Contextual Fear Generalization Research Articles

GABAergic interneurons in the hippocampal CA1 mediate contextual fear generalization in PTSD rats.

Fear overgeneralization is widely accepted as a pathogenic marker of post-traumatic stress disorder (PTSD). Recently, GABAergic interneurons have been regarded as key players in the regulation of fear memory. The role of hippocampal GABAergic interneurons in contextual fear generalization of PTSD remains incompletely understood. In the present study, we established a rat model of PTSD with inescapable foot shocks (IFS) and observed the loss of GABAergic interneuron phenotype in the hippocampal cornu ammonis-1 (CA1) subfield. To determine whether the loss of GABAergic interneuron phenotype was associated with fear generalization in PTSD rats, we used adeno-associated virus (AAV) to reduce the expression of GAD67 in CA1 and observed its effect on fear generalization. The results showed that the reduction of GAD67 in CA1 enhanced contextual fear generalization in rats. We investigated whether the PERK pathway was involved in the GABAergic interneuron injury. Increased expression of p-PERK, CHOP, and Caspase12 in GABAergic interneurons of PTSD rats was observed. Then, we used salubrinal, an endoplasmic reticulum stress inhibitor, to modulate the PERK pathway. The salubrinal treatment significantly protected the GABAergic interneurons and relieved fear generalization in PTSD rats. In addition, the results showed that salubrinal down-regulated the expression of CHOP and Caspase12 in GABAergic interneurons of PTSD rats. In conclusion, this study provided evidence that the loss of GABAergic interneuron phenotype in CA1 may contribute to contextual fear generalization in PTSD. The PERK pathway is involved in the GABAergic interneuron injury of PTSD rats and modulating it can protect GABAergic interneurons and constrain contextual fear generalization.

Emotional Value of Fear Memory and the Role of the Ventral Hippocampus in Systems Consolidation

Recent studies have explored the circuitry involving the ventral hippocampus (vHPC), the amygdala, and the prefrontal cortex, a pathway mainly activated to store contextual information efficiently. Lesions in the vHPC impair remote memory, but not in the short term. However, how the vHPC is affected by distinct memory strength or its role in systems consolidation has not yet been elucidated. Here, we investigated how distinct training intensities, with strong or weak contextual fear conditioning, affect activation of the dorsal hippocampus (dHPC) and the vHPC. We found that the time course of memory consolidation differs in fear memories of different training intensities in both the dHPC and vHPC. Our results also indicate that memory generalization happens alongside greater activation of the vHPC, and these processes occur faster with stronger fear memories. The vHPC is required for the expression of remote fear memory and may control contextual fear generalization, a view corroborated by the fact that inactivation of the vHPC suppresses generalized fear expression, making memory more precise again. Systems consolidation occurs concomitantly with greater activation of the vHPC, which is accelerated in stronger fear memories. These findings lead us to propose that greater activation of the vHPC could be used as a marker for memory generalization.

Adenosine A2A receptors control generalization of contextual fear in rats

Fear learning is essential to survival, but traumatic events may lead to abnormal fear consolidation and overgeneralization, triggering fear responses in safe environments, as occurs in post-traumatic stress disorder (PTSD). Adenosine A2A receptors (A2AR) control emotional memory and fear conditioning, but it is not known if they affect the consolidation and generalization of fear, which was now investigated. We now report that A2AR blockade through systemic administration of the A2AR antagonist SCH58261 immediately after contextual fear conditioning (within the consolidation window), accelerated fear generalization. Conversely, A2AR activation with CGS21680 decreased fear generalization. Ex vivo electrophysiological recordings of field excitatory post-synaptic potentials (fEPSPs) in CA3-CA1 synapses and of population spikes in the lateral amygdala (LA), showed that the effect of SCH58261 is associated with a reversion of fear conditioning-induced decrease of long-term potentiation (LTP) in the dorsal hippocampus (DH) and with increased amplitude of LA LTP in conditioned animals. These data suggest that A2AR are engaged during contextual fear consolidation, controlling long-term potentiation mechanisms in both DH and LA during fear consolidation, impacting on fear generalization; this supports targeting A2AR during fear consolidation to control aberrant fear processing in PTSD and other fear-related disorders.

CB1R blockade unmasks TRPV1-mediated contextual fear generalization in female, but not male rats.

Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, but many of the specific mechanisms underlying these sex differences have not been fully defined. Here we investigated potential sex differences in endocannabinoid (eCB) modulation of Pavlovian fear conditioning and extinction, examining multiple defensive behaviors, including shock responsivity, conditioned freezing, and conditioned darting. We found that while systemic administration of drugs acting on eCB receptors did not influence the occurrence of darting, females that were classified as Darters responded differently to the drug administration than those classified as Non-darters. Most notably, CB1R antagonist AM251 produced an increase in cue-elicited freezing and context generalization selectively in female Non-darters that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. To identify a potential synaptic mechanism for these sex differences, we next employed biochemical and neuroanatomical tracing techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, focusing on the ventral hippocampus (vHip) given its known role in mediating contextual fear generalization. These assays identified sex-specific effects of both fear conditioning-elicited AEA release and vHip-BLA circuit structure. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked.

Generalization of contextual fear is sex-specifically affected by high salt intake.

A hallmark symptom of many anxiety disorders, and multiple neuropsychiatric disorders more broadly, is generalization of fearful responses to non-fearful stimuli. Anxiety disorders are often comorbid with cardiovascular diseases. One established, and modifiable, risk factor for cardiovascular diseases is salt intake. Yet, investigations into how excess salt consumption affects anxiety-relevant behaviors remains little explored. Moreover, no studies have yet assessed how high salt intake influences generalization of fear. Here, we used adult C57BL/6J mice of both sexes to evaluate the influence of two or six weeks of high salt consumption (4.0% NaCl), compared to controls (0.4% NaCl), on contextual fear acquisition, expression, and generalization. Further, we measured osmotic and physiological stress by quantifying serum osmolality and corticosterone levels, respectively. Consuming excess salt did not influence contextual fear acquisition nor discrimination between the context used for training and a novel, neutral context when training occurred 48 prior to testing. However, when a four week delay between training and testing was employed to induce natural fear generalization processes, we found that high salt intake selectively increases contextual fear generalization in females, but the same diet reduces contextual fear generalization in males. These sex-specific effects were independent of any changes in serum osmolality nor corticosterone levels, suggesting the behavioral shifts are a consequence of more subtle, neurophysiologic changes. This is the first evidence of salt consumption influencing contextual fear generalization, and adds information about sex-specific effects of salt that are largely missing from current literature.

Ventral hippocampal diacylglycerol lipase-alpha deletion decreases avoidance behaviors and alters excitation-inhibition balance

The endogenous cannabinoid, 2-arachidonoylglycerol (2-AG), plays a key role in the regulation of anxiety- and stress-related behavioral phenotypes and may represent a novel target for the treatment of anxiety disorders. However, recent studies have suggested a more complex role for 2-AG signaling in the regulation of stress responsivity, including increases in acute fear responses after 2-AG augmentation under some conditions. Thus, 2-AG signaling within distinct brain regions and circuits could regulate anxiety-like behavior and stress responsivity in opposing manners. The ventral hippocampus (vHPC) is a critical region for emotional processing, anxiety-like behaviors, and stress responding. Here, we use a conditional knock-out of the 2-AG synthesis enzyme, diacylglycerol lipase α (DAGLα), to study the role of vHPC 2-AG signaling in the regulation of affective behavior. We show that vHPC DAGLα deletion decreases avoidance behaviors both basally and following an acute stress exposure. Genetic deletion of vHPC DAGLα also promotes stress resiliency, with no effect on fear acquisition, expression, or contextual fear generalization. Using slice electrophysiology, we demonstrate that vHPC DAGLα deletion shifts vHPC activity towards enhanced inhibition. Together, these data indicate endogenous 2-AG signaling in the vHPC promotes avoidance and increases stress reactivity, confirming the notion that 2-AG signaling within distinct brain regions may exert divergent effects on anxiety states and stress adaptability.

Sex differences in contextual fear learning and generalization: a behavioral and computational analysis of hippocampal functioning.

There are sex differences in anxiety disorders with regard to occurrence and severity of episodes such that females tend to experience more frequent and more severe episodes. Contextual fear learning and generalization are especially relevant to anxiety disorders, which are often defined by expressing fear and/or anxiety in safe contexts. In contextual fear conditioning, a representation of the context must first be created, and then that representation must be paired with an aversive consequence. With some variation, the experiments presented here use a 3-d procedure in which day 1 consists of pre-exposure to the to-be-shocked context, day 2 consists of a single context-shock pairing after some placement-to-shock interval (PSI), and day 3 consists of testing in either the same or a novel context. With shorter pre-exposure periods, male rats showed more contextual fear, consistent with previous literature; however, after longer pre-exposure periods, female rats showed greater contextual fear. Additionally, while pre-exposure and PSI are both periods of time prior to the shock, it was found that they were not equivalent to each other. Animals with 120 sec of pre-exposure and a 30-sec PSI show a differential level and time course of fear expression than animals who received no pre-exposure and a 150-sec PSI, and this further depended on sex of the rat. Additionally, an experiment comparing recently versus remotely acquired contextual fear was run. Males were again shown to have greater contextual fear at both time points, and this contextual fear incubated/increased over time in males but not females. To facilitate identification of what processes caused sex differences, we used BaconX, a conceptual and computational model of hippocampal contextual learning. Computational simulations using this model predicted many of our key findings. Furthermore, these simulations suggest potential mechanisms with regard to hippocampal computation; namely, an increased feature sampling rate in males, which may account for the sex differences presented here and in prior literature.

The effects of excess salt consumption on contextual fear generalization in mice

Excess salt (NaCl) consumption is prevalent worldwide. Most Americans consume more than twice the daily recommended amount of sodium (Na; 2300mg). Excessive salt intake is a risk factor for cardiovascular pathology such as hypertension. Anxiety is often comorbid with cardiovascular diseases. Research has been done to examine the relationship between salt consumption and cardiovascular and other related peripheral diseases. However, far less research has been done to examine the relationship between salt consumption and brain health or anxiety. To examine this, mice were used because their salt intake and stress exposure can be rigorously controlled, unlike in humans. Specifically, we focused on fear generalization, a key characteristic of anxiety symptoms involving contextually inappropriate fear of situations not currently present. We hypothesized that mice consuming a diet with excess salt would exhibit higher levels of fear generalization relative to mice consuming a low salt diet. Adult (9 wk old) male and female C57BL/6J mice were given either a low (0.4% NaCl, w/w) or high (4.0% NaCl) salt diet ad libitum. Mice underwent contextual fear training 2 or 6 wks after commencing diet manipulation. Testing for contextually appropriate (fear expression) and contextually inappropriate (fear generalization) fear behavior occurred either 2 days (2 and 6 wk groups) or 4 wks (6 wk group) after training. Regardless of diet condition, mice of both sexes acquired contextual fear similarly when trained at the 2 wk timepoint, and exhibited equivalent fear expression and contextual discrimination when tested 2 days later. Similarly, and as expected, male mice consuming a low salt diet expressed normal levels of fear generalization at 4 wks. Contrary to our hypothesis, however, male mice consuming a high salt diet did not exhibit fear generalization at 4 wks. Female tests of 4 wk fear generalization, and both sexes in the 6 wk group being tested after 2 days, are ongoing. Thus far, our findings indicate that male mice consuming a high salt diet do not generalize their contextual fear after a standard time delay (4 wks), or alternatively display enhanced contextual discrimination at the 4 wk timepoint post‐training. Our findings suggest intake of salt, a non‐caloric dietary component, can have a significant effect on a measure directly related to anxiety symptoms. Future experiments will evaluate the contribution of neuroinflammation to these behavioral changes, as well as investigate cued fear responses in both sexes.

Anterior cingulate cortex projections to the dorsal hippocampus positively control the expression of contextual fear generalization.

Fear generalization is one of the main symptoms of posttraumatic stress disorder. In rodents, the anterior cingulate cortex (ACC) and the hippocampus (HPC) control the expression of contextual fear memory generalization. Consistently, ACC projections to the ventral HPC contribute to contextual fear generalization. However, the roles of ACC projections to the dorsal HPC (dHPC) in fear generalization are unclear, although the dHPC is required for the retrieval of recent contextual fear memory. To investigate these roles, we examined the effects of optogenetic silencing and stimulation of these projections in contextual fear generalization at the recent and remote time points. Mice underwent contextual fear conditioning and, at 1 or 28 d later, were tested in the conditioned chamber, a novel context, or a similar context. Optogenetic activation of these projections induced higher freezing in mice in the novel context compared with the control group at a recent (1-d), but not remote (28-d), time point following conditioning, suggesting that activation of this pathway enhances contextual fear generalization. In contrast, optogenetic inactivation of these projections induced lower freezing in the similar context compared with the control group at a recent, but not remote, time point, suggesting that inactivation of this pathway impaired contextual fear generalization. These observations suggest that the ACC to the dHPC projections positively regulate the expression of contextual fear generalization when contextual fear memory is recent.

Fear learning, avoidance, and generalization are more context-dependent for adults than adolescents

This study examined developmental differences in contextual and perceptual generalization of fear and avoidance learning. Adults (N = 39) and adolescents (N = 44) completed differential fear acquisition wherein each conditional stimulus (CS) appeared in a background context. In the dangerous context, one stimulus (CS+) predicted an aversive sound, and the other stimulus (CS-) did not. In the safe context, the aversive sound was never administered with either CS. During fear generalization, participants were presented with three generalization stimuli (GSs), ranging on a perceptual continuum from threat to safety stimuli, in both contexts. Participants then completed avoidance conditioning and avoidance generalization phases, allowing them to actively avoid the upcoming aversive sound by pressing an avoidance button. Developmental differences emerged in threat perception, physiological arousal, avoidance behavior, and eye movements during contextual fear learning and generalization. Adolescents showed less discrimination between stimuli and contexts than adults, resulting primarily from their elevated fear responses to safety and generalized stimuli. Developmental differences in fear learning should be further explored in future research, as they could explain why adolescence is a sensitive developmental period for anxiety.

Generalization of Contextual Fear Depends on the Activity of the Serotonin System of the Medial Prefrontal Cortex

Generalization of Contextual Fear Depends on the Activity of the Serotonin System of the Medial Prefrontal Cortex

Corticosterone differentially modulates time-dependent fear generalization following mild or moderate fear conditioning training in rats

Stressful and emotionally arousing experiences create strong memories that seem to lose specificity over time. It is uncertain, however, how the stress system contributes to the phenomenon of time-dependent fear generalization. Here, we investigated whether post-training corticosterone (CORT-HBC) injections, given after different training intensities, affect contextual fear memory specificity at several time points. We trained male Wistar rats on the contextual fear conditioning (CFC) task using two footshock intensities (mild CFC, 3 footshocks of 0.3 mA, or moderate CFC, 3x 0.6 mA) and immediately after the training session we administered CORT-HBC systemically. We first tested the animals in a novel context and then in the training context at different intervals following training (2, 14, 28 or 42 days). By measuring freezing in the novel context and then contrasting freezing times shown in both contexts, we inferred contextual fear generalization for each rat, classifying them into Generalizers or Discriminators. Following mild CFC training, the glucocorticoid injection promoted an accurate contextual memory at the recent time point (2 days), and increase the contextual memory accuracy 28 days after training. In contrast, after the moderate CFC training, CORT-HBC facilitated contextual generalization at 14 days, compared to the control group that maintained contextual discrimination at this timepoint. For this training intensity, however, CORT-HBC did not have any effect on recent memory specificity. These findings indicate that treatment with CORT-HBC immediately after the encoding of mild or moderately arousing experiences may differentially modulate memory consolidation and time-dependent fear generalization.

Decreased cued fear discrimination learning in female rats as a function of estrous phase.

Relative to males, female rats can show enhanced contextual fear generalization (demonstrating a fear response in a safe or neutral context) dependent on estrogen receptor activation. The current experiment aimed to extend this finding to cued fear conditioning. Females in low-estrogen phases of the estrous cycle showed good discrimination, similar to males, between a conditional stimulus that predicted shock (CS+) and an equally familiar one that did not (CS−), while females in the proestrus (high estrogen) phase demonstrated similar levels of fear between the CS+ and CS−. These results demonstrate that cued fear generalization is similarly influenced by endogenous estrogens.

Effects of Alcohol and Cocaine in a Mutant Mouse Model of Predisposition to Post-Traumatic Stress Disorder.

Comorbidity between drug abuse and post-traumatic stress disorder (PTSD), a stress-related dysregulation of fear responses, is very high. While some drugs are known to increase fear and anxiety, there are only few data regarding interactions between voluntary drug consumption and fear memory. The spontaneous chronic consumption of either alcohol or cocaine under a 3-week free-choice progressive paradigm of alcohol (3/6/10%) or cocaine (0.2/0.4/0.6 mg/ml), was assessed in VGV transgenic mice, having full 5-HT2C receptor editing and displaying PTSD-like behaviors. The consequences of these drug consumptions on the potentiated contextual and cued fear conditioning responses of VGV mice were assessed. The effects of drugs on hippocampal brain-derived neurotrophic factor (Bdnf) mRNA were measured as its expression was previously found to be decreased in VGV mice. Chronic alcohol consumption was similar in WT and VGV mice. In the alcohol condition, fear acquisition was not different at the end of the learning session and cue-fear extinction was facilitated. Regarding cocaine, in contrast to WT mice, VGV mice did not increase their drug consumption along with increasing doses, an effect that might be related with enhanced drug stimuli discrimination via increased 5-HT2C receptors. Cocaine-intake VGV mice did not display the contextual fear generalization usually observed in control VGV mice. In addition, Bdnf expression was upregulated after either chronic alcohol or cocaine intake. Altogether, these results suggest that both chronic alcohol and cocaine voluntary oral consumptions can exert some therapeutic-like effects in a mutant model of PTSD predisposition.

Corticosterone Treatment and Incubation Time After Contextual Fear Conditioning Synergistically Induce Fear Memory Generalization in Neuropeptide S Receptor-Deficient Mice.

Fear memory generalization is a learning mechanism that promotes flexible fear responses to novel situations. While fear generalization has adaptive value, overgeneralization of fear memory is a characteristic feature of the pathology of anxiety disorders. The neuropeptide S (NPS) receptor (NPSR) has been shown to be associated with anxiety disorders and has recently been identified as a promising target for treating anxiety disorders. Moreover, stress hormones play a role in regulating both physiological and pathological fear memories and might therefore also be involved in anxiety disorders. However, little is known about the interplay between stress hormone and the NPS system in the development of overgeneralized fear. Here, we hypothesize that NPSR-deficient mice with high corticosterone (CORT) levels during the fear memories consolidation are more prone to develop generalized fear. To address this hypothesis, NPSR-deficient mice were submitted to a contextual fear conditioning procedure. Immediately after conditioning, mice received CORT injections (2.5 or 5 mg/kg). One day and 1 month later, the mice were tested for the specificity and strength of their fear memory, their anxiety level, and their startle response. Moreover, CORT blood levels were monitored throughout the experiment. Using this protocol, a specific contextual fear memory was observed in all experimental groups, despite the 5-mg/kg CORT-treated NPSR-deficient mice. This group of mice showed a generalization of contextual fear memory and a decreased startle response, and the females of this group had significantly less body weight gain. These findings indicate that interplay between CORT and the NPS system during the consolidation of fear memories is critical for the generalization of contextual fear.

Генерализация страха обстановки зависит от активности серотониновой системы медиальной префронтальной коры

The medial prefrontal cortex (mPFC) is involved in the regulation of fear generalization. We have previously shown that generalized fear formation to auditory cues is under the control of the mPFC serotonin system, as its pharmacological activation during a conditioned fear response acquisition (CFR - a fear model) enhances fear expression to safe auditory stimuli during testing. The aim of this work was to find out whether the mPFC serotonin system is involved in the contextual fear generalization. Using in vivo intracranial microdialysis in Sprague-Dawley rats, it was shown that placing animals in a potentially dangerous box A, in which they had previously acquired CFR (the paired presentation of a conditioned cue and inescapable footshock) was accompanied by an increase in the mPFC extracellular serotonin level and caused animals animals' freezing (a contextual fear measure). Placing the same animals in a safe differentiation box B also led to the extracellular serotonin level rise in the mPFC and caused animals' freezing (a measure of contextual fear generalization). The intra-mPFC infusion of a selective serotonin reuptake inhibitor fluoxetine (1 μM) during the fear response elaboration enhanced, during testing, animals' freezing in differentiation box B but did not affect animals' freezing in box A. The fluoxetine administration during training did not change the testing-induced extracellular serotonin levels rise in the mPFC in boxes A and B. The data indicate that activation of the mPFC serotonin system during fear formation affects the degree of contextual fear generalization. In addition, they suggest that one of the neurochemical manifestations of contextual fear generalization may be an increased activity of the mPFC serotonin system in the safe environment.

Activity of the Serotonin System of the Prefrontal Cortex in Rats with High and Low Level of Contextual Fear Generalization

Intracerebral microdialysis studies in rats showed that acquisition of a conditioned fear reaction (combination of conditioned sound signal with pain reinforcement in chamber A) was accompanied by serotonin release in the medial prefrontal cortex, which did not occur in animals of the control group (same procedure but without pain stimulation). The level of serotonin release seen during acquisition of the fear reaction was high in animals demonstrating significant freezing (a measure of fear) in chamber B, not associated with pain reinforcement (high level of contextual fear generalization). Serotonin release in rats with low levels of freezing in chamber B (low contextual fear generalization) was low and did not correlate with freezing in chamber A. These data provide evidence that activation of the serotoninergic input of the medial prefrontal cortex on acquisition of the conditioned reflex fear reaction predicted the level of generalization of the contextual component of this reaction.

Contextual Fear Conditioning and Fear Generalization in Individuals With Panic Attacks.

Context conditioning is characterized by unpredictable threat and its generalization may constitute risk factors for panic disorder (PD). Therefore, we examined differences between individuals with panic attacks (PA; N = 21) and healthy controls (HC, N = 22) in contextual learning and context generalization using a virtual reality (VR) paradigm. Successful context conditioning was indicated in both groups by higher arousal, anxiety and contingency ratings, and increased startle responses and skin conductance levels (SCLs) in an anxiety context (CTX+) where an aversive unconditioned stimulus (US) occurred unpredictably vs. a safety context (CTX−). PA compared to HC exhibited increased differential responding to CTX+ vs. CTX− and overgeneralization of contextual anxiety on an evaluative verbal level, but not on a physiological level. We conclude that increased contextual conditioning and contextual generalization may constitute risk factors for PD or agoraphobia contributing to the characteristic avoidance of anxiety contexts and withdrawal to safety contexts and that evaluative cognitive process may play a major role.

Anterior Cingulate Cortex to Ventral Hippocampus Circuit Mediates Contextual Fear Generalization.

Contextual fear memory becomes less context-specific over time, a phenomenon referred to as contextual fear generalization. Overgeneralization of contextual fear memory is a core symptom of post-traumatic stress disorder (PTSD), but circuit mechanisms underlying the generalization remain unclear. We show here that neural projections from the anterior cingulate cortex (ACC) to ventral hippocampus (vHPC) mediate contextual fear generalization in male mice. Retrieval of contextual fear in a novel context at a remote time point activated cells in the ACC and vHPC, as indicated by significantly increased C-fos+ cells. Using chemogenetic or photogenetic manipulations, we observed that silencing the activity of ACC or vHPC neurons reduced contextual fear generalization at the remote time point, whereas stimulating the activity of ACC or vHPC neurons facilitated contextual fear generalization at a recent time point. We found that ACC neurons projected to the vHPC unidirectionally, and importantly, silencing the activity of projection fibers from the ACC to vHPC inhibited contextual fear generalization at the remote time point. Together, our findings reveal an ACC to vHPC circuit that controls expression of fear generalization and may offer new strategies to prevent or reverse contextual fear generalization in subjects with anxiety disorders, especially in PTSD.SIGNIFICANCE STATEMENT Overgeneralization of contextual fear memory is a cardinal feature of PTSD, but circuit mechanisms underlying it remain unclear. Our study indicates that neural projections from the anterior cingulate cortex to ventral hippocampus control the expression of contextual fear generalization. Thus, manipulating the circuit may prevent or reverse fear overgeneralization in subjects with PTSD.

Uncoupling nNOS-PSD-95 in the ACC can inhibit contextual fear generalization

A typical feature of the contextual fear memory is increased fear generalization with time. Though much attention has been given to the neural structures that underlie the long-term consolidation of a contextual fear memory, the molecular mechanisms regulating fear generalization remain unclear. We observed that retrieval of contextual fear in a novel context at a remote time point increased coupling of neuronal nitric oxide synthase (nNOS) with postsynaptic density-95 (PSD-95) and c-Fos expression in the anterior cingulate cortex (ACC). Disrupting nNOS-PSD-95 coupling in the ACC decreased the expression of Histone deacetylase 2 (HDAC2), and inhibited contextual fear generalization at a remote time point. Together, our findings reveal nNOS-PSD-95 interaction in the ACC could be a promising target to prevent or reverse contextual fear generalization.