Contextual Fear Conditioning In Rats Research Articles

Sex and the facilitation of cued fear by prior contextual fear conditioning in rats.

Previous studies have shown that the formation of new memories can be influenced by prior experience. This includes work using Pavlovian fear conditioning in rodents that has shown that an initial fear conditioning experience can become associated with and facilitate the acquisition of new fear memories, especially when they occur close together in time. However, most of the prior studies used only males as subjects, resulting in questions about the generalizability of the findings from this work. Here we tested whether prior contextual fear conditioning would facilitate later learning of cued fear conditioning in both male and female rats, and if there were differences based on the interval between the two conditioning episodes. Our results showed that levels of cued fear were not influenced by prior contextual fear conditioning or by the interval between training; however, females showed lower levels of cued fear. Freezing behavior in the initial training context differed by sex, with females showing lower levels of contextual fear, and by the type of initial training, with rats given delayed shock showing higher levels of fear than rats given immediate shock during contextual fear conditioning. These results indicate that contextual fear conditioning does not prime subsequent cued fear conditioning and that female rats express lower levels of cued and contextual fear conditioning than males.

Sex and the facilitation of cued fear by prior contextual fear conditioning in rats.

Previous studies have shown that the formation of new memories can be influenced by prior experience. This includes work using pavlovian fear conditioning in rodents that have shown that an initial fear conditioning experience can become associated with and facilitate the acquisition of new fear memories, especially when they occur close together in time. However, most of the prior studies used only males as subjects resulting in questions about the generalizability of the findings from this work. Here we tested whether prior contextual fear conditioning would facilitate later learning of cued fear conditioning in both male and female rats, and if there were differences based on the interval between the two conditioning episodes. Our results showed that levels of cued fear were not influenced by prior contextual fear conditioning or by the interval between training, however, females showed lower levels of cued fear. Freezing behavior in the initial training context differed by sex, with females showing lower levels of contextual fear, and by the type of initial training, with rats given delayed shock showing higher levels of fear than rats given immediate shock during contextual fear conditioning. These results indicate that contextual fear conditioning does not prime subsequent cued fear conditioning and that female rats express lower levels of cued and contextual fear conditioning than males.

Generalization and recovery of post-retrieval amnesia.

Selective amnesia for previously established memories can be induced by administering drugs that impair protein synthesis shortly after memory reactivation. Competing theoretical accounts attribute this selective post-retrieval amnesia to drug-induced engram degradation (reconsolidation blockade) or to incorporation of sensory features of the reactivation experience into the memory representation, hampering later retrieval in a drug-free state (memory integration). Here we present evidence that critically challenges both accounts. In contextual fear conditioning in rats, we find that amnesia induced by administration of midazolam (MDZ) after reexposure to the training context A generalizes readily to a similar context B. Amnesia is also observed when animals are exposed to the similar context B prior to MDZ administration and later tested for fear to context B but recovers when instead testing for fear to the original training context A or an equally similar but novel context C. Next to their theoretical implications for the nature of forgetting, our findings raise important questions about the viability of reconsolidation-based interventions for the treatment of emotional disorders. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Spaced Training Enhances Contextual Fear Memory via Activating Hippocampal 5-HT2A Receptors.

Spaced training is robustly superior to massed training, which is a well-documented phenomenon in humans and animals. However, the mechanisms underlying the spacing effect still remain unclear. We have reported previously that spacing training exerts memory-enhancing effects by inhibiting forgetting via decreasing hippocampal Rac1 activity. Here, using contextual fear conditioning in rat, we found that spaced but not massed training increased hippocampal 5-HT2A receptors’ expression. Furthermore, hippocampal administration of 5-HT2A receptor antagonist MDL11939 before spaced training blocked the enhanced memory, while hippocampal administration of 5-HT2A receptor agonist TCB-2 before massed training promoted the memory. Moreover, MDL11939 activated hippocampal Rac1, while TCB-2 decreased hippocampal Rac1 activity in naïve rats. These results indicated the possibility of interaction between 5-HT2A receptors and Rac1, which was demonstrated by co-immunoprecipitation experiments. Our study first demonstrates that activation of hippocampal 5-HT2A is a mechanism underlying the spacing effect, and forgetting related molecular Rac1 is engaged in this process through interacting with 5-HT2A receptors, which suggest a promising strategy to modulate abnormal learning in cognitive disorders.

Nucleus reuniens mediates the extinction of contextual fear conditioning

Learning and remembering the context in which events occur requires interactions between the hippocampus (HPC) and medial prefrontal cortex (mPFC). The nucleus reuniens (RE) is a ventral midline thalamic nucleus that coordinates activity in the mPFC and HPC and is involved in spatial and contextual memory. We recently found that the RE is critical for contextual fear conditioning in rats, a form of learning that involves interactions between the HPC and mPFC. Here we examined whether the RE mediates the extinction of contextual fear. After contextual fear conditioning, rats underwent an extinction procedure in which they were merely exposed to the conditioning context; freezing behavior during the extinction procedure and during a retrieval test 24 h later served as an index of conditioned fear. Muscimol inactivation of the RE prior to extinction impaired the acquisition of both short- and long-term extinction memories. Similarly, inactivation of the RE prior to the extinction retrieval test also impaired the expression of extinction; this effect was not state-dependent. Taken together, these results reveal that the extinction of contextual fear memories requires the RE, which is consistent with a broader role for the RE in forms of learning that require HPC-mPFC interactions.

Chronic fluoxetine prevents fear memory generalization and enhances subsequent extinction by remodeling hippocampal dendritic spines and slowing down systems consolidation

Fear memory overgeneralization contributes to the genesis and persistence of anxiety disorders and is a central hallmark in the pathophysiology of post-traumatic stress disorder (PTSD). Recent findings suggest that fear generalization is closely related to hippocampal dependency during retrieval. The selective serotonin reuptake inhibitor (SSRI) fluoxetine has been used as a first-line treatment for PTSD; however, how it exerts its therapeutic effect remains a matter of debate. Here, using contextual fear conditioning in rats, we show that chronic fluoxetine treatment prevents fear generalization and enhances subsequent extinction. Moreover, fluoxetine treatment after extinction prevents spontaneous recovery. The mechanism through which fluoxetine affects generalization and extinction seems to be through the postponement of systems consolidation, thereby maintaining hippocampal involvement during retrieval. Such an effect relies on a remodeling of dendritic spines in the hippocampus, as well as the number of mature, mushroom-type spines promoted by fluoxetine treatment. In order to further investigate whether fear generalization is a potential predictor of extinction effectiveness, we categorized a large naive population according to their generalization rate. We found that discriminator rats showed a better extinction profile compared to generalizers, suggesting that the generalization rate predicts extinction effectiveness. Hence, we propose that the therapeutic strategy of choice should take into account the extension of memory generalization, in which therapies based on extinction could induce a better outcome in patients who present less fear overgeneralization. These results open new avenues for the development of interventions that prevent fear generalization by maintaining memory dependency of the hippocampus.

P.2.20 Neuropeptide Y in the dorsal dentate gyrus is an important protective factor that mediate trauma resilience

Neuropeptide Y (NPY) is the most abundant peptide in the central nervous system (CNS) and is densely localized in the brain regions involved in stress, memory, fear and anxiety. Although previous research supports a role for NPY in the mediation of rodent and human emotional behavior, there is currently a lack of information on the effects of low doses of NPY that could have a potential therapeutic advantage, minimizing side-effects such as cognition impairment or sedation. Herein, we assessed the effects of intracerebroventricular (i.c.v.) administration of low doses of NPY, and of the Y1-agonist Leu31Pro34-NPY (LP-NPY) on contextual fear conditioning (CFC), as they have no effect on unconditioned anxiety-like, locomotor activity and non-emotional memory. NPY (3 pmol) and LP-NPY (1 pmol) inhibited freezing behavior when administered in the acquisition or consolidation stages, indicating a reduction of fear. When injected in the extinction phase, only NPY inhibited freezing behavior on CFC. Pre-treatment with the Y1-antagonist BIBO3304 before NPY and LP-NPY was able to prevent the inhibition of fear responses induced by both NPY agonists. Taken together, our results demonstrate robust fear-inhibiting effects of i.c.v. injection of NPY on contextual fear conditioning in rats, a response that is mediated, at least in part, by the Y1 receptor. Moreover, these treatments were unable to change locomotor activity or to show an anxiolytic-like effect, as evaluated in an open-field and an elevated plus-maze. This specific fear reduction effect may underlie resilience systems in the CNS and has potential therapeutic relevance in PTSD.

Periodical reactivation under the effect of caffeine attenuates fear memory expression in rats

In the last decade, several studies have shown that fear memories can be attenuated by interfering with reconsolidation. However, most of the pharmacological agents used in preclinical studies cannot be administered to humans. Caffeine is one of the world’s most popular psychoactive drugs and its effects on cognitive and mood states are well documented. Nevertheless, the influence of caffeine administration on fear memory processing is not as clear. We employed contextual fear conditioning in rats and acute caffeine administration under a standard memory reconsolidation protocol or periodical memory reactivation. Additionally, potential rewarding/aversion and anxiety effects induced by caffeine were evaluated by conditioning place preference or open field, respectively. Caffeine administration was able to attenuate weak fear memories in a standard memory reconsolidation protocol; however, periodical memory reactivation under caffeine effect was necessary to attenuate strong and remote memories. Moreover, caffeine promoted conditioned place preference and anxiolytic-like behavior, suggesting that caffeine weakens the initial learning during reactivation through counterconditioning mechanisms. Thus, our study shows that rewarding and anxiolytic effects of caffeine during fear reactivation can change the emotional valence of fear memory. It brings a new promising pharmacological approach based on drugs widely used such as caffeine to treat fear-related disorders.

Effect of deep brain stimulation of the amygdala on post-traumatic stress disorder syndrome-induced by contextual fear conditioning in rat: Role of c-Fos protein and corticosterone hormone

Effect of deep brain stimulation of the amygdala on post-traumatic stress disorder syndrome-induced by contextual fear conditioning in rat: Role of c-Fos protein and corticosterone hormone

Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual fear conditioning in rats: Involvement of local glutamatergic/nitrergic and GABAergic neurotransmissions

The cannabinoid receptor type 1 (CB1) is highly expressed in the dorsal portion of hippocampus – a brain region that has been involved in the control of conditioned emotional response (CER) in the contextual fear conditioning (CFC) model. These responses are characterized by increased freezing behavior and autonomic parameters. Moreover, CB1 receptors activation negatively modulate the release of several neurotransmitters, including glutamate and GABA, which also have been related to modulation of CER. Therefore, our aim was to investigate the involvement of CB1 receptors in the dorsal hippocampus on CER expression. Independent groups of male Wistar rats submitted to the contextual fear conditioning received bilateral intra-hippocampal injections (500 nL/side) of the following drugs or vehicle before re-exposure to the aversive context: AM251 (CB1 antagonist; 0.1, 0.3 and 1nmol); AP7 (NMDA antagonist; 1nmol)+AM251 (0.3nmol); NPLA (0.01nmol; nNOS inhibitor)+AM251 (0.3nmol); Bicuculline (1.3pmol; GABAA antagonist)+AM251 (0.1 and 1nmol). In the present paper, AM251 (0.3nmol) increased CER, while this response was prevented by both AP7 and NPLA pretreatment. After pretreatment with Bicuculline, the lower and higher ineffective doses of AM251 were able to increase the CER, supporting the balance between GABAergic and glutamatergic mechanisms controlling this response. Our results suggest that increased CER evoked by CB1 blockade in the dorsal hippocampus depends on NMDA receptor activation and NO formation. Moreover, a fine-tune control promoted by GABAergic and glutamatergic mechanisms in this brain area modulate the CER after CB1 blockade.

Role of the orexin (hypocretin) system in contextual fear conditioning in rats

Orexin (hypocretin) neurons located in the posterior hypothalamus send projections to multiple areas of the brain involved in arousal and experimental evidence indicates that these neurons play a role in the physiological and behavioral responses to stress. This study was done to determine if the orexin system was involved in mediating the fear associated with shock context (5×2s of 1.5mA). First, real-time RT-PCR was used to examine changes in the mRNA levels for prepro-orexin (ppOX), the orexin-1 receptor (OX1R) and the orexin-2 receptor (OX2R) at two weeks post-shock. We found that the mRNA levels for ppOX and OX1R were increased in the posterior hypothalamus of shocked rats. In contrast, no significant difference was found in the midline thalamus or the locus coeruleus/parabrachial region. Second, the study examined if systemic injections of antagonists for orexin receptors attenuated the freezing related to contextual fear. The OX1R antagonist SB334867 (20 or 30mg/kg; i.p.) decreased freezing while the same doses of the OX2R antagonist TCSOX229 had no effect. The dual orexin antagonist TCS1102 (20mg/kg; i.p.) also decreased the freezing to the shock context. The results of the present study show upregulation of orexin activity and of the OX1R in the hypothalamus following exposure of rats to footshocks and highlight a specific role of OX1R in contextual fear.

Bed nucleus of the stria terminalis NMDA receptors and nitric oxide modulate contextual fear conditioning in rats

The bed nucleus of the stria terminalis (BNST) modulates anxiety-like responses, including conditioned emotional responses. Evidence suggests that glutamatergic neurotransmission in the BNST plays a role in the modulation of defensive responses. However, little is known about the involvement of glutamate NMDA receptor activation within the BNST, and its resultant increase in nitric oxide (NO) levels, in the expression of contextual fear conditioning (CFC). We investigated whether the antagonism of NMDA receptors or the reduction of NO levels in the BNST would attenuate behavioral and autonomic responses (i.e. increase in arterial pressure and heart rate, and decrease in tail cutaneous temperature) of rats submitted to a CFC paradigm. Intra-BNST infusion of AP7, an NMDA receptor antagonist, attenuated both behavioral and autonomic changes induced by CFC. Similar results were observed with NPLA and c-PTIO, an nNOS inhibitor and an NO scavenger, respectively. A positive correlation between BNST NO levels and the time spent in freezing behavior was also observed for animals submitted to the CFC. These findings indicate that the expression of CFC involves a facilitation of BNST NMDA receptor-NO signaling.This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’.

The flavonoid baicalein rescues synaptic plasticity and memory deficits in a mouse model of Alzheimer’s disease

Increasing evidence suggests that disruptions of synaptic functions correlate with the severity of cognitive deficit in Alzheimer’s disease (AD). Our previous study demonstrated that baicalein enhances long-term potentiation (LTP) in acute rat hippocampal slices and improves hippocampus-dependent contextual fear conditioning in rats. Given that baicalein possess various biological activities, especially its effects on synaptic plasticity and cognitive function, we examined the effect of baicalein on synaptic function both in vitro and in vivo in AD model. The effect of baicalein on Aβ42 oligomer impaired LTP was investigated by electrophysiological methods. Baicalein was administered orally via drinking water to the APP/PS1 mice and sex- and age-matched wild-type mice. Treatment started at 5 months of age and mice were assessed for cognition and AD-like pathology at 7-month-old. Cognition was analyzed by Morris water maze test, fear conditioning test, and novel object recognition test. Changes in hippocampal 12/15 Lipoxygenase (12/15LO) and glycogen synthase kinase 3β (GSK3β) activity, Aβ production, tau phosphorylation, synaptic plasticity, and dendritic spine density were evaluated. Baicalein prevented Aβ-induced impairments in hippocampal LTP through activation of serine threonine Kinase (Akt) phosphorylation. Long-term oral administration of baicalein inhibited 12/15LO and GSK3β activity, reduced β-secretase enzyme (BACE1), decreased the concentration of total Aβ, and prevented phosphorylation of tau in APP/PS1 mice. Meanwhile, baicalein restored spine number, synaptic plasticity, and memory deficits. Our results strengthen the potential of the flavonoid baicalein as a novel and promising oral bioactive therapeutic agent that prevents memory deficits in AD.

Mirtazapine exerts an anxiolytic-like effect through activation of the median raphe nucleus-dorsal hippocampal 5-HT pathway in contextual fear conditioning in rats

The functional role of serotonergic projections from the median raphe nucleus (MRN) to the dorsal hippocampus (DH) in anxiety remains understood poorly. The purpose of the present research was to examine the functional role of this pathway, using the contextual fear conditioning (CFC) model of anxiety. We show that intra-MRN microinjection of mirtazapine, a noradrenergic and specific serotonergic antidepressant, reduced freezing in CFC without affecting general motor activity dose-dependently, suggesting an anxiolytic-like effect. In addition, intra-MRN microinjection of mirtazapine dose-dependently increased extracellular concentrations of serotonin (5-HT) but not dopamine in the DH. Importantly, intra-DH pre-microinjection of WAY-100635, a 5-HT1A antagonist, significantly attenuated the effect of mirtazapine on freezing. These results, for the first time, suggest that activation of the MRN-DH 5-HT1A pathway exerts an anxiolytic-like effect in CFC. This is consistent with the literature that the hippocampus is essential for retrieval of contextual memory and that 5-HT1A receptor activation in the hippocampus primarily exerts an inhibitory effect on the neuronal activity.

The cannabinoid system in the retrosplenial cortex modulates fear memory consolidation, reconsolidation, and extinction.

Despite the fact that the cannabinoid receptor type 1 (CB1R) plays a pivotal role in emotional memory processing in different regions of the brain, its function in the retrosplenial cortex (RSC) remains unknown. Here, using contextual fear conditioning in rats, we showed that a post-training intra-RSC infusion of the CB1R antagonist AM251 impaired, and the agonist CP55940 improved, long-term memory consolidation. Additionally, a post-reactivation infusion of AM251 enhanced memory reconsolidation, while CP55940 had the opposite effect. Finally, AM251 blocked extinction, whereas CP55940 facilitated it and maintained memory extinguished over time. Altogether, our data strongly suggest that the cannabinoid system of the RSC modulates emotional memory.

Systemic administration of riluzole enhances recognition memory and facilitates extinction of fear memory in rats

Strategies to enhance recognition memory and facilitate extinction of fear memory have attracted increasing attention for enhancing the effectiveness of exposure therapy for anxiety disorders. Previously, we demonstrated that systemic administration of riluzole has clear anxiolytic-like effects, without impairing memory, in rats. In the present study, we examined whether riluzole could have therapeutic potential for anxiety disorders when combined with exposure therapy. Both riluzole and d-cycloserine enhanced recognition memory in the novel object recognition test and facilitated extinction learning in the contextual fear conditioning in rats. Interestingly, the facilitatory effect of riluzole on extinction learning was clearly observed even after a short re-exposure to the context, while d-cycloserine was ineffective at facilitating extinction when a short duration exposure session was given. In contrast, diazepam impaired both recognition memory and the extinction of fear memory. Our findings strongly suggest that systemic administration of riluzole may have therapeutic efficacy when combined with exposure therapy for treating a range of anxiety disorders. Clinical trials to examine the efficacy of riluzole in combination with exposure therapy in these patients are warranted.

Sex differences in the behavioural and hypothalamic–pituitary–adrenal response to contextual fear conditioning in rats

In recent years, special attention is being paid to sex differences in susceptibility to disease. In this regard, there is evidence that male rats present higher levels of both cued and contextual fear conditioning than females. However, little is known about the concomitant hypothalamic–pituitary–adrenal (HPA) axis response to those situations which are critical in emotional memories. Here, we studied the behavioural and HPA responses of male and female Wistar rats to context fear conditioning using electric footshock as the aversive stimulus. Fear-conditioned rats showed a much greater ACTH and corticosterone response than those merely exposed to the fear conditioning chamber without receiving shocks. Moreover, males presented higher levels of freezing whereas HPA axis response was greater in females. Accordingly, during the fear extinction tests, female rats consistently showed less freezing and higher extinction rate, but greater HPA activation than males. Exposure to an open-field resulted in lower activity/exploration in fear-conditioned males, but not in females, suggesting greater conditioned cognitive generalization in males than females. It can be concluded that important sex differences in fear conditioning are observed in both freezing and HPA activation, but the two sets of variables are affected in the opposite direction: enhanced behavioural impact in males, but enhanced HPA responsiveness in females. Thus, the role of sex differences on fear-related stimuli may depend on the variables chosen to evaluate it, the greater responsiveness of the HPA axis in females perhaps being an important factor to be further explored.

The expression of contextual fear conditioning involves activation of a NMDA receptor-nitric oxide-cGMP pathway in the dorsal hippocampus of rats

The dorsal portion of the hippocampus is a limbic structure that is involved in fear conditioning modulation in rats. Moreover, evidence shows that the local dorsal hippocampus glutamatergic system, nitric oxide (NO) and cGMP modulate behavioral responses during aversive situations. Therefore, the present study investigated the involvement of dorsal hippocampus NMDA receptors and the NO/cGMP pathway in contextual fear conditioning expression. Male Wistar rats were submitted to an aversive contextual conditioning session and 48h later they were re-exposed to the aversive context in which freezing, cardiovascular responses (increase of both arterial pressure and heart rate) and decrease of tail temperature were recorded. The intra-dorsal hippocampus administration of the NMDA receptor antagonist AP7, prior to the re-exposure to the aversive context, attenuated fear-conditioned responses. The re-exposure to the context evoked an increase in NO concentration in the dorsal hippocampus of conditioned animals. Similar to AP7 administration, we observed a reduction of contextual fear conditioning after dorsal hippocampus administration of either the neuronal NO synthase inhibitor N-propyl-l-arginine, the NO scavenger c-PTIO or the guanylate cyclase inhibitor ODQ. Therefore, the present findings suggest the possible existence of a dorsal hippocampus NMDA/NO/cGMP pathway modulating the expression of contextual fear conditioning in rats.

Long-term effects of traumatic stress on subsequent contextual fear conditioning in rats

Exposure to stressful events affects subsequent sensitivity to fear. We investigated the long-term effects of a traumatic experience on subsequent contextual fear conditioning and anxiety-like behaviors in rats (Experiment 1). In addition, we tested whether the administration of the glucocorticoid synthesis inhibitor metyrapone (MET) attenuated the sensitization of fear induced by traumatic stress (Experiment 2). Male rats were subjected to a multiple stress (MS) session, which consisted of 4 foot shocks (1mA, 1s) and forced swimming for 20min, followed by exposure to a situational reminder 7days after the MS session. MET (25 or 100mg/kg, intraperitoneal) was administered 30min before MS. The contextual fear conditioning was performed 14days after MS. MS enhanced the conditioned fear response for at least 14days after the conditioning, and pretreatment with MET did not affect the enhancement of conditioned fear. These results suggest that glucocorticoid secretion triggered by MS is not involved in regulating the long-term stress-induced sensitization of fear.

Memory destabilization is critical for the success of the reactivation-extinction procedure.

It has been suggested that, unlike pure extinction which typically results in the return of the fear response under a variety of circumstances, memory reactivation followed by extinction can attenuate the reemergence of conditioned fear. The reactivation–extinction procedure has attracted the attention of basic and clinical researchers due to its potential clinical value for the treatment of psychiatric conditions, such as anxiety and drug abuse disorders. However, mixed results have been achieved so far in replicating and understanding this paradigm. It has been proposed that memory destabilization could be critical in this sense. Using contextual fear conditioning in rats and midazolam as an amnesic agent, we first determined what reactivation conditions are necessary to destabilize the mnemonic trace. After establishing the conditions for memory destabilization, a series of experiments was conducted to determine if destabilization is critical for the success of the reactivation–extinction procedure. Data confirmed the importance of memory destabilization prior to extinction inside the reconsolidation window to attenuate spontaneous recovery and retard reacquisition of conditioned fear. The present report offers a candidate explanation of the discrepancy in results obtained with the reactivation–extinction procedure by different laboratories.