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Doublecortin (DCX) has long been implicated in, and employed as a marker for, neurogenesis, yet little is known about its function in non-neurogenic brain regions, including the amygdala. This study sought first to explore, in rodents, whether fear learning and extinction modulate amygdala DCX expression and, second, to assess the utility of peripheral DCX correlates as predictive biomarkers of trauma response in rodents and humans. Pavlovian conditioning was found to alter DCX protein levels in mice 24 h later, resulting in higher DCX expression associated with enhanced learning in paradigms examining both the acquisition and extinction of fear (p

Previous studies have shown that activating the attachment system attenuates fear learning. This study aimed to explore whether attachment priming can also impact on fear extinction processes, which underpin the management of anxiety disorders. In this study, 81 participants underwent a standard fear conditioning and extinction protocol on day 1 and returned 24 h later for an extinction recall and reinstatement test. Half the participants were primed to imagine their closest attachment figure prior to undergoing extinction training, while the other half were instructed to imagine a positive situation. Fear-potentiated startle and subjective expectancies of shock were measured as the primary indicators of fear. Attachment priming led to less relapse during the reinstatement test at the physiological but not subjective levels. These findings have translational potential to imply that activating awareness of attachment figures might augment long-term safety memories acquired in existing treatments to reduce relapse of fear.

Background Recent studies have found that attachment security primes can inhibit fear acquisition. This current study aimed to examine whether a brief imaginal prime of one's attachment figure could impact on fear consolidation. Methods A total of 75 participants underwent fear conditioning on Day 1 and fear recall was tested on Day 2. Immediately following conditioning, half the participants were instructed to imagine an attachment figure while the other half imagined a nonattachment positive situation. Fear-potentiated startle and subjective expectancy of shock ratings were used as the measures of fear learning across trials. Results The attachment group showed significantly lower levels of fear recall on Day 2 at both physiological and subjective levels. Furthermore, this effect was moderated by attachment anxiety, such that it was greatest for individuals who were securely attached. Conclusions These findings suggest that attachment relationships are protective during the consolidation of fear memories, and may have implications for how social attachments may impact how anxiety disorders can develop.

Overgeneralized fear (OGF), or indiscriminate fear responses to signals of threat and nonthreat, is a well-studied cognitive mechanism in human anxiety. Anxiety-related OGF has been studied primarily through fear-learning paradigms and conceptualized as overly exaggerated learning of cues signaling imminent threat. However, the role of safety learning in OGF has not only received much less empirical attention but has been fundamentally conceptualized as learning about the absence of threat rather than the presence of safety. As a result, the relative contributions of exaggerated fear learning and weakened safety learning to anxiety-related OGF remain poorly understood, as do the potentially unique biological and behavioral underpinnings of safety learning. The present review outlines these gaps by, first, summarizing animal and human research on safety learning related to anxiety and OGF. Second, we outline innovations in methods to tease apart unique biological and behavioral contributions of safety learning to OGF. Lastly, we describe clinical and treatment implications of this framework for translational research relevant to human anxiety.

Drug-induced neuroadaptations in the mPFC have been implicated in addictive behaviors. Repeated cocaine exposure has been shown to increase pyramidal neuron excitability in the prelimbic (PL) region of the mouse mPFC, an adaptation attributable to a suppression of G protein-gated inwardly rectifying K+(GIRK) channel activity. After establishing that this neuroadaptation is not seen in adjacent GABA neurons, we used viral GIRK channel ablation and complementary chemogenetic approaches to selectively enhance PL pyramidal neuron excitability in adult mice, to evaluate the impact of this form of plasticity on PL-dependent behaviors. GIRK channel ablation decreased somatodendritic GABABreceptor-dependent signaling and rheobase in PL pyramidal neurons. This manipulation also enhanced the motor-stimulatory effect of cocaine but did not impact baseline activity or trace fear learning. In contrast, selective chemogenetic excitation of PL pyramidal neurons, or chemogenetic inhibition of PL GABA neurons, increased baseline and cocaine-induced activity and disrupted trace fear learning. These effects were mirrored in male mice by selective excitation of PL pyramidal neurons projecting to the VTA, but not NAc or BLA. Collectively, these data show that manipulations enhancing the excitability of PL pyramidal neurons, and specifically those projecting to the VTA, recapitulate behavioral hallmarks of repeated cocaine exposure in mice.SIGNIFICANCE STATEMENTProlonged exposure to drugs of abuse triggers neuroadaptations that promote core features of addiction. Understanding these neuroadaptations and their implications may suggest interventions capable of preventing or treating addiction. While previous work showed that repeated cocaine exposure increased the excitability of pyramidal neurons in the prelimbic cortex (PL), the behavioral implications of this neuroadaptation remained unclear. Here, we used neuron-specific manipulations to evaluate the impact of increased PL pyramidal neuron excitability on PL-dependent behaviors. Acute or persistent excitation of PL pyramidal neurons potentiated cocaine-induced motor activity and disrupted trace fear conditioning, effects replicated by selective excitation of the PL projection to the VTA. Our work suggests that hyperexcitability of this projection drives key behavioral hallmarks of addiction.

A previously acquired fear response often spreads to perceptually or conceptually close stimuli or contexts. This process, known as fear generalization, facilitates the avoidance of danger, and dysregulations in this process play an important role in anxiety disorders. Oxytocin (OT) has been shown to modulate fear learning, yet effects on fear generalization remain unknown. We employed a randomized, placebo-controlled, double-blind, between-subject design during which healthy male participants received either intranasal OT or placebo (PLC) following fear acquisition and before fear generalization with concomitant acquisition of skin conductance responses (SCRs). Twenty-four to 72 h before the fear learning and immediately after the fear generalization task, participants additionally complete a discrimination threshold task. Relative to PLC, OT significantly reduced perceived risk and SCRs towards the CS+ and GS1 (the generalization stimulus that is most similar to CS+) during fear generalization, whereas the discrimination threshold was not affected. Together, the results suggest that OT can attenuate fear generalization in the absence of effects on discrimination threshold. This study provides the first evidence for effects of OT on fear generalization in humans and suggests that OT may have therapeutic potential in anxiety disorders characterized by dysregulated fear generalization.

Several labels, such as neuroticism, negative emotionality, and dispositional negativity, indicate a broad dimension of psychopathology. However, largely separate, often disorder-specific research lines have developed that focus on different cognitive and affective characteristics that are associated with this dimension, such as perseverative cognition (worry, rumination), reduced autobiographical memory specificity, compromised fear learning, and enhanced somatic-symptom reporting. In this article, we present a theoretical perspective within a predictive-processing framework in which we trace these phenotypically different characteristics back to a common underlying “better-safe-than-sorry” processing strategy. This implies information processing that tends to be low in sensory-perceptual detail, which allows threat-related categorical priors to dominate conscious experience and for chronic uncertainty/surprise because of a stagnated error-reduction process. This common information-processing strategy has beneficial effects in the short term but important costs in the long term. From this perspective, we suggest that the phenomenally distinct cognitive and affective psychopathological characteristics mentioned above represent the same basic processing heuristic of the brain and are only different in relation to the particular type of information involved (e.g., in working memory, in autobiographical memory, in the external and internal world). Clinical implications of this view are discussed. ispartof: Perspectives in Psychological Science vol:16 issue:2 pages:1-22 ispartof: location:United States status: Published online

Aberrant fear learning processes are assumed to be a key factor in the pathogenesis of anxiety disorders. Thus, effective behavioral interventions to reduce dysfunctional fear responding are needed. Beyond passive extinction learning, instrumental control over threatening events is thought to diminish fear. However, the neural mechanisms underlying instrumental control—and to what extent these differ from extinction—are not well understood. We therefore contrasted the neural signatures of instrumental control and passive extinction using an aversive learning task, relative to a control condition. Participants (n = 64) could either learn to exert instrumental control over electric shocks, received a yoked number and sequence of shocks without instrumental control or did not receive any shocks. While both passive extinction and instrumental control reduced threat-related skin conductance responses (SCRs) relative to pre-extinction/control, instrumental control resulted in a significantly more pronounced decrease of SCRs. Instrumental control was further linked to decreased striatal activation and increased cross talk of the ventromedial prefrontal cortex (vmPFC) with the amygdala, whereas passive extinction was associated with increased vmPFC activation. Our findings demonstrate that instrumental learning processes may shape Pavlovian fear responses and that the neural underpinnings of instrumental control are critically distinct from those of passive extinction learning.

Disruption of persistent, stress-associated memories is relevant for treating posttraumatic stress disorder (PTSD) and related syndromes, which develop in a subset of individuals following a traumatic event. We previously developed a stress-enhanced fear learning (SEFL) paradigm in inbred mice that produces PTSD-like characteristics in a subset of mice, including persistently enhanced memory and heightened cFos in the basolateral amygdala complex (BLC) with retrieval of the remote (30-day-old) stress memory. Here, the contribution of BLC microRNAs (miRNAs) to stress-enhanced memory was investigated because of the molecular complexity they achieve through their ability to regulate multiple targets simultaneously. We performed small-RNA sequencing (smRNA-Seq) and quantitative proteomics on BLC tissue collected from mice 1 month after SEFL and identified persistently changed microRNAs, including mir-135b-5p, and proteins associated with PTSD-like heightened fear expression. Viral-mediated overexpression of mir-135b-5p in the BLC of stress-resilient animals enhanced remote fear memory expression and promoted spontaneous renewal 14 days after extinction. Conversely, inhibition of BLC mir-135b-5p in stress-susceptible animals had the opposite effect, promoting a resilient-like phenotype. mir-135b-5p is highly conserved across mammals and was detected in post mortem human amygdala, as well as human serum samples. The mir-135b passenger strand, mir-135b-3p, was significantly elevated in serum from PTSD military veterans, relative to combat-exposed control subjects. Thus, miR-135b-5p may be an important therapeutic target for dampening persistent, stress-enhanced memory and its passenger strand a potential biomarker for responsivity to a mir-135-based therapeutic.

BACKGROUND: The amygdala is a subcortical structure involved in socioemotional and associative fear learning processes relevant for understanding the mechanisms of posttraumatic stress disorder (PTSD). Research in animals indicates that the amygdala is a heterogeneous structure in which the basolateral and centromedial divisions are susceptible to stress. While the amygdala complex is implicated in the pathophysiology of PTSD, little is known about the specific contributions of the individual nuclei that constitute the amygdala complex. METHODS: Military veterans (n = 355), including military veterans with PTSD (n = 149) and trauma-exposed control subjects without PTSD (n = 206), underwent high-resolution T1-weighted anatomical scans. Automated FreeSurfer segmentation of the amygdala yielded 9 structures: basal, lateral, accessory basal, anterior amygdaloid, and central, medial, cortical, and paralaminar nuclei, along with the corticoamygdaloid transition zone. Subregional volumes were compared between groups using ordinary-least-squares regression with relevant demographic and clinical regressors followed by 3-dimensional shape analysis of whole amygdala. RESULTS: PTSD was associated with smaller left and right lateral and paralaminar nuclei, but with larger left and right central, medial, and cortical nuclei (p < .05, false discovery rate corrected). Shape analyses revealed lower radial distance in anterior bilateral amygdala and lower Jacobian determinant in posterior bilateral amygdala in PTSD compared with control subjects. CONCLUSIONS: Alterations in select amygdala subnuclear volumes and regional shape distortions are associated with PTSD in military veterans. Volume differences of the lateral nucleus and the centromedial complex associated with PTSD demonstrate a subregion-specific pattern that is consistent with their functional roles in fear learning and fear expression behaviors.

While results from many past studies have implicated the bed nucleus of the stria terminalis (BNST) in mediating the expression of sustained negative affect, recent studies have highlighted a more complex role for BNST that includes aspects of fear learning in addition to defensive responding. As BNST is thought to encode ambiguous or unpredictable threat, it seems plausible that it may be involved in encoding early cued fear learning, especially immediately following a first tone-shock pairing when the conditioned stimulus–unconditioned stimulus (CS–US) contingency is not fully apparent. To investigate this, we conducted in vivo electrophysiological recording studies to examine neural dynamics of BNST units during cued fear acquisition and recall. We identified two functionally distinct subpopulations of BNST neurons that encode the intertrial interval (ITI) and may contribute to within- and across-session fear learning. “Ramping” cell activity during cued fear acquisition parallels the increase in freezing expression as mice learn the CS–US contingency, while “Phasic” cells encode postshock (USpost) periods (30 sec following encounter with footshock) only during early trials. Importantly, the magnitude of Phasic unit responsivity to the first USpost period predicted not only freezing expression in response to the subsequent CS during acquisition, but also CS freezing evoked 24 h later during CS retrieval. These findings suggest for the first time that BNST activity may serve as an instructive signal during cued fear learning.

Many contemporary studies of human fear conditioning exclude participants who fail to show differential electrodermal responding during late stages of acquisition training, deeming them to be non-Learners. The current study examined whether non-Learners, defined as those who fail to show larger electrodermal first interval responses to CS+ than to CS- in the second half of acquisition, show differential electrodermal responding early during acquisition or during extinction or evidence of fear-learning on other measures, including rated CS valence and contingency report. In a sample of 351 participants who completed a standard differential fear-conditioning paradigm that employed electrodermal first and second interval responses (FIR, SIR), continuous CS evaluations, and post-experimental contingency reports to assess fear-learning, 74 participants were identified as non-Learners. These non-Learners displayed overall smaller electrodermal responses but showed evidence for differential conditioning during acquisition in electrodermal FIR (block1) and SIR (blocks 2-3) and in CS evaluations during acquisition (blocks 2-4) and extinction (blocks 1-4). Fifty-nine non-Learners correctly reported the contingencies. A lack of differential electrodermal first interval responding during the second half of acquisition does not indicate the absence of fear-learning. Rather, this criterion appears to capture participants who exhibit low physiological arousal and performance decrements toward the end of acquisition. Applying criteria based on "end of acquisition" electrodermal responding to determine "non-learning" results in the exclusion of participants who display fear-learning at other experimental stages or in other measures.

The ability to learn to differentiate safety from danger matures gradually, particularly when such learning occurs over an extended time period. And yet, most research on fear learning examines the early phases of such learning and mainly in adults. The current study examined fear conditioning and extinction, as well as one form of extended learning, return of fear (ROF). Thirty-three typically developing children (age range: 7-14 years) completed fear conditioning and extinction; self-reports and psychophysiological indices were measured at this point. Two weeks later, children completed a ROF test (n = 23), and event-related potentials (ERPs) were recorded. Results indicated successful fear acquisition and extinction. Moreover, participants reported greater fear of the conditioned stimulus (CS+) than the safety stimulus (CS-) in the ROF test 2 weeks later. In electrophysiology data, ROF manifested as a larger late positive potential (LPP) response to the CS+ than the CS-. Finally, these differences in LPP responses were positively correlated with poorer extinction, as indicated by the GSR responses 2 weeks earlier. This is the first ERP study to demonstrate ROF in children. The LPP measure may index an interplay between inhibitory and excitatory brain-related processes underlying the long-term effects of fear learning.

BACKGROUND: Traumatic experiences, such as conditioned threat, are coded as enduring memories that are frequently subject to generalization, which is characterized by (re-) expression of fear in safe environments. However, the neurobiological mechanisms underlying threat generalization after a traumatic experience and the role of stress hormones in this process remain poorly understood.METHODS: We examined the influence of glucocorticoid hormones on the strength and specificity of conditioned fear memory at the level of sparsely distributed dentate gyrus (DG) engram cells in male mice.RESULTS: We found that elevating glucocorticoid hormones after fear conditioning induces a generalized contextual fear response. This was accompanied by a selective and persistent increase in the excitability and number of activated DG granule cells. Selective chemogenetic suppression of these sparse cells in the DG prevented glucocorticoid-induced fear generalization and restored contextual memory specificity, while leaving expression of auditory fear memory unaffected.CONCLUSIONS: These results implicate the sparse ensemble of DG engram cells as a critical cellular substrate underlying fear generalization induced by glucocorticoid stress hormones.

Recent studies suggest that the Zona Incerta (ZI) plays a role in fear learning and recall. However, there is a clear gap in knowledge as to whether the ZI can encode fear evoking threats and cues that predict them. Here, we subject mice to a classical fear conditioning paradigm while recording the in-vivo calcium dynamics of ZI neurons. We observed that ZI neurons can encode not only a fear evoking stimulus, but can also learn to encode predictive cues, associate them with the unconditioned fear evoking stimulus, and discriminate them from neutral non-predictive cues. While the ZI across all mice learned to become mainly excited by fear predictive cues, only mice that did not generalize fear became largely inhibited by non-predictive cues and better discriminated the two. Together, we provide extensive evidence that the ZI differentially encode fear predictive and non-predictive cues in a generalization dependent manner.

The inability to extinguish a traumatic memory is a key aspect of post-traumatic stress disorder (PTSD). While PTSD affects 10–20% of individuals who experience a trauma, women are particularly susceptible to developing the disorder. Despite this notable female vulnerability, few studies have investigated this particular resistance to fear extinction observed in females. Similar to humans, rodent models of Pavlovian fear learning and extinction show a wide range of individual differences in fear learning and extinction, although female rodents are considerably understudied. Therefore, the present study examined individual differences in fear responses, including freezing behavior and ultrasonic vocalizations (USVs), of female Long–Evans rats during acquisition of fear conditioning and cued fear extinction. Similar to prior studies in males, female rats displayed individual variation in freezing during cued fear extinction and were divided into extinction competent (EC) and extinction resistant (ER) phenotypes. Differences in freezing between ER and EC females were accompanied by shifts in rearing during extinction, but no darting was seen in any trial. Freezing behavior during fear learning did not differ between the EC and ER females. Vocalizations emitted in the 22 and 50 kHz ranges during fear learning and extinction were also examined. Unlike vocalizations seen in previous studies in males, very few 22 kHz distress vocalizations were emitted by female rats during fear acquisition and extinction, with no difference between ER and EC groups. Interestingly, all female rats produced significant levels of 50 kHz USVs, and EC females emitted significantly more 50 kHz USVs than ER rats. This difference in 50 kHz USVs was most apparent during initial exposure to the testing environment. These results suggest that like males, female rodents show individual differences in both freezing and USVs during fear extinction, although females appear to vocalize more in the 50 kHz range, especially during initial periods of exposure to the testing environment, and emit very few of the 22 kHz distress calls that are typically observed in males during fear learning or extinction paradigms. Overall, these findings show that female rodents display fear behavior repertoires divergent from males.

Discrimination between sensory stimuli associated with safety and threat is crucial for behavioral decisions. Discriminative conditioning paradigms with two acoustic conditioned stimuli (one paired with shock [CS+], the other unpaired with shock [CS−]) have been widely used as an experimental model for fear learning. However, no attention has been paid to the effect of the CS− on safety in the paradigms, because the CS− served as a neutral cue or elevated the freezing level due to fear generalization although less effectively than the CS+. By using a noise and a tone as two acoustic CSs in a discriminative auditory fear conditioning (AFC) paradigm, here we demonstrate that mice learn safety for the CS− while showing fear for the CS+ with opposing emotional behaviors. We found that after learning mice exhibited a significant suppression of context-dependent freezing during the CS−, but not during the CS+, indicating learned safety without fear generalization for the CS−. In contrast, the mice showed an enhanced level of freezing during the CS+ even in a novel spatial context, indicating cued fear for the CS+. Moreover, the CS+ also induced rapid defensive behaviors, whereas the CS− disinhibited normal exploratory behaviors. On the other hand, mice showed no significant suppression of contextual fear during the CS− in a paradigm with a pair of tone CSs at different frequencies, although they clearly discriminated the two tones. These results suggest our AFC paradigm with the noise and tone CSs as a useful experimental model for cue-dependent discriminative learning of safety and threat.

In classical fear conditioning, neutral conditioned stimuli that have been paired with aversive physical unconditioned stimuli eventually trigger fear responses. Here, we tested whether aversive mental images systematically paired with a conditioned stimulus also cause de novo fear learning in the absence of any external aversive stimulation. In two experiments ( N = 45 and N = 41), participants were first trained to produce aversive, neutral, or no imagery in response to three different visual-imagery cues. In a subsequent imagery-based differential-conditioning paradigm, each of the three cues systematically coterminated with one of three different neutral faces. Although the face that was paired with the aversive-imagery cue was never paired with aversive external stimuli or threat-related instructions, participants rated it as more arousing, unpleasant, and threatening and displayed relative fear bradycardia and fear-potentiated startle. These results could be relevant for the development of fear and related disorders without trauma.

How and where sensory stimuli, such as tones or lights, are linked to valence is an important unresolved question in the field of neuroscience. The auditory cortex is essential to analyse the identity and the behavioural importance of tones paired with emotional events. On the contrary, whether the auditory cortex may also encode information on the emotional-motivational valence of sounds is much more controversial. Here, we reviewed recent studies showing that the activity of cortical neurons reflects information about the content of emotional stimuli paired with tones. Critically, the blockade of these neuronal processes prevents animals from recognising sounds as aversive or pleasant. Based on these findings, we proposed a conceptual model in which the auditory cortex may incorporate ascending information from subcortical nuclei about the valence of sounds in sound representations and may consequently drive the activity of subcortical structures towards emotionally laden tones. This hypothesis may also have important implications in the characterisation of neural circuits engaged by maladaptive affective disorders, such as phobias.

Anxiety disorders are characterised by the perception of fear and threat in the presence of stimuli that are neutral or ambiguous. Attempts in previous research to explain the relationship between anxiety and fear learning have been inconsistent, possibly due to the influence of an unmeasured mechanism that mediates the relationship between them. Working memory capacity has been suggested as one such mechanism. The current study investigated the influence of anxiety-based individual differences upon associative fear learning, while accounting for individual differences in working memory. We hypothesised that individuals high in both anxiety and working memory would show unimpaired fear learning whereas individuals high in anxiety and low in working memory would exhibit dysfunctional fear learning. Sixty participants completed a battery of anxiety and working memory tests, as well as a fear conditioning experiment that tested for blocking, conditioned inhibition and fear discrimination. We found that anxious individuals were more likely to show impaired fear discrimination only if they also had a low working memory capacity. Furthermore, anxiety was particularly associated with poorer learning about safety cues. Such relationships were not observed for blocking and conditioned inhibition. These results suggest that the relationship between anxiety and fear learning is complex and warrants further investigation of the potential mediating role of higher-order cognitive faculties.

Zusammenfassung. Die Begriffe Nahrungsaversion und Nahrungsvermeidung werden in der Literatur häufig synonym gebraucht. Obwohl sie eng verwandt sind, bezeichnen sie doch zwei verschiedene Konstrukte, die das Resultat verschiedener Prozesse sind. Bislang wird angenommen, dass Nahrungseinschränkung und Nahrungsvermeidung bei Patientinnen mit Anorexia nervosa, und Bulimia nervosa mit der Angst vor Gewichtszunahme oder der Angst vor Unwohlsein (z.B, Bauchschmerzen) nach der Nahrungseinnahme zusammenhängt. Studien zeigen jedoch, dass Nahrungsvermeidung nicht nur durch Vermeidungslernen bedingt ist, bei dem der Geschmack der Nahrung zum konditionierten Reiz für Gefahr wird. Vielmehr spielt das Geschmacksaversionslernen eine wichtige Rolle. Nahrungsvermeidung, der eine Geschmacksaversion zugrunde liegt, ist auf eine negative Veränderung der Valenz des Geschmacks zurückzuführen, die durch ein Erlebnis starker Übelkeit hervorgerufen wird. Beide Formen der Nahrungsvermeidung haben verschiedene Verhaltensmerkmale und unterscheiden sich bezüglich der gelernten Inhalte. Zudem sind sie mit anderen Gehirnregionen und Neuromodulatoren assoziiert. Dies ist relevant für das Verständnis und die Behandlung von Essstörungen, insbesondere für ihre bedrohlichste Manifestation: Der extremen Vermeidung der Nahrungseinnahme. Klinische Implikationen dieser Unterscheidung und Vorschläge für weitere Forschung werden diskutiert.

Over the course of evolution the human brain has been shaped to prioritize cues that signal potential danger. Thereby, the brain does not only favor specie-specific prepared stimulus sets such as snakes or spiders, but can learn associations between new cues and aversive outcomes. One important mechanism to achieve this is associated with learning induced plasticity changes in sensory cortex that optimizes the representation of motivationally relevant sensory stimuli. Animal studies have shown that the modulation of gamma band oscillations predicts cholinergic driven plasticity changes in sensory cortices shifting neurons’ responses to fear relevant features as acquired by Pavlovian fear conditioning. Here, we report conditioned excitatory and inhibitory gamma band modulations in humans during fear conditioning of orthogonally oriented sinus gratings representing fear relevant and irrelevant conditioned cues, respectively. Thereby, pairing of a sinus grating with an aversive loud noise not only increased short latency (during the first 180 ms) evoked visual gamma band responses, but was also accompanied by strong gamma power reductions for the fear irrelevant control grating. The current findings will be discussed in the light of recent neurobiological models of cholinergic driven plasticity changes in sensory cortices and classic learning models such as the Rescorla-Wagner framework.

Fear conditioning (FC) is a widely accepted tool for the assessment of learning and memory processes in rodents related to normal and dysregulated acquired fear. The study of sex differences in fear learning and memory is vast and currently increasing. Sex hormones have proven to be crucial for fear memory formation in males and females, and several methods have been developed to assess this hormonal state in rats and mice. Herein, we explain a routine FC and extinction protocol, together with the evaluation of sex hormonal state in male and female rodents. We explain three protocols for the evaluation of this hormonal state directly from blood samples extracted during the procedure or indirectly through histological verification of the estrous cycle for females or behavioral assessment of social hierarchies in males. Although females have typically been considered to present great variability in sex hormones, it is highlighted that sex hormone assessment in males is as variable as in females and equally important for fear memory formation. The readout of these protocols has had a great impact on different fields of fear learning and memory study and appears essential when studying FC. The proven interaction with drugs involved in the modulation of these processes makes sex hormone assessment during FC a valuable tool for the development of effective treatments for fear-related disorders in men and women. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Fear conditioning and fear extinction Basic Protocol 2: Blood collection for direct measurement of sex hormone levels in fear conditioning Basic Protocol 3: Indirect measurement of sex hormones in females during fear conditioning Basic Protocol 4: Assessment of dominance status in males before a fear conditioning protocol Support Protocol: Construction of a confrontation tube.

The ability to recognize motivationally salient events and adaptively respond to them is critical for survival. Here, we tested whether dopamine (DA) neurons in the dorsal raphe nucleus (DRN) contribute to this process in both male and female mice. Population recordings of DRNDAneurons during associative learning tasks showed that their activity dynamically tracks the motivational salience, developing excitation to both reward-paired and shock-paired cues. The DRNDAresponse to reward-predicting cues was diminished after satiety, suggesting modulation by internal states. DRNDAactivity was also greater for unexpected outcomes than for expected outcomes. Two-photon imaging of DRNDAneurons demonstrated that the majority of individual neurons developed activation to reward-predicting cues and reward but not to shock-predicting cues, which was surprising and qualitatively distinct from the population results. Performing the same fear learning procedures in freely-moving and head-fixed groups revealed that head-fixation itself abolished the neural response to aversive cues, indicating its modulation by behavioral context. Overall, these results suggest that DRNDAneurons encode motivational salience, dependent on internal and external factors.SIGNIFICANCE STATEMENTDopamine (DA) contributes to motivational control, composed of at least two functional cell types, one signaling for motivational value and another for motivational salience. Here, we demonstrate that DA neurons in the dorsal raphe nucleus (DRN) encode the motivational salience in associative learning tasks. Neural responses were dynamic and modulated by the animal's internal state. The majority of single-cells developed responses to reward or paired cues, but not to shock-predicting cues. Additional experiments with freely-moving and head-fixed mice showed that head-fixation abolished the development of cue responses during fear learning. This work provides further characterization on the functional roles of overlooked DRNDApopulations and an example that neural responses can be altered by head-fixation, which is commonly used in neuroscience.

G-protein coupled receptor 139 (GPR139) is an evolutionarily conserved orphan receptor, predominantly expressing in the habenula of vertebrate species. The habenula has recently been implicated in aversive response and its associated learning. Here, we tested the hypothesis that GPR139 signalling in the habenula may play a role in fear learning in the zebrafish. We examined the effect of intraperitoneal injections of a human GPR139-selective agonist (JNJ-63533054) on alarm substance-induced fear learning using conditioned place avoidance paradigm, where an aversive stimulus is paired with one compartment, while its absence is associated with the other compartment of the apparatus. The results indicate that fish treated with 1 µg/g body weight of GPR139 agonist displayed no difference in locomotor activity and alarm substance-induced fear response. However, avoidance to fear-conditioned compartment was diminished, which suggests that the agonist blocks the consolidation of contextual fear memory. On the other hand, fish treated with 0.1 µg/g body weight of GPR139 agonist spent a significantly longer time in the unconditioned neutral compartment as compared to the conditioned (punished and unpunished) compartments. These results suggest that activation of GPR139 signalling in the habenula may be involved in fear learning and the decision-making process in the zebrafish.

This research was supported by the German Research Foundation (DFG) Grant to Florian Bublatzky (BU 3255/1-1 and 1-2)., Significant others provide individuals with a sense of safety and security. However, the mechanisms that underlie attachment-induced safety are hardly understood. Recent research has shown beneficial effects when viewing pictures of the romantic partner, leading to reduced pain experience and defensive responding. Building upon this, we examined the inhibitory capacity of loved face pictures on fear learning in an instructed threat paradigm. Pictures of loved familiar or unknown individuals served as signals for either threat of electric shocks or safety, while a broad set of psychophysiological measures was recorded. We assumed that a long-term learning history of beneficial relations interferes with social threat learning. Nevertheless, results yielded a typical pattern of physiological defense activation towards threat cues, regardless of whether threat was signaled by an unknown or a loved face. These findings call into question the notion that pictures of loved individuals are shielded against becoming threat cues, with implications for attachment and trauma research., German Research Foundation (DFG) BU 3255/1-1 BU 3255/1-2

Post-traumatic stress disorder (PTSD) can develop after exposure to traumatic events and severely impacts the quality of life. PTSD is frequently comorbid with substance use disorders, with alcoholism being particularly common. However, not everyone who experiences trauma develops PTSD and the factors that render individuals susceptible or resilient to the effects of stress are unknown although gender appears to play an important role. Rodent models of stress exposure such as stress-enhanced fear learning (SEFL) recapitulate some aspects of PTSD symptomology, making them an invaluable tool for studying this disorder. This study examined whether exposure to a modified version of the SEFL procedure (4 footshocks instead of the standard 15 over 90min) would reveal both susceptible and resilient subjects. Following stress exposure, distinct susceptible and resilient groups emerged that differed in fear learning and anxiety-related behavior as well as voluntary alcohol intake. Some aspects of stress susceptibility manifested differently in males compared to females, with susceptibility associated with increased alcohol intake in males and increased baseline anxiety in females.

Rats can acquire fear by observing conspecifics that express fear in the presence of conditioned fear stimuli. This process is called observational fear learning and is based on the social transmission of the demonstrator rat’s emotion and the induction of an empathy-like or anxiety state in the observer. The aim of the present study was to investigate the role of trait anxiety and ultrasonic vocalization in observational fear learning. Two experiments with male Wistar rats were performed. In the first experiment, trait anxiety was assessed in a light–dark box test before the rats were submitted to the observational fear learning procedure. In the second experiment, ultrasonic vocalization was recorded throughout the whole observational fear learning procedure, and 22 kHz and 50 kHz calls were analyzed. The results of our study show that trait anxiety differently affects direct fear learning and observational fear learning. Direct fear learning was more pronounced with higher trait anxiety, while observational fear learning was the best with a medium-level of trait anxiety. There were no indications in the present study that ultrasonic vocalization, especially emission of 22 kHz calls, but also 50 kHz calls, are critical for observational fear learning.

The pivotal role of the periaqueductal grey (PAG) in fear learning is reinforced by the identification of neurons in rat ventral PAG (vPAG) that encode fear memory through signalling the onset and offset of an auditory conditioned stimulus during presentation of the unreinforced conditioned tone during retrieval. Some units only display CS+ onset or offset responses and the two signals differ in extinction sensitivity, suggesting they are independent of each other. In addition, understanding cerebellar contributions to survival circuits is advanced by the discovery that: (i) reversible inactivation of the medial cerebellar nucleus (MCN) during fear consolidation leads in subsequent retrieval to: (a) disruption of the temporal precision of vPAG offset, but not onset responses to the unreinforced conditioned tone, and (b) an increase in duration of freezing behaviour. And (ii) chemogenetic manipulation of the MCN-vPAG projection during fear acquisition: (a) reduces the occurrence of fear- related ultrasonic vocalisations and (b) during subsequent retrieval, slows the extinction rate of fear- related freezing. These findings show that the cerebellum is part of the survival network that regulates fear memory processes at multiple timescales and in multiple ways; raising the possibility that dysfunctional interactions in the cerebellar-survival network may underlie fear-related disorders and comorbidities.Impact StatementCerebellar-periaqueductal grey interactions contribute to fear conditioned processes and, as such, provide a novel target for treating psychological conditions including post-traumatic stress disorder.

Current models of human fear learning processes emphasize a primary contribution of the ventromedial prefrontal cortex (vmPFC) to fear inhibition (i.e., fear extinction). Here, we discuss crucial findings from recent brain imaging studies that highlight the role of vmPFC and its subregions in how fear is acquired (i.e., fear conditioning).

BackgroundAnxiety disorders are highly prevalent with an early age of onset. Understanding the aetiology of disorder emergence and recovery is important for establishing preventative measures and optimising treatment. Experimental approaches can serve as a useful model for disorder and recovery relevant processes. One such model is fear conditioning. We conducted a remote fear conditioning paradigm in monozygotic and dizygotic twins to determine the degree and extent of overlap between genetic and environmental influences on fear acquisition and extinction.Methods1937 twins aged 22-25 years, including 538 complete pairs from the Twins Early Development Study (TEDS) took part in a fear conditioning experiment delivered remotely via the Fear Learning and Anxiety Response (FLARe) smartphone app.In the fear acquisition phase participants were exposed to two neutral shape stimuli, one of which was repeatedly paired with a loud aversive noise, while the other was never paired with anything aversive. In the extinction phase the shapes were repeatedly presented again, this time without the aversive noise. Outcomes were participant ratings of how much they expected the aversive noise to occur when they saw either shape, throughout each phase.ResultsTwin analyses indicated a significant contribution of genetic effects to the initial acquisition and consolidation of fear, and the extinction of fear (15%, 30% and 15% respectively) with the remainder of variance due to the non-shared environment. Multivariate analyses revealed that the development of fear and fear extinction show moderate genetic overlap (genetic correlations .4-.5).ConclusionsFear acquisition and extinction are heritable, and share some, but not all of the same genetic influences.

Survival depends on the ability of animals to select the appropriate behavior in response to threat and safety sensory cues. However, the synaptic and circuit mechanisms by which the brain learns to encode accurate predictors of threat and safety remain largely unexplored. Here, we show that frontal association cortex (FrA) pyramidal neurons integrate auditory cues and basolateral amygdala (BLA) inputs non-linearly in a NMDAR-dependent manner. We found that the response of FrA pyramidal neurons was more pronounced to Gaussian noise than to pure 8 kHz tone, and that the activation of BLA-to-FrA axons was the strongest during safe periods in between conditioning pairings. Blocking BLA-to-FrA signaling specifically at the time of presentation of Gaussian noise (but not 8 kHz tone) as a safety signal impaired the formation of auditory fear memories. Taken together, our data reveal a circuit mechanism that facilitates the formation of fear traces in the FrA, thus providing a new framework for probing discriminative learning and related disorders.; Survival depends on the ability of animals to select the appropriate behavior in response to threat and safety sensory cues. However, the synaptic and circuit mechanisms by which the brain learns to encode accurate predictors of threat and safety remain largely unexplored. Here, we show that frontal association cortex (FrA) pyramidal neurons integrate auditory cues and basolateral amygdala (BLA) inputs non-linearly in a NMDAR-dependent manner. We found that the response of FrA pyramidal neurons was more pronounced to Gaussian noise than to pure 8 kHz tone, and that the activation of BLA-to-FrA axons was the strongest during safe periods in between conditioning pairings. Blocking BLA-to-FrA signaling specifically at the time of presentation of Gaussian noise (but not 8 kHz tone) as a safety signal impaired the formation of auditory fear memories. Taken together, our data reveal a circuit mechanism that facilitates the formation of fear traces in the FrA, thus providing a new framework for probing discriminative learning and related disorders.

Learning to anticipate potentially dangerous contexts is an adaptive behavioral response to coping with stressors. An animal’s stress coping style (e.g. proactive-reactive axis) is known to influence how it encodes salient events. However, the neural and molecular mechanisms underlying these stress coping style differences in learning are unknown. Further, while a number of neuroplasticity-related genes have been associated with alternative stress coping styles, it is unclear if these genes may bias the development of conditioned behavioral responses to stressful stimuli, and if so, which brain regions are involved. Here, we trained adult zebrafish to associate a naturally aversive olfactory cue with a given context. Next, we investigated if expression of two neural plasticity and neurotransmission-related genes (npas4a and gabbr1a) were associated with the contextual fear conditioning differences between proactive and reactive stress coping styles. Reactive zebrafish developed a stronger conditioned fear response and showed significantly higher npas4a expression in the medial and lateral zones of the dorsal telencephalon (Dm, Dl), and the supracommissural nucleus of the ventral telencephalon (Vs). Our findings suggest that the magnitude of expression of activity-dependent genes like npas4a may be differentially expressed across several interconnected forebrain regions in response to fearful stimuli and promote biases in fear learning among different stress coping styles.

Neural circuits in the basolateral amygdala (BLA) play a pivotal role in the learning and memory formation, and processing of emotionally salient experiences, particularly aversive ones. A diverse population of GABAergic neurons present in the BLA orchestrate local circuits to mediate emotional memory functions. Targeted manipulation of GABAergic neuronal subtypes has shed light on cell-type specific functional roles in the fear learning and memory, revealing organizing principles for the operation of inhibitory circuit motifs in the BLA.

Anxiety disorders are characterized by difficulty distinguishing safe contexts from previous or imagined threats. Conditioned fears spread beyond what is reasonable or adaptive, leading to broad and interfering anxieties when people overgeneralize their fears. Difficulties with mnemonic discrimination, a component process of memory supporting the integration of old and new experiences, may foster overgeneralization and increase risk for anxiety disorders. Individuals along a spectrum of anxiety severity (n = 117) completed a differential fear conditioning paradigm and the computerized Mnemonic Similarity Task. The task measures mnemonic discrimination by requiring individuals to differentiate between highly similar old and new entities. We predicted that low mnemonic discrimination would be associated with overgeneralization, i.e., flatter slopes of change in response to stimuli increasingly dissimilar to the conditioned stimulus. Conditional growth models showed that as expected, participants with the highest mnemonic discrimination scores also exhibited the steepest declines in fear ratings as stimuli increasingly differed from the conditioned stimulus. Results were unchanged after adjusting for recognition memory, self-reported anxiety, and clinical diagnoses and symptoms. Results support the hypothesis that memory interference (i.e., low mnemonic discrimination) could increase vulnerability for overgeneralization. Findings justify additional exploration of mnemonic discrimination and its role in anxious psychopathology.

Observational fear learning can contribute to the development of fear-related psychopathologies, such as anxiety disorders and post-traumatic stress disorder. Observational fear learning is especially relevant during childhood. Parent-child attachment and anxiety sensitivity modulate fear reactions and fear learning but their impact on observational fear learning has not been investigated. This study investigated how these factors contribute to observational fear learning in children. We examined this question among 55 healthy parent-child dyads. Children (8–12 years old) watched a video of their parent undergoing a direct fear conditioning protocol, where one stimulus (CS+Parent) was paired with a shock and one was not (CS−), and a video of a stranger for whom a different stimulus was reinforced (CS+Stranger). Subsequently, all stimuli were presented to children (without shocks) while skin conductance responses were recorded to evaluate fear levels. Our results showed that children more sensitive to anxiety and who had lower father-child relationship security levels exhibited higher skin conductance responses to the CS+Parent. Our data suggest that the father-child relationship security influences vicarious fear transmission in children who are more sensitive to anxiety. This highlights the importance of the father-child relationship security as a potential modulator of children’s vulnerability to fear-related psychopathologies.

Learning to avoid threats often occurs by observing others. Most previous research on observational fear learning (OFL) in humans has used pre-recorded standardized video of an actor and thus lacked ecological validity. Here, we aimed to enhance ecological validity of the OFL by engaging participants in a real-time observational procedure (35 pairs of healthy male friends, age 18–27). One of the participants watched the other undergo a differential fear conditioning task, in which a conditioned stimulus (CS+) was paired with an aversive electric shock and another stimulus (CS−) was always safe. Subsequently, the CS+ and CS− were presented to the observer to test the OFL. While the friend’s reactions to the shock elicited strong skin conductance responses (SCR) in all observers, subsequent differential SCRs (CS+ > CS−) were found only when declarative knowledge of the CS+/US contingency (rated by the participants) was acquired. Contingency-aware observers also showed elevated fear potentiated startle responses during both CS+ and CS− compared to baseline. We conclude that our real-time procedure can be effectively used to study OFL. The procedure allowed for dissecting two components of the OFL: an automatic emotional reaction to the response of the demonstrator and learning about stimulus contingency.

The biological mechanisms involved in fear transmission within families have been scarcely investigated in humans. Here we studied (1) how children acquired conditioned fear from observing their parent, or a stranger, being exposed to a fear conditioning paradigm, and (2) the subsequent fear extinction process in these children. Eighty-three child-parent dyads were recruited. The parent was filmed while undergoing a conditioning procedure where one cue was paired with a shock (CS + Parent) and one was not (CS −). Children (8 to 12 years old) watched this video and a video of an adult stranger who underwent conditioning with a different cue reinforced (CS + Stranger). Children were then exposed to all cues (no shocks were delivered) while skin conductance responses (SCR) were recorded. Children exhibited higher SCR to the CS + Parent and CS + Stranger relative to the CS −. Physiological synchronization between the child’s SCR during observational learning and the parent’s SCR during the actual process of fear conditioning predicted higher SCR for the child to the CS + Parent. Our data suggest that children acquire fear vicariously and this can be measured physiologically. These data lay the foundation to examine observational fear learning mechanisms that might contribute to fear and anxiety disorders transmission in clinically affected families.

Animals, including humans, use prediction error to guide learning about danger in the environment. The basolateral amygdala (BLA) is obligatory for this learning and BLA excitatory projection neurons are instructed by aversive prediction error to form fear associations. Complex networks of inhibitory interneurons, dominated by parvalbumin (PV) expressing GABAergic neurons, form the intrinsic microcircuitry of the BLA to control projection neuron activity. Whether BLA PV interneurons are also sensitive to prediction error and how they use this error to control fear learning remains unknown. We used PV cell-type specific recording and manipulation approaches in male transgenic PV-Cre rats to address these issues. We show that BLA PV neurons control fear learning about aversive events but not learning about their omission. Furthermore, during fear learning BLA PV neurons express the activity signatures of aversive prediction error: greater activity to unexpected than expected aversive events and greater activity to better rather than poorer predictors of these events. Crucially, we show that BLA PV neurons act to limit fear learning across these variations in prediction error. Together, this demonstrates that prediction error instructs and regulates BLA fear association formation in a cell-type specific manner. Whereas BLA projection neurons use prediction error signals to form and store fear associations, BLA PV interneurons use prediction error signals to constrain fear association formation.Significance StatementThe capacity to predict sources of danger in the environment is essential for survival. This capacity is supported by associative learning mechanisms that are triggered when the danger experienced is greater than the danger expected. Here we show that the activity of parvalbumin positive GABAergic interneurons in the rat basolateral amygdala neurons report this difference between the danger expected and the danger experienced and that they use this difference to limit the amount of fear which is learned.

Emerging evidence suggests that histone variants are novel epigenetic regulators of memory, whereby histone H2A.Z suppresses fear memory. However, it is not clear if altered fear memory can also modify risk for PTSD, and whether these effects differ in males and females. Using conditional-inducible H2A.Z knockout (cKO) mice, we showed that H2A.Z binding is higher in females and that H2A.Z cKO enhanced fear memory only in males. However, H2A.Z cKO improved memory on the non-aversive object-in-place task in both sexes, suggesting that H2A.Z suppresses non-stressful memory irrespective of sex. Given that risk for fear-related disorders, such as PTSD, is biased toward females, we examined whether H2A.Z cKO also has sex-specific effects on fear sensitization in the stress-enhanced fear learning (SEFL) model of PTSD, as well as associated changes in pain sensitivity. We found that H2A.Z cKO reduced stress-induced sensitization of fear learning and pain responses preferentially in female mice, indicating that the effects of H2A.Z depend on sex and the type of task, and are influenced by history of stress. These data suggest that H2A.Z may be a sex-specific epigenetic risk factor for PTSD susceptibility, with implications for developing sex-specific therapeutic interventions.

Risk for adverse outcomes, including the onset of mental illness, increases during adolescence. This increase may be linked to both new exposures, such as violence at home or in the community, or to physiological changes driven by puberty. There are significant sex differences in adolescent risk, for instance, anxiety disorders are significantly more prevalent in girls than boys. Fear learning is linked to mental health and may develop during adolescence, but the role of puberty in adolescent-specific change has not yet been systematically evaluated. We conducted a longitudinal study of fear learning that tested fear-potentiated startle (FPS) in 78 children (40 girls) aged 8-16 years. Participants completed two to three visits that included a differential fear conditioning task and self-report of both pubertal status and violence exposure. We tested for effects of sex, pubertal status, and violence exposure on FPS over time with latent growth curve models. We also examined the association between FPS and later anxiety symptoms. We found significant changes in FPS to the threat cue, but not the safety cue, across visits. Higher pubertal status was significantly associated with increased FPS to threat cues at each visit, whereas sex and violence exposure were not. FPS to threat during the baseline visit also predicted later anxiety symptoms. These findings suggest that puberty drives increased fear response to threat cues similarly for girls and boys, and that this effect may not be significantly impacted by individual differences in violence exposure during early adolescence.

Excessive fear learning and generalized, extinction-resistant fear memories are core symptoms of anxiety and trauma-related disorders. Despite significant evidence from clinical studies reporting hyperactivity of the bed nucleus of stria terminalis (BNST) under these conditions, the role of BNST in fear learning and expression is still not clarified. Here, we tested how BNST modulates fear learning in male mice using a chemogenetic approach. Activation of GABAergic neurons of BNST during fear conditioning or memory consolidation resulted in enhanced cue-related fear recall. Importantly, BNST activation had no acute impact on fear expression during conditioning or recalls, but it enhanced cue-related fear recall subsequently, potentially via altered activity of downstream regions. Enhanced fear memory consolidation could be replicated by selectively activating somatostatin (SOM), but not corticotropin-releasing factor (CRF), neurons of the BNST, which was accompanied by increased fear generalization. Our findings suggest the significant modulation of fear memory strength by specific circuits of the BNST.SIGNIFICANCE STATEMENTThe bed nucleus of stria terminalis (BNST) mediates different defensive behaviors, and its connections implicate its integrative modulatory role in fear memory formation; however, the involvement of BNST in fear learning has yet to be elucidated in detail. Our data highlight that BNST stimulation enhances fear memory formation without direct effects on fear expression. Our study identified somatostatin (SOM) cells within the extended amygdala as specific neurons promoting fear memory formation. These data underline the importance of anxiety circuits in maladaptive fear memory formation, indicating elevated BNST activity as a potential vulnerability factor to anxiety and trauma-related disorders.

The ability to recognize motivationally salient events and respond to them adaptively is critical for survival. Here we tested whether dopamine (DA) neurons in the dorsal raphe nucleus (DRN) contribute to this process. Population recordings of DRNDA neurons during associative learning tasks showed that their activity dynamically tracks salience, developing excitation to both reward- and punishment-paired cues. The DRNDA response to reward-predicting cues was diminished after satiety, suggesting modulation by internal states. DRNDA activity was also greater for unexpected outcomes than for expected outcomes. Two-photon imaging of DRNDA neurons demonstrated that the majority of individual neurons developed activation to reward-predicting cues but not to punishment-predicting cues, which was surprising and qualitatively distinct from the population results. Head-fixation during fear learning abolished the neural response to aversive cues, indicating modulation by behavioral context. Overall, these results suggest that DRNDA neurons encode motivational salience, dependent on internal and external factors.

Early life stress (ELS) experiences can cause changes in cognitive and affective functioning. This study examined the persistent effects of a single traumatic event in infancy on several adult behavioral outcomes in male and female C57BL/6J mice. Mice received 15 footshocks in infancy and were tested for stress-enhanced fear learning, extinction learning, discrimination and reversal learning, and novel object recognition. Infant trauma potentiated fear learning in adulthood and produced resistance to extinction but did not influence other behaviors, suggesting restricted effects of infant trauma on behaviors reliant on cortico-amygdala circuitry.

The perirhinal cortex (PER) receives multimodal and unimodal sensory information from all modalities. In addition, the PER is anatomically connected with several brain regions that support fear learning. Several studies suggest that the PER is involved in fear conditioning to discontinuous auditory cues but not to continuous auditory cues. To date, studies examining the role of the PER in fear conditioning has largely focused on auditory and contextual stimuli. The present study assessed whether the role of the PER in fear conditioning would extend to visual modalities. Rodents were randomly assigned to one of four conditioned stimuli, which consisted of either a tone or a light stimulus that was either continuous or discontinuous. Pre-training excitotoxic lesions to the PER significantly reduced freezing to auditory and visual cues during the acquisition phase regardless of stimulus continuity. During subsequent testing, perirhinal lesions produced significant decreases in freezing levels to both continuous and discontinuous tones but not to either of the light CS groups. These results suggest that the PER is involved in the acquisition of fear across multiple cue modalities. However, the PER may have a more limited role in the retrieval of the fear memory dependent upon the cue modality.