consolidation of extinction memory. Although orexin antagonists increase sleep, including REM sleep, the possible contribution of sleep to the effects of orexin antagonists on extinction memory has not been examined. Therefore, this study examined the effects of suvorexant, a dual orexin receptor antagonist, on extinction memory and sleep and their associations in mice. C57BL/6 mice underwent sleep recording for 24 h before and after contextual fear conditioning with footshocks and extinction learning during the early light phase or early dark phase. Mice were systemically injected with either 25 mg/kg of suvorexant or vehicle immediately after the extinction session. We found that suvorexant neither altered sleep nor improved extinction memory recall compared with vehicle. The higher percentages of REM sleep during the post-extinction dark phase were associated with lower extinction memory recall and greater freezing responses to the fear context. Results also indicate that animals did not reach complete extinction of fear with the fear extinction training protocol used in this study. These findings suggest that promoting REM sleep may not enhance fear extinction memory when extinction of fear is incomplete.

No influence of threat uncertainty on fear generalization.

Fear overgeneralization and perceived uncertainty about future outcomes have been suggested as risk factors for clinical anxiety. However, little is known regarding how they influence each other. In this study, we investigated whether different levels of threat uncertainty influence fear generalization. Three groups of healthy participants underwent a differential fear conditioning protocol followed by a generalization test. All groups learned to associate one female face (conditioned stimulus, CS+) with a female scream (unconditioned stimulus, US), whereas the other face (CS-) was not associated with the scream. In order to manipulate threat uncertainty, one group (low uncertainty, n = 26) received 80%, the second group (moderate uncertainty, n = 32) received 60%, and the third group (high uncertainty, n = 30) 40% CS-US contingency. In the generalization test, all groups saw CS+ and CS- again along with four morphs resembling the CSs in steps of 20%. Subjective (expectancy, valence, and arousal ratings), psychophysiological (skin conductance response, SCR), and visuocortical (steady-state visual evoked potentials, ssVEPs) indices of fear were registered. Participants expected the US according to their reinforcement schedules and the discriminative responses to CS+/CS- increased with more uncertainty in skin conductance. However, acquisition of conditioned fear was not evident in ssVEPs. During the generalization test, we found no effect of threat uncertainty in any of the measured variables, but the strength of generalization for threat expectancy ratings was positively correlated with dispositional intolerance of uncertainty. This study suggests that mere threat uncertainty does not modulate fear generalization.

The influence of instructions on reversing the generalization of valence, US expectancy, and electrodermal responding in fear conditioning.

Pairing a conditional stimulus (CS) with an aversive unconditional stimulus (US) causes negative valence and US expectancy to generalize to stimuli that are perceptually and/or conceptually similar to the CS. Past research has shown that instructing participants that the US is more likely to follow stimuli that are dissimilar to the CS reversed the generalization of US expectancy but left generalized valence unchanged. Here, we examined whether instructions about the relationship between stimuli that are perceptually similar would affect the generalization of valence. A picture of an alien (CS+) was paired with an electric stimulus, while a perceptually different alien stimulus (CS-) was presented alone. After conditioning, valence, US expectancy, and electrodermal responses generalized to different aliens that were perceptually similar (by color and shape) to the CS+ and CS-. Participants were then instructed that aliens perceptually similar to the CS+ belonged to the same group as the CS- and that aliens perceptually similar to the CS- belonged to the same group as the CS+. The instructions caused an elimination (but not a reversal) of generalized expectancy and valence but did not affect generalized electrodermal responses. This suggests that evaluations of generalization stimuli are sensitive to instructions about their relationship to the CS and that dissociations reported in the literature between valence and expectancy after instructions may occur due to the type of instruction used.

Sex divergent behavioral responses in platform-mediated avoidance and glucocorticoid receptor blockade.

Women are more likely than men to develop anxiety or stress-related disorders. A core behavioral symptom of all anxiety disorders is avoidance of fear or anxiety eliciting cues. Recent rodent models of avoidance show reliable reproduction of this behavioral phenomenon in response to learned aversive associations. Here, a modified version of platform-mediated avoidance that lacked an appetitive task was utilized to investigate the learning and extinction of avoidance in male and female C57BL6/J mice. Here, we found a robust sex difference in the acquisition and extinction of platform-mediated avoidance. Across three experiments, 63.7% of female mice acquired avoidance according to our criterion, whereas 83.8% of males acquired it successfully. Of those females that acquired avoidance, they displayed persistent avoidance after extinction compared to males. Given their role in regulating stress responses and habitual behaviors, we investigated if glucocorticoid receptors (GR) mediated avoidance learning in males and females. We found that a subcutaneous injection (25 mg/kg) of the GR antagonist, RU486 (Mifepristone), significantly reduced persistent avoidance in females but did not further reduce avoidance in males after extinction. These data suggest that GR activation during avoidance learning may contribute to persistent avoidance in females that is resistant to extinction.

Rats respond to aversive emotional arousal of human handlers with the activation of the basolateral and central amygdala.

Reading danger signals may save an animal's life, and learning about threats from others allows avoiding first-hand aversive and often fatal experiences. Fear expressed by other individuals, including those belonging to other species, may indicate the presence of a threat in the environment and is an important social cue. Humans and other animals respond to conspecifics' fear with increased activity of the amygdala, the brain structure crucial for detecting threats and mounting an appropriate response to them. It is unclear, however, whether the cross-species transmission of threat information involves similar mechanisms, e.g., whether animals respond to the aversively induced emotional arousal of humans with activation of fear-processing circuits in the brain. Here, we report that when rats interact with a human caregiver who had recently undergone fear conditioning, they show risk assessment behavior and enhanced amygdala activation. The amygdala response involves its two major parts, the basolateral and central, which detect a threat and orchestrate defensive responses. Further, we show that humans who learn about a threat by observing another aversively aroused human, similar to rats, activate the basolateral and centromedial parts of the amygdala. Our results demonstrate that rats detect the emotional arousal of recently aversively stimulated caregivers and suggest that cross-species social transmission of threat information may involve similar neural circuits in the amygdala as the within-species transmission.

Nigrostriatal dopamine modulates the striatal-amygdala pathway in auditory fear conditioning.

The auditory striatum, a sensory portion of the dorsal striatum, plays an essential role in learning and memory. In contrast to its roles and underlying mechanisms in operant conditioning, however, little is known about its contribution to classical auditory fear conditioning. Here, we reveal the function of the auditory striatum in auditory-conditioned fear memory. We find that optogenetically inhibiting auditory striatal neurons impairs fear memory formation, which is mediated through the striatal-amygdala pathway. Using calcium imaging in behaving mice, we find that auditory striatal neuronal responses to conditioned tones potentiate across memory acquisition and expression. Furthermore, nigrostriatal dopaminergic projections plays an important role in modulating conditioning-induced striatal potentiation. Together, these findings demonstrate the existence of a nigro-striatal-amygdala circuit for conditioned fear memory formation and expression.

Rapid withdrawal from a threatening animal is movement-specific and mediated by reflex-like neural processing.

Responses to potentially dangerous stimuli are among the most basic animal behaviors. While research has shown that threats automatically capture the attention of human participants, research has failed to demonstrate automatic behavioral responses to threats in humans. Using a novel naturalistic paradigm, we show that two species of animals humans often report fearing trigger rapid withdrawal responses: participants withdrew their arm from photos of snakes and spiders faster, and with higher acceleration when compared to bird and butterfly stimuli. The behavior was specific to withdrawal as approach movements or button-press/release tasks failed to detect a similar difference. Moreover, between-participant differences in how aversive they found the stimuli predicted the participant's withdrawal speed, indicating that the paradigm was also sensitive to trait-level differences between individuals. Using electroencephalography (EEG), we show that the fast withdrawal was mediated by two attentional processes. First, fast withdrawal responses were associated with early amplification of sensory signals (40-110 ms after stimulus). Second, a later correlate of feature-based attention (early posterior negativity, EPN, 200-240 ms after stimulus) revealed the opposite pattern: Stronger EPN was associated with slower behavioral responses, suggesting that the deployment of attention towards the threatening stimulus features, or failure to "disengage" attention from the stimulus, was detrimental for withdrawal speed. Altogether, the results suggest that rapid behavioral withdrawal from a threatening animal is mediated by reflex-like attentional processing, and later, conscious attention to stimulus features may hinder escaping the treat.

Brain activation during fear extinction recall in unmedicated patients with obsessive-compulsive disorder.

Specific brain activation patterns during fear conditioning and the recall of previously extinguished fear responses have been associated with obsessive-compulsive disorder (OCD). However, further replication studies are necessary. We measured skin-conductance response and blood oxygenation level-dependent responses in unmedicated adult patients with OCD (n = 27) and healthy participants (n = 22) submitted to a two-day fear-conditioning experiment comprising fear conditioning, extinction (day 1) and extinction recall (day 2). During conditioning, groups differed regarding the skin conductance reactivity to the aversive stimulus (shock) and regarding the activation of the right opercular cortex, insular cortex, putamen, and lingual gyrus in response to conditioned stimuli. During extinction recall, patients with OCD had higher responses to stimuli and smaller differences between responses to conditioned and neutral stimuli. For the entire sample, the higher the response delta between conditioned and neutral stimuli, the greater the dACC activation for the same contrast during early extinction recall. While activation of the dACC predicted the average difference between responses to stimuli for the entire sample, groups did not differ regarding the activation of the dACC during extinction recall. Larger unmedicated samples might be necessary to replicate the previous findings reported in patients with OCD.

Conditioned flight response in female rats to naturalistic threat is estrous-cycle dependent.

Despite the prevalent expression of freezing behavior following Pavlovian fear conditioning, a growing body of literature suggests potential sex differences in defensive responses. Our study investigated how female defensive behaviors are expressed in different threat situations and modulated by the estrous cycle. We aimed to compare freezing and flight-like responses during the acquisition and retrieval of fear conditioning using two distinct unconditioned stimuli (US) in two different spatial configurations: (1) electrical footshock (FUS) in a small, conventional enclosure with a grid floor, and (2) a predator-like robot (PUS) in a spacious, open arena. Fear conditioning with FUS showed no substantial differences between male and female rats of two different estrous cycles (proestrus and diestrus) in the levels of freezing and flight. However, when PUS was employed, proestrus female rats showed significantly more flight responses to the CS during both acquisition and the retrieval compared to the male and diestrus female rats. Taken together, our findings suggest that hormonal influences on the choice of defensive strategies in threat situations are significantly modulated by both the type of US and the spatial configuration of the environment.

Brn3b regulates the formation of fear-related midbrain circuits and defensive responses to visual threat.

Defensive responses to visually threatening stimuli represent an essential fear-related survival instinct, widely detected across species. The neural circuitry mediating visually triggered defensive responses has been delineated in the midbrain. However, the molecular mechanisms regulating the development and function of these circuits remain unresolved. Here, we show that midbrain-specific deletion of the transcription factor Brn3b causes a loss of neurons projecting to the lateral posterior nucleus of the thalamus. Brn3b deletion also down-regulates the expression of the neuropeptide tachykinin 2 (Tac2). Furthermore, Brn3b mutant mice display impaired defensive freezing responses to visual threat precipitated by social isolation. This behavioral phenotype could be ameliorated by overexpressing Tac2, suggesting that Tac2 acts downstream of Brn3b in regulating defensive responses to threat. Together, our experiments identify specific genetic components critical for the functional organization of midbrain fear-related visual circuits. Similar mechanisms may contribute to the development and function of additional long-range brain circuits underlying fear-associated behavior.

Pavlovian Fear Conditioning Is More than You Think It Is.

A common neuroscience application of Pavlovian fear conditioning is to manipulate neuron-type activity, pair a cue with foot shock, then measure cue-elicited freezing in a novel context. If the manipulation reduces freezing, the neuron type is implicated in Pavlovian fear conditioning. This application reduces Pavlovian fear conditioning to a single concept. In this Viewpoint, I describe experiments supporting the view that Pavlovian fear conditioning refers to three distinct concepts: procedure, process, and behavior. An experimenter controls procedure, observes behavior, but infers process. Distinguishing these concepts is essential because: (1) a shock-paired cue can engage numerous processes and behaviors; (2) experimenter decisions about procedure influence the processes engaged and behaviors elicited; and (3) many processes are latent, imbuing the cue with properties that only manifest outside of the original conditioning setting. This means we could understand the complete neural basis of freezing, yet know little about the neural basis of fear. Neuroscientists can choose to use a variety of procedures to study a diversity of processes and behaviors. Manipulating neuron-type activity in multiple procedures can reveal specific, general, or complex neuron-type contributions to cue-elicited processes and behaviors. The results will be a broader and more detailed neural basis of fear with greater relevance to the spectrum of symptoms defining anxiety and stressor-related disorders.

Fear response of rat pups to a non-aversive social stimulus: Evidence for the involvement of memory processes.

Learning processes in rats during early development are importantly mediated by the mother, which represents the primary source of environmental information. This study aimed to determine whether aversive early experiences can induce the expression of pups' fear responses toward a non-aversive stimulus as a consequence of a memory process. First, we determined pups' fear responses toward an anesthetized female after being exposed to this stimulus or an empty cage together with their mothers from Postnatal Day (PNDs) 1 to 4. Second, we evaluated if the administration of the protein synthesis inhibitor cycloheximide (CHX; 0.2 mg/kg, subcutaneously (sc).) disrupted the reconsolidation processes and abolished the fear response on PND 9. Only female pups previously exposed to the female intruder expressed fear responses toward an anesthetized female on PND 8. CHX administration to female pups immediately after exposure to an anesthetized female on PND 8 suppressed fear responses on PND 9, indicating that the fear expression was the result of a memory process, probably mediated by the mother. These findings demonstrated that early experiences can shape responses to social stimuli in a sex-dependent manner and emphasize the critical role of the mother in influencing fear learning in a social context.

Incidental learning of faces during threat: No evidence for enhanced physiological responses to former threat identities.

Remembering an unfamiliar person and the contextual conditions of that encounter is important for adaptive future behavior, especially in a potentially dangerous situation. Initiating defensive behavior in the presence of former dangerous circumstances can be crucial. Recent studies showed selective electrocortical processing of faces that were previously seen in a threat context compared to a safety context, however, this was not reflected in conscious recognition performance. Here, we investigated whether previously seen threat-faces, that could not be remembered, were capable to activate defensive psychophysiological response systems. During an encoding phase, 50 participants with low to moderate levels of anxiety viewed 40 face pictures with neutral expressions (6 s each), without an explicit learning instruction (incidental learning task). Each half of the faces were presented with contextual background colors that signaled either threat-of-shock or safety. In the recognition phase, all old and additional new faces (total of 60) were presented intermixed without context information. Participants had to decide whether a face was new or had been presented previously in a threatening or a safe context. Results show moderate face recognition independent of context conditions. Startle reflex and skin conductance responses (SCR) were more pronounced for threat compared to safety during encoding. For SCR, this differentiation was enhanced with higher levels of depression and anxiety. There were no differential startle reflex or SCR effects during recognition. From a clinical perspective, these findings do not support the notion that perceptual biases and physiological arousal directly relate to threat-associated identity recognition deficits in healthy and clinical participants with anxiety and trauma-related disorders.

Developmental changes in functional connectivity between the prefrontal cortex and amygdala following fear extinction.

The amygdala and prefrontal cortex (PFC) undergo dramatic changes in structure, function, and regional connectivity in early life, ultimately stabilizing in early adulthood. Pathways between these two structures underlie many forms of emotional learning, including the extinction of conditioned fear. Here we sought to characterize changes in extinction-related medial PFC (mPFC) â†’ amygdala functional connectivity across development that might explain adolescent impairments in extinction. The retrograde tracer Fluorogold was infused into the amygdala of postnatal day (P)22-23 (juvenile), P31-32 (adolescent), or ≥ P69 (adult) rats, which were then exposed to fear conditioning and extinction training. Brains were collected following extinction or context exposure and processed for expression of pMAPK (as a marker of learning-dependent plasticity) in prelimbic (PL) and infralimbic (IL) amygdala-projecting neurons. Consistent with previous findings, amygdala-projecting mPFC neurons were located primarily in layers (L)II/III and V of the mPFC. We noted that mPFC LII/III projected predominantly to the ipsilateral basolateral amygdala, whereas LV projected bilaterally and targeted multiple amygdalar nuclei. Extinction was not associated with changes in extinction-related plasticity in the PL-amygdala pathways in any age group. No changes were seen in LII/III of the IL, but extinction-related plasticity in LV amygdala-projecting IL neurons decreased linearly across development. These findings suggest that extinction-related functional connectivity between the IL and the amygdala undergoes fundamental changes across development that may contribute to alterations in fear suppression across development.

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Fear, a negative emotion triggered by dangerous stimuli, can lead to psychiatric disorders such as phobias, anxiety disorders, and depression. Investigating the neural circuitry underlying congenital fear can offer insights into the pathophysiological mechanisms of related psychiatric conditions. Research on innate fear primarily centers on the response mechanisms to various sensory signals, including olfactory, visual and auditory stimuli. Different types of fear signal inputs are regulated by distinct neural circuits. The neural circuits of the main and accessory olfactory systems receive and process olfactory stimuli, mediating defensive responses like freezing. Escape behaviors elicited by visual stimuli are primarily regulated through the superior colliculus and hypothalamic projection circuits. Auditory stimuli-induced responses, including escape, are mainly mediated through auditory cortex projection circuits. In this article, we review the research progress on neural circuits of innate fear defensive behaviors in animals. We further discuss the different sensory systems, especially the projection circuits of olfactory, visual and auditory systems, to provide references for the mechanistic study of related mental disorders.

The Role of Dorsal Raphe Nucleus Serotonergic Systems in Emotional Learning and Memory in Male BALB/c Mice.

Selective serotonin reuptake inhibitors (SSRIs) are the first-line pharmacological treatment for a variety of anxiety-, trauma- and stressor-related disorders. Although they are efficacious, therapeutic improvements require several weeks of treatment and are often associated with an initial exacerbation of symptoms. The dorsal raphe nucleus (DR) has been proposed as an important target for the modulation of emotional responses and the therapeutic effects of SSRIs. Using a fear-conditioning paradigm we aimed to understand how SSRIs affect emotional learning and memory, and their effects on serotonergic circuitry. Adult male BALB/c mice were treated with vehicle (n = 16) or the SSRI fluoxetine (18 mg/kg/d) acutely (n = 16), or chronically (21d, n = 16), prior to fear conditioning. Treatment was stopped, and half of the mice (n = 8/treatment group) were exposed to cued fear memory recall 72 h later. Activation of DR serotonergic neurons during fear conditioning (Experiment 1) or fear memory recall (Experiment 2), was measured using dual-label immunohistochemistry for Tph2 and c-Fos. Acute and chronic fluoxetine treatment reduced associative fear learning without affecting memory recall and had opposite effects on anxiety-like behaviour. Acute fluoxetine decreased serotonergic activity in the DR, while chronic treatment led to serotonergic activity that was indistinguishable from that of control levels in DRD and DRV subpopulations. Chronic fluoxetine facilitated fear extinction, which was associated with rostral DRD inhibition. These findings provide further evidence that SSRIs can alter aspects of learning and memory processes and are consistent with a role for discrete populations of DR serotonergic neurons in regulating fear- and anxiety-related behaviours.

Thalamic nucleus reuniens coordinates prefrontal-hippocampal synchrony to suppress extinguished fear.

Traumatic events result in vivid and enduring fear memories. Suppressing the retrieval of these memories is central to behavioral therapies for pathological fear. The medial prefrontal cortex (mPFC) and hippocampus (HPC) have been implicated in retrieval suppression, but how mPFC-HPC activity is coordinated during extinction retrieval is unclear. Here we show that after extinction training, coherent theta oscillations (6-9 Hz) in the HPC and mPFC are correlated with the suppression of conditioned freezing in male and female rats. Inactivation of the nucleus reuniens (RE), a thalamic hub interconnecting the mPFC and HPC, reduces extinction-related Fos expression in both the mPFC and HPC, dampens mPFC-HPC theta coherence, and impairs extinction retrieval. Conversely, theta-paced optogenetic stimulation of RE augments fear suppression and reduces relapse of extinguished fear. Collectively, these results demonstrate a role for RE in coordinating mPFC-HPC interactions to suppress fear memories after extinction.

Fear Conditioning by Proxy: The Role of High Affinity Nicotinic Acetylcholine Receptors.

Observational fear-learning studies in genetically modified animals enable the investigation of the mechanisms underlying the social transmission of fear-related information. Here, we used a three-day protocol to examine fear conditioning by proxy (FCbP) in wild-type mice (C57BL/6J) and mice lacking the ß2-subunit of the nicotinic acetylcholine receptor (nAChR). Male animals of both genotypes were exposed to a previously fear-conditioned (FC) cage mate during the presentation of the conditioned stimulus (CS, tone). On the following day, observer (FCbP) mice were tested for fear reactions to the tone: none of the ß2-KO mice froze to the stimulus, while 30% of the wild-type mice expressed significant freezing. An investigation of the possible factors that predicted the fear response revealed that only wild-type mice that exhibited enhanced and more flexible social interaction with the FC cage mate during tone presentations (Day 2) expressed fear toward the CS (Day-3). Our results indicate that (i) FCbP is possible in mice; (ii) the social transmission of fear depends on the interaction pattern between animals during the FCbP session and (iii) ß2-KO mice display a more rigid interaction pattern compared to wild-type mice and are unable to acquire such information. These data suggest that ß2-nAChRs influence observational fear learning indirectly through their effect on social behaviour.

Presence of a remote fear memory engram in the central amygdala.

Fear memory formation and recall are highly regulated processes, with the central amygdala (CeA) contributing to fear memory-related behaviors. We recently reported that a remote fear memory engram is resident in the anterior basolateral amygdala (aBLA). However, the extent to which downstream neurons in the CeA participate in this engram is unknown. We tested the hypothesis that CeA neurons activated during fear memory formation are reactivated during remote memory retrieval such that a CeA engram participates in remote fear memory recall and its associated behavior. Using contextual fear conditioning in TRAP2;Ai14 mice, we identified, by persistent Cre-dependent tdTomato expression (i.e., "TRAPing"), CeA neurons that were c-fos-activated during memory formation. Twenty-one days later, we quantified neurons activated during remote memory recall using Fos immunohistochemistry. Dual labeling was used to identify the subpopulation of CeA neurons that was both activated during memory formation and reactivated during recall. Compared with their context-conditioned (no shock) controls, fear-conditioned (electric shock) mice (n = 5/group) exhibited more robust fear memory-related behavior (freezing) as well as larger populations of activated (tdTomato+) and reactivated (dual-labeled) CeA neurons. Most neurons in both groups were mainly located in the capsular CeA subdivision (CeAC). Notably, however, only the size of the TRAPed population distributed throughout the CeA was significantly correlated with time spent freezing during remote fear memory recall. Our findings indicate that fear memory formation robustly activates CeA neurons and that a subset located mainly in the CeAC may contribute to both remote fear memory storage/retrieval and the resulting fear-like behavior.

A thalamic-hippocampal CA1 signal for contextual fear memory suppression, extinction, and discrimination.

The adaptive regulation of fear memories is a crucial neural function that prevents inappropriate fear expression. Fear memories can be acquired through contextual fear conditioning (CFC) which relies on the hippocampus. The thalamic nucleus reuniens (NR) is necessary to extinguish contextual fear and innervates hippocampal CA1. However, the role of the NR-CA1 pathway in contextual fear is unknown. We developed a head-restrained virtual reality CFC paradigm, and demonstrate that mice can acquire and extinguish context-dependent fear responses. We found that inhibiting the NR-CA1 pathway following CFC lengthens the duration of fearful freezing epochs, increases  fear generalization, and delays fear extinction. Using in vivo imaging, we recorded NR-axons innervating CA1 and found that NR-axons become tuned to fearful freezing following CFC. We conclude that the NR-CA1 pathway actively suppresses fear by disrupting contextual fear memory retrieval in CA1 during fearful freezing behavior, a process that also reduces fear generalization and accelerates extinction.