Threat Memory Research Articles

Stress disrupts engram ensembles in lateral amygdala to generalize threat memory in mice.

Stress induces aversive memory overgeneralization, a hallmark of many psychiatric disorders. Memories are encoded by a sparse ensemble of neurons active during an event (an engram ensemble). We examined the molecular and circuit processes mediating stress-induced threat memory overgeneralization in mice. Stress, acting via corticosterone, increased the density of engram ensembles supporting a threat memory in lateral amygdala, and this engram ensemble was reactivated by both specific and non-specific retrieval cues (generalized threat memory). Furthermore, we identified a critical role for endocannabinoids, acting retrogradely on parvalbumin-positive (PV+) lateral amygdala interneurons in the formation of a less-sparse engram and memory generalization induced by stress. Glucocorticoid receptor antagonists, endocannabinoid synthesis inhibitors, increasing PV+ neuronal activity, and knocking down cannabinoid receptors in lateral amygdala PV+ neurons restored threat memory specificity and a sparse engram in stressed mice. These findings offer insights into stress-induced memory alterations, providing potential therapeutic avenues for stress-related disorders.

Brain imaging and machine learning reveal uncoupled functional network for contextual threat memory in long sepsis.

Positron emission tomography (PET) utilizes radiotracers like [18F]fluorodeoxyglucose (FDG) to measure brain activity in health and disease. Performing behavioral tasks between the FDG injection and the PET scan allows the FDG signal to reflect task-related brain networks. Building on this principle, we introduce an approach called behavioral task-associated PET (beta-PET) consisting of two scans: the first after a mouse is familiarized with a conditioning chamber, and the second upon recall of contextual threat. Associative threat conditioning occurs between scans. Beta-PET focuses on brain regions encoding threat memory (e.g., amygdala, prefrontal cortex) and contextual aspects (e.g., hippocampus, subiculum, entorhinal cortex). Our results show that beta-PET identifies a biologically defined network encoding contextual threat memory and its uncoupling in a mouse model of long sepsis. Moreover, machine learning algorithms (linear logistic regression) and ordinal trends analysis demonstrate that beta-PET robustly predicts the behavioral defense response and its breakdown during long sepsis.

Brain imaging and machine learning reveal uncoupled functional network for contextual threat memory in long sepsis.

Positron emission tomography (PET) is a highly sensitive tool for studying physiology and metabolism through positron-emitting radionuclides that label molecular targets in the body with unparalleled specificity, without disturbing their biological function. Here, we introduce a small-animal technique called behavioral task-associated PET (beta-PET) consisting of two scans: the first after a mouse is familiarized with a conditioning chamber, and the second upon recall of contextual threat. Associative threat conditioning occurs between the scans. Beta-PET focuses on brain regions encoding threat memory (e.g., amygdala, prefrontal cortex) and contextual aspects (e.g., hippocampus, subiculum, entorhinal cortex). Our results show that beta-PET identifies a biologically defined functional network encoding contextual threat memory and its uncoupling in a mouse model of long sepsis. Moreover, machine learning algorithms (linear logistic regression) and ordinal trends analysis demonstrate that beta-PET robustly predicts the behavioral defense response and its breakdown during long sepsis.

Handholding reduces the recovery of threat memories and magnifies prefrontal hemodynamic responses

Human touch is a powerful means of social and affective regulation, promoting safety behaviors. Yet, despite its importance across human contexts, it remains unknown how touch can promote the learning of new safety memories and what neural processes underlie such effects. The current study used measures of peripheral physiology and brain activity to examine the effects of interpersonal touch during safety learning (extinction) on the recovery of previously learned threat. We observed that handholding during extinction significantly reduced threat recovery, which was reflected in enhanced prefrontal hemodynamic responses. This effect was absent when learners were instructed to hold a rubber ball, independent of the presence of their partners. Our findings indicate that social touch contributes to safety learning, potentially influencing threat memories via prefrontal circuitry.

Semantic structures facilitate threat memory integration throughout the medial temporal lobe and medial prefrontal cortex

Emotional experiences can profoundly impact our conceptual model of the world, modifying how we represent and remember a host of information even indirectly associated with that experienced in the past. Yet, how a new emotional experience infiltrates and spreads across pre-existing semantic knowledge structures (e.g., categories) is unknown. We used a modified aversive sensory preconditioning paradigm in fMRI (n= 35) to investigate whether threat memories integrate with a pre-established category to alter the representation of the entire category. We observed selective but transient changes in the representation of conceptually related items in the amygdala, medial prefrontal cortex, and occipitotemporal cortex following threat conditioning to a simple cue (geometric shape) pre-associated with a different, but related, set of category exemplars. These representational changes persisted beyond 24h in the hippocampus and perirhinal cortex. Reactivation of the semantic category during threat conditioning, combined with activation of the hippocampus or medial prefrontal cortex, was predictive of subsequent amygdala reactivity toward novel category members at test. This provides evidence for online integration of emotional experiences into semantic categories, which then promotes threat generalization. Behaviorally, threat conditioning by proxy selectively and retroactively enhanced recognition memory and increased the perceived typicality of the semantic category indirectly associated with threat. These findings detail a complex route through which new emotional learning generalizes by modifying semantic structures built up over time and stored in memory as conceptual knowledge.

Higher-order interactions between hippocampal CA1 neurons are disrupted in amnestic mice.

Across systems, higher-order interactions between components govern emergent dynamics. Here we tested whether contextual threat memory retrieval in mice relies on higher-order interactions between dorsal CA1 hippocampal neurons requiring learning-induced dendritic spine plasticity. We compared population-level Ca2+ transients as wild-type mice (with intact learning-induced spine plasticity and memory) and amnestic mice (TgCRND8 mice with high levels of amyloid-β and deficits in learning-induced spine plasticity and memory) were tested for memory. Using machine-learning classifiers with different capacities to use input data with complex interactions, our findings indicate complex neuronal interactions in the memory representation of wild-type, but not amnestic, mice. Moreover, a peptide that partially restored learning-induced spine plasticity also restored the statistical complexity of the memory representation and memory behavior in Tg mice. These findings provide a previously missing bridge between levels of analysis in memory research, linking receptors, spines, higher-order neuronal dynamics and behavior.

Medial anterior prefrontal cortex stimulation downregulates implicit reactions to threats and prevents the return of fear.

Downregulating emotional overreactions toward threats is fundamental for developing treatments for anxiety and post-traumatic disorders. The prefrontal cortex (PFC) is critical for top-down modulatory processes, and despite previous studies adopting repetitive transcranial magnetic stimulation (rTMS) over this region provided encouraging results in enhancing extinction, no studies have hitherto explored the effects of stimulating the medial anterior PFC (aPFC, encompassing the Brodmann area 10) on threat memory and generalization. Here we showed that rTMS over the aPFC applied before threat memory retrieval immediately decreases implicit reactions to learned and novel stimuli in humans. These effects enduringly persisted 1 week later in the absence of rTMS. No effects were detected on explicit recognition. Critically, rTMS over the aPFC resulted in a more pronounced reduction of defensive responses compared to rTMS targeting the dorsolateral PFC. These findings reveal a previously unexplored prefrontal region, the modulation of which can efficiently and durably inhibit implicit reactions to learned threats. This represents a significant advancement toward the long-term deactivation of exaggerated responses to threats.

Sleep deprivation increases the generalization of perceptual and concept-based fear: An fNIRS study

Insufficient sleep can initiate or exacerbate anxiety by triggering excessive fear generalization. In this study, a de novo paradigm was developed and used to examine the neural mechanisms governing the effects of sleep deprivation on processing perceptual and concept-based fear generalizations. A between-subject design was adopted, wherein a control group (who had a typical night's sleep) and a one-night sleep deprivation group completed a fear acquisition task at 9:00 PM on the first day and underwent a generalization test the following morning at 7:00 AM. In the fear acquisition task, navy blue and olive green were used as perceptual cues (P+ and P−, respectively), while animals and furniture items were used as conceptual cues (C+ and C−, respectively). Generalization was tested for four novel generalized categories (C+P+, C+P−, C−P+, and C−P−). Shock expectancy ratings, skin conductance responses, and functional near-infrared spectroscopy were recorded during the fear acquisition and generalization processes. Compared with the group who had a typical night's sleep, the sleep deprived group showed higher shock expectancy ratings (especially for P+ and C−), increased oxygenated hemoglobin in the dorsolateral prefrontal cortex, and increased activation in the triangular inferior frontal gyrus during the generalization test. These findings suggest that sleep deprivation increases the generalization of threat memories, thus providing insights into the overgeneralization characteristics of anxiety and fear-related disorders.

Comparing behaviours induced by natural memory retrieval and optogenetic reactivation of an engram ensemble in mice.

Memories are thought to be stored within sparse collections of neurons known as engram ensembles. Neurons active during a training episode are allocated to an engram ensemble ('engram neurons'). Memory retrieval is initiated by external sensory or internal cues present at the time of training reactivating engram neurons. Interestingly, optogenetic reactivation of engram ensemble neurons alone in the absence of external sensory cues is sufficient to induce behaviour consistent with memory retrieval in mice. However, there may exist differences between the behaviours induced by natural retrieval cues or artificial engram reactivation. Here, we compared two defensive behaviours (freezing and the syllable structure of ultrasonic vocalizations, USVs) induced by sensory cues present at training (natural memory retrieval) and optogenetic engram ensemble reactivation (artificial memory retrieval) in a threat conditioning paradigm in the same mice. During natural memory recall, we observed a strong positive correlation between freezing levels and distinct USV syllable features (characterized by an unsupervised algorithm, MUPET (Mouse Ultrasonic Profile ExTraction)). Moreover, we observed strikingly similar behavioural profiles in terms of freezing and USV characteristics between natural memory recall and artificial memory recall in the absence of sensory retrieval cues. Although our analysis focused on two behavioural measures of threat memory (freezing and USV characteristics), these results underscore the similarities between threat memory recall triggered naturally and through optogenetic reactivation of engram ensembles. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

‘Nip it in the bud’: Low-frequency rTMS of the prefrontal cortex disrupts threat memory consolidation in humans

It is still unclear how the human brain consolidates aversive (e.g., traumatic) memories and whether this process can be disrupted. We hypothesized that the dorsolateral prefrontal cortex (dlPFC) is crucially involved in threat memory consolidation. To test this, we used low-frequency repetitive transcranial magnetic stimulation (LF-rTMS) within the memory stabilization time window to disrupt the expression of threat memory. We combined a differential threat-conditioning paradigm with LF-rTMS targeting the dlPFC in the critical condition, and occipital cortex stimulation, delayed dlPFC stimulation, and sham stimulation as control conditions. In the critical condition, defensive reactions to threat were reduced immediately after brain stimulation, and 1 h and 24 h later. In stark contrast, no decrease was observed in the control conditions, thus showing both the anatomical and temporal specificity of our intervention. We provide causal evidence that selectively targeting the dlPFC within the early consolidation period prevents the persistence and return of conditioned responses. Furthermore, memory disruption lasted longer than the inhibitory window created by our TMS protocol, which suggests that we influenced dlPFC neural activity and hampered the underlying, time-dependent consolidation process. These results provide important insights for future clinical applications aimed at interfering with the consolidation of aversive, threat-related memories.

Excitability mediates allocation of pre-configured ensembles to a hippocampal engram supporting contextual conditioned threat in mice

Little is understood about how engrams, sparse groups of neurons that store memories, are formed endogenously. Here, we combined calcium imaging, activity tagging, and optogenetics to examine the role of neuronal excitability and pre-existing functional connectivity on the allocation of mouse cornu ammonis area 1 (CA1) hippocampal neurons to an engram ensemble supporting a contextual threat memory. Engram neurons (high activity during recall or TRAP2-tagged during training) were more active than non-engram neurons 3h (but not 24h to 5days) before training. Consistent with this, optogenetically inhibiting scFLARE2-tagged neurons active in homecage 3 h, but not 24 h, before conditioning disrupted memory retrieval, indicating that neurons with higher pre-training excitability were allocated to the engram. We also observed stable pre-configured functionally connected sub-ensembles of neurons whose activity cycled over days. Sub-ensembles that were more active before training were allocated to the engram, and their functional connectivity increased at training. Therefore, both neuronal excitability and pre-configured functional connectivity mediate allocation to an engram ensemble.

The ventral hippocampus is activated in olfactory but not auditory threat memory.

Hippocampal networks required for associative memory formation are involved in cue- and context-dependent threat conditioning. The hippocampus is functionally heterogeneous at its dorsal and ventral poles, and recent investigations have focused on the specific roles required from each sub-region for associative conditioning. Cumulative evidence suggests that contextual and emotional information is processed by the dorsal and ventral hippocampus, respectively. However, it is not well understood how these two divisions engage in threat conditioning with cues of different sensory modalities. Here, we compare the involvement of the dorsal and ventral hippocampus in two types of threat conditioning: olfactory and auditory. Our results suggest that the dorsal hippocampus encodes contextual information and is activated upon recall of an olfactory threat memory only if contextual cues are relevant to the threat. Overnight habituation to the context eliminates dorsal hippocampal activation, implying that this area does not directly support cue-dependent threat conditioning. The ventral hippocampus is activated upon recall of olfactory, but not auditory, threat memory regardless of habituation duration. Concurrent activation of the piriform cortex is consistent with its direct connection with the ventral hippocampus. Together, our study suggests a unique role of the ventral hippocampus in olfactory threat conditioning.

Functionally refined encoding of threat memory by distinct populations of basal forebrain cholinergic projection neurons.

Neurons of the basal forebrain nucleus basalis and posterior substantia innominata (NBM/SIp) comprise the major source of cholinergic input to the basolateral amygdala (BLA). Using a genetically encoded acetylcholine (ACh) sensor in mice, we demonstrate that BLA-projecting cholinergic neurons can 'learn' the association between a naive tone and a foot shock (training) and release ACh in the BLA in response to the conditioned tone 24 hr later (recall). In the NBM/SIp cholinergic neurons express the immediate early gene, Fos following both training and memory recall. Cholinergic neurons that express Fos following memory recall display increased intrinsic excitability. Chemogenetic silencing of these learning-activated cholinergic neurons prevents expression of the defensive behavior to the tone. In contrast, we show that NBM/SIp cholinergic neurons are not activated by an innately threatening stimulus (predator odor). Instead, VP/SIa cholinergic neurons are activated and contribute to defensive behaviors in response to predator odor, an innately threatening stimulus. Taken together, we find that distinct populations of cholinergic neurons are recruited to signal distinct aversive stimuli, demonstrating functionally refined organization of specific types of memory within the cholinergic basal forebrain of mice.

Olfactory threat extinction in the piriform cortex: An age-dependent employment of NMDA receptor-dependent long-term depression.

Extinction of threat memory is a measure of behavioral flexibility. In the absence of additional reinforcement, the extinction of learned behaviors allows animals and humans to adapt to their changing environment. Extinction mechanisms and their therapeutic implications for maladaptive learning have been extensively studied. However, how aging affects extinction learning is much less understood. Using a rat model of olfactory threat extinction, we show that the extinction of olfactory threat memory is impaired in aged Sprague-Darley rats. Following extinction training, long-term depression (LTD) in the piriform cortex (PC) was inducible ex vivo in aged rats and was NMDA receptor (NMDAR)-independent. On the other hand, adult rats acquired successful olfactory threat extinction, and LTD was not inducible following extinction training. Neuronal cFos activation in the posterior PC correlated with learning and extinction performance in rats. NMDAR blockade either systemically or locally in the PC during extinction training prevented successful extinction in adult rats, following which NMDAR-dependent LTD became inducible ex vivo. This suggests that extinction learning employs NMDAR-dependent LTD mechanisms in the PC of adult rats, thus occluding further LTD induction ex vivo. The rescue of olfactory threat extinction in aged rats by D-cycloserine, a partial NMDAR agonist, suggests that the impairment in olfactory threat extinction of aged animals may relate to NMDAR hypofunctioning and a lack of NMDAR-dependent LTD. These findings are consistent with an age-related switch from NMDAR-dependent to NMDAR-independent LTD in the PC. Optimizing NMDAR function in sensory cortices may improve learning and flexible behavior in the aged population.

Sex differences in avoidance behavior and cued threat memory dynamics in mice: Interactions between estrous cycle and genetic background

Anxiety disorders are the most prevalent mental illnesses worldwide, exhibit high heritability, and affect twice as many women as men. To evaluate potential interactions between genetic background and cycling ovarian hormones on sex differences in susceptibility to negative valence behaviors relevant to anxiety disorders, we assayed avoidance behavior and cued threat memory dynamics in gonadally-intact adult male and female mice across four common inbred mouse strains: C57Bl/6J, 129S1/SVlmJ, DBA/2J, and BALB/cJ. Independent of sex, C57Bl/6J mice exhibited low avoidance but high threat memory, 129S1/SvlmJ mice high avoidance and high threat memory, DBA/2J mice low avoidance and low threat memory, and BALB/cJ mice high avoidance but low threat memory. Within-strain comparisons revealed reduced avoidance behavior in the high hormone phase of the estrous cycle (proestrus) compared to all other estrous phases in all strains except DBA/2J, which did not exhibit cycle-dependent behavioral fluctuations. Robust and opposing sex differences in threat conditioning and extinction training were found in the C57Bl/6J and 129S1/SvlmJ lines, whereas no sex differences were observed in the DBA/2J or BALB/cJ lines. C57Bl/6J males exhibited enhanced acute threat memory, whereas 129S1/SvlmJ females exhibited enhanced sustained threat memory, compared to their sex-matched littermates. These effects were not mediated by estrous cycle stage or sex differences in active versus passive defensive behavioral responses. Our data demonstrate that core features of behavioral endophenotypes relevant to anxiety disorders, such as avoidance and threat memory, are genetically driven yet dissociable and can be influenced further by cycling ovarian hormones.

Potentiation of cholinergic and corticofugal inputs to the lateral amygdala in threat learning

The amygdala, cholinergic basal forebrain, and higher-order auditory cortex (HO-AC) regulate brain-wide plasticity underlying auditory threat learning. Here, we perform multi-regional extracellular recordings and optical measurements of acetylcholine (ACh) release to characterize the development of discriminative plasticity within and between these brain regions as mice acquire and recall auditory threat memories. Spiking responses are potentiated for sounds paired with shock (CS+) in the lateral amygdala (LA) and optogenetically identified corticoamygdalar projection neurons, although not in neighboring HO-AC units. Spike- or optogenetically triggered local field potentials reveal enhanced corticofugal-but not corticopetal-functional coupling between HO-AC and LA during threat memory recall that is correlated with pupil-indexed memory strength. We also note robust sound-evoked ACh release that rapidly potentiates for the CS+ in LA but habituates across sessions in HO-AC. These findings highlight a distributed and cooperative plasticity in LA inputs as mice learn to reappraise neutral stimuli as possible threats.

The Basolateral Amygdala: The Core of a Network for Threat Conditioning, Extinction, and Second-Order Threat Conditioning.

Threat conditioning, extinction, and second-order threat conditioning studied in animal models provide insight into the brain-based mechanisms of fear- and anxiety-related disorders and their treatment. Much attention has been paid to the role of the basolateral amygdala (BLA) in such processes, an overview of which is presented in this review. More recent evidence suggests that the BLA serves as the core of a greater network of structures in these forms of learning, including associative and sensory cortices. The BLA is importantly regulated by hippocampal and prefrontal inputs, as well as by the catecholaminergic neuromodulators, norepinephrine and dopamine, that may provide important prediction-error or learning signals for these forms of learning. The sensory cortices may be required for the long-term storage of threat memories. As such, future research may further investigate the potential of the sensory cortices for the long-term storage of extinction and second-order conditioning memories.

Sex differences in socioemotional behavior and changes in ventral hippocampal transcription across aging in C57Bl/6J mice

Socioemotional health is positively correlated with improved cognitive and physical aging. Despite known sex differences in socioemotional behaviors and the trajectory of aging, the interactive effects between sex and aging on socioemotional outcomes are poorly understood. We performed the most comprehensive assessment of sex differences in socioemotional behaviors in C57Bl/6J mice across aging to date. Compared to males, females exhibited decreased anxiety-like behavior and social preference but increased social recognition. With age, anxiety-like behavior, cued threat memory generalization, and social preference increased in both sexes. To investigate potential neural mechanisms underlying these behavioral changes, we analyzed transcriptional neuropathology markers in the ventral hippocampus and found age-related changes in genes related to activated microglia, angiogenesis, and cytokines. Sex differences emerged in the timing, direction, and magnitude of these changes, independent of reproductive senescence in aged females. Interestingly, female-specific upregulation of autophagy-related genes correlated with age-related behavioral changes selectively in females. These novel findings reveal critical sex differences in trajectories of ventral hippocampal aging that may contribute to sex- and age-related differences in socioemotional outcomes.

Evidence for Different Roles of Inhibitory and Prospective Intolerance of Uncertainty During Threat Discrimination Learning

Uncertainty is a core component of threat and associated learning processes. One methodological factor impacting uncertainty in threat learning paradigms is the threat reinforcement rate, which refers to the proportion of times a cue is reinforced with an aversive stimulus. This study tested the effect of partial vs continuous threat reinforcement on threat / safety discrimination learning, as indexed by skin conductance response (SCR). Using a within-participants design, fifty-nine participants completed a task in which three colored shapes were paired with electric shock at reinforcement schedules of 100% (CS+), 50% (CS+) and 0% (CS-). In addition, the study examined the relationship between the Intolerance of Uncertainty scale (IU) and two subscales – inhibitory and prospective IU – with threat discrimination learning. The data show heightened SCR in the continuous vs partial reinforcement condition to all stimuli, but limited evidence of enhanced discrimination learning. Furthermore, no association was observed between total IU score and threat-safety discrimination. However, using a two-factor model of IU, findings showed higher inhibitory IU and higher prospective IU were associated with diminished and heightened threat discrimination, respectively. These results contribute to a fast-growing literature exploring how the uncertainty inherent to predictors of threat, individual differences in sensitivity to uncertainty, and interactions between these two factors, can shape the acquisition of threat memory.

Threat Memory in the Sensory Cortex: Insights from Olfaction

The amygdala has long held the center seat in the neural basis of threat conditioning. However, a rapidly growing literature has elucidated extra-amygdala circuits in this process, highlighting the sensory cortex for its critical role in the mnemonic aspect of the process. While this literature is largely focused on the auditory system, substantial human and rodent findings on the olfactory system have emerged. The unique nature of the olfactory neuroanatomy and its intimate association with emotion compels a review of this recent literature to illuminate its special contribution to threat memory. Here, integrating recent evidence in humans and animal models, we posit that the olfactory (piriform) cortex is a primary and necessary component of the distributed threat memory network, supporting mnemonic ensemble coding of acquired threat. We further highlight the basic circuit architecture of the piriform cortex characterized by distributed, auto-associative connections, which is prime for highly efficient content-addressable memory computing to support threat memory. Given the primordial role of the piriform cortex in cortical evolution and its simple, well-defined circuits, we propose that olfaction can be a model system for understanding (transmodal) sensory cortical mechanisms underlying threat memory.