Extinction Of Auditory Fear Conditioning Research Articles

Involvement of canonical Wnt/β-catenin signaling in the extinction of auditory fear conditioning in male mice

The Wnt signaling pathway plays a critical role in activity-dependent plasticity processes such as long-term potentiation, learning and memory. However, the role of the Wnt signaling pathway in adult extinction is still not well understood. In this study, we aimed to investigate the roles and mechanisms of the canonical Wnt/β-catenin signaling pathway in the extinction of auditory fear conditioning (AFC) in adult mice. We found that AFC extinction training induced a significant decrease in p-GSK3β and nuclear β-catenin in the medial prefrontal cortex (mPFC). Micro-infusion of the canonical Wnt inhibitor Dkk1 into the mPFC before AFC extinction training facilitated AFC extinction, suggesting that the Wnt/β-catenin pathway is involved in AFC extinction. To determine how Dkk1 affects canonical Wnt/β-catenin signaling in AFC extinction, the protein levels of p-GSK3β and β-catenin were measured. We found that DKK1 produces a decrease in p-GSK3β and β-catenin. Moreover, we found that upregulating the Wnt/β-catenin pathway using LiCl (2 µg/side) impaired AFC extinction. These findings may help us understand the role of canonical Wnt signaling pathway in memory extinction and suggest that appropriate manipulating the Wnt/β-catenin signaling pathway might be a suitable way of therapeutically treating psychiatric disorders.

β-adrenoceptors of the infra-limbic cortex mediate corticosterone-induced enhancement of acquisition and consolidation of fear memory extinction in rats

The extinction of auditory fear conditioning (AFC) refers to reducing the fear responses induced following repeated presentation of a conditioned stimulus (tone) in the absence of an unconditioned stimulus (electric foot shock). Glucocorticoid receptors (GRs) play an important role in extinction, but the underlying neurobiological mechanisms are unclear. This study aimed to investigate the interaction between glucocorticoids and β-adrenoceptors of the infra-limbic cortex (IL) in regulating the acquisition and consolidation of fear memory extinction in rats. Male rats were trained to AFC and received three trial tones (30 s, 4 kHz, 80 dB) co-terminated with a footshock (0.8 mA, 1 s; unconditioned stimulus). Extinction trials were conducted over 3 days after training (Ext 1–3). In experiment 1, rats received clenbuterol (0.25 mg/kg/2 ml, IP) as a β2-adrenoceptor agonist or propranolol (2.5 mg/kg/2 ml, IP) as a β-adrenoceptors antagonist before Ext 1 and immediately after Ext 1 and Ext 2 followed by systemic injection of corticosterone (3 mg/kg/2 ml, IP). In Experiment 2, separate groups of rats received a bilateral intra-IL injection of clenbuterol (50 ng/0.5 µl/side) or propranolol (500 ng/0.5 µl/side) followed by a systemic injection of corticosterone (3 mg/kg/2 ml) before Ext 1 and immediately after Ext 1 and Ext 2. Results indicated that systemic and intra-IL injections of clenbuterol and propranolol inhibited and increased the facilitative effects of corticosterone on fear memory extinction, respectively. These findings show that activating β-adrenergic receptors in the IL mediates glucocorticoid effects on the acquisition and consolidation of auditory-conditioned fear memory extinction.

Effects of sex and retention interval on the retrieval and extinction of auditory fear conditioning.

Fear memory retrieval is relevant to psychiatric disorders such as post-traumatic stress disorder (PTSD). One of the hallmark symptoms of PTSD is the repeated retrieval and re-experiencing of the initial fear memory even long after the traumatic event has occurred. Women are nearly twice as likely to develop PTSD following a trauma than men, thus sex differences in the retrieval of fear memories is highly relevant for understanding the development and maintenance of PTSD. In the current study, we aimed to examine sex differences in the retrieval and extinction of either recent or remote fear memories. To do so, we conditioned male and female rats either 1 day (recent) or 28 days (remote) prior to testing retrieval and extinction. While there was no effect of sex or retention interval on initial retrieval, we found that remotely conditioned females exhibited higher rates of freezing than remotely conditioned males in later retrieval/extinction sessions, suggesting a sex difference in the retrieval and/or extinction of remote, but not recent, fear memories. Overall, these results are the first to demonstrate a sex difference in the extinction of remote fear memory, and this may contribute to the differential expression of fear-related disorders like PTSD in men and women.

Daily Optogenetic Stimulation of the Left Infralimbic Cortex Reverses Extinction Impairments in Male Rats Exposed to Single Prolonged Stress.

Post-traumatic stress disorder (PTSD) is associated with decreased activity in the prefrontal cortex. PTSD-like pathophysiology and behaviors have been observed in rodents exposed to a single prolonged stress (SPS) procedure. When animals are left alone for 7 days after SPS treatment, they show increased anxiety-like behavior and impaired extinction of conditioned fear, and reduced activity in the prefrontal cortex. Here, we tested the hypothesis that daily optogenetic stimulation of the infralimbic region (IL) of the medial prefrontal cortex (mPFC) during the 7 days after SPS would reverse SPS effects on anxiety and fear extinction. Male Sprague-Dawley rats underwent SPS and then received daily optogenetic stimulation (20 Hz, 2 s trains, every 10 s for 15 min/day) of glutamatergic neurons of the left or right IL for seven days. After this incubation period, rats were tested in the elevated plus-maze (EPM). Twenty-four hours after the EPM test, rats underwent auditory fear conditioning (AFC), extinction training and a retention test. SPS increased anxiety-like behavior in the EPM task and produced a profound impairment in extinction of AFC. Optogenetic stimulation of the left IL, but not right, during the 7-day incubation period reversed the extinction impairment. Optogenetic stimulation did not reverse the increased anxiety-like behavior, suggesting that the extinction effects are not due to a treatment-induced reduction in anxiety. Results indicate that increased activity of the left IL after traumatic experiences can prevent development of extinction impairments. These findings suggest that non-invasive brain stimulation may be a useful tool for preventing maladaptive responses to trauma.

Vagus nerve stimulation promotes extinction generalization across sensory modalities

Traumatic experiences involve complex sensory information, and individuals with trauma-related psychological disorders, such as posttraumatic stress disorder (PTSD), can exhibit abnormal fear to numerous different stimuli that remind them of the trauma. Vagus nerve stimulation (VNS) enhances extinction of auditory fear conditioning in rat models for PTSD. We recently found that VNS-paired extinction can also promote extinction generalization across different auditory cues. Here we tested whether VNS can enhance extinction of olfactory fear and promote extinction generalization across auditory and olfactory sensory modalities. Male Sprague Dawley rats were implanted with a stimulating cuff on the cervical vagus nerve. Rats then received two days of fear conditioning where olfactory (amyl acetate odor) and auditory (9 kHz tones) stimuli were concomitantly paired with footshock. Twenty-four hours later, rats were given three days of sham or VNS-paired extinction (5 stimulations, 30-sec trains at 0.4 mA) overlapping with presentation of either the olfactory or the auditory stimulus. Two days later, rats were given an extinction retention test where avoidance of the olfactory stimulus or freezing to the auditory stimulus were measured. VNS-paired with exposure to the olfactory stimulus during extinction reduced avoidance of the odor in the retention test. VNS-paired with exposure to the auditory stimulus during extinction also decreased avoidance of the olfactory cue, and VNS paired with exposure to the olfactory stimulus during extinction reduced freezing when the auditory stimulus was presented in the retention test. These results indicate that VNS enhances extinction of olfactory fear and promotes extinction generalization across different sensory modalities. Extinction generalization induced by VNS may therefore improve outcomes of exposure-based therapies.

Memory reconsolidation and extinction of fear conditioning induced different Arc/Arg3.1 expression.

Re-exposure to the conditioned stimulus (CS) during which the threat was experienced leads to two different processes: brief CS presentation initiates reconsolidation to re-stabilize the original fear memory, whereas prolonged CS presentation leads to extinction to form an inhibitory memory. The two processes used distinct mechanisms, but the difference in neuronal activation during these processes still remains to be addressed. Using the auditory fear-conditioning model, we analyzed Arc (activity-regulated cytoskeletal-associated protein) expression after brief or prolonged CS presentation at several time points. Here, we reported that Arc expression increased in the amygdala and infralimbic prefrontal cortex from 2 to 6 h after 20 trials of CS presentation, whereas the Arc expression increased mainly in the basolateral amygdala (BLA) 2 h after one trial of CS presentation. The Arc neurons in BLA induced by brief or prolonged CS presentation were non-GABAergic, suggesting that the activation of the non-GABAergic neurons in BLA might be involved in memory reconsolidation and extinction processes of the auditory fear condition. Our results also suggest that memory reconsolidation or extinction of auditory fear conditioning induced Arc activation in distinct neural circuits.

CXCL12 impairs the acquisition and extinction of auditory fear conditioning in rats via crosstalk with GABAergic system

ObjectiveChemokines, such as CXCL12, are signaling molecules playing an important role in immune regulations. Chemokine upsurge has also been associated with neuroinflammatory conditions characterized with cognitive impairments. Recently, some in-vitro data suggests that CXCL12 is a potential neuromodulator and interacts with GABAergic system, but, so far, whether these effects translate into alterations in neural and behavioral functions has not been investigated. MethodsIn the present study, we used auditory fear conditioning as a model to define the contribution of CXCL12/CXCR4 on fear-related cognitive disorders. We microinjected different dosages of CXCL12 into the bilateral amygdala of rats to investigate their behavioral effects on the acquisition and extinction of conditioned fear memory. Moreover, we pretreated the rats with the selective CXCR4 receptor antagonist (AMD3100), GABAA antagonist (bicuculline) and GABAB antagonist (CGP55845) to examine whether the CXCL12 induced changes could be reversed. ResultsWe found that intra-amygdala infusion of CXCL12 impaired the acquisition and extinction of conditioned fear response. Pretreatment with AMD3100, rescued the CXCL12 induced impairments, indicating that CXCL12 produced the effects by activating CXCR4 receptors. Furthermore, both bicuculline and CGP55845 prevented CXCL12 from impairing the rat's ability of conditioned learning, indicating a crosstalk between CXCL12/CXCR4 and GABAergic system. ConclusionOur data suggest that the chemokine CXCL12 is able to regulate neurotransmitter mechanisms involved in associative learning functions, and the effect of GABAergic agents on CXCL12/CXCR4 may be new therapeutic potentials for neuroinflammatory diseases.

Voluntary exercise during extinction of auditory fear conditioning reduces the relapse of fear associated with potentiated activity of striatal direct pathway neurons

Relapse of previously extinguished fear presents a significant, pervasive obstacle to the successful long-term treatment of anxiety and trauma-related disorders. Thus, identification of a novel means to enhance fear extinction to stand the passage of time and generalize across contexts is of the utmost importance. Acute bouts of exercise can be used as inexpensive, noninvasive treatment strategies to reduce anxiety, and have been shown to enhance memory for extinction when performed in close temporal proximity to the extinction session. However, it is unclear whether acute exercise can be used to prevent relapse of fear, and the neural mechanisms underlying this potential effect are unknown. The current study therefore examined whether acute exercise during extinction of auditory fear can protect against the later relapse of fear. Male F344 rats lacking an extended history of wheel running were conditioned to fear a tone CS and subsequently extinguished within either a freely mobile running wheel, a locked wheel, or a control context lacking a wheel. Rats exposed to fear extinction within a freely mobile wheel ran during fear extinction, and demonstrated reduced fear as well as attenuated corticosterone levels during re-exposure to the extinguished CS during the relapse test in a novel context 1week later. Examination of cfos mRNA patterns elicited by re-exposure to the extinguished CS during the relapse test revealed that acute exercise during extinction decreased activation of brain circuits classically involved in driving fear expression and interestingly, increased activity within neurons of the direct striatal pathway involved in reward signaling. These data suggest that exercise during extinction reduces relapse through a mechanism involving the direct pathway of the striatum. It is suggested that a positive affective state could become associated with the CS during exercise during extinction, thus resulting in a relapse-resistant extinction memory.

Revisiting the role of infralimbic cortex in fear extinction with optogenetics.

Previous rodent studies have implicated the infralimbic (IL) subregion of the medial prefrontal cortex in extinction of auditory fear conditioning. However, these studies used pharmacological inactivation or electrical stimulation techniques, which lack temporal precision and neuronal specificity. Here, we used an optogenetic approach to either activate (with channelrhodopsin) or silence (with halorhodopsin) glutamatergic IL neurons during conditioned tones delivered in one of two phases: extinction training or extinction retrieval. Activating IL neurons during extinction training reduced fear expression and strengthened extinction memory the following day. Silencing IL neurons during extinction training had no effect on within-session extinction, but impaired the retrieval of extinction the following day, indicating that IL activity during extinction tones is necessary for the formation of extinction memory. Surprisingly, however, silencing IL neurons optogenetically or pharmacologically during the retrieval of extinction 1 day or 1 week following extinction training had no effect. Our findings suggest that IL activity during extinction training likely facilitates storage of extinction in target structures, but contrary to current models, IL activity does not appear to be necessary for retrieval of extinction memory.

Intra-amygdala microinjection of TNF-α impairs the auditory fear conditioning of rats via glutamate toxicity

During an inflammatory or infectious process, innate immune cells produce large amount of pro-inflammatory cytokines that act on the brain to cause cognitive dysfunctions. Tumor necrosis factor alpha (TNF-α) is one of the main pro-inflammatory cytokines. Thus, it is important to study how the excessive TNF-α affects the cognitive functions of central nervous system and possible antagonists to its effects. In the present study, we conducted behavioral experiments of rats to determine whether murine TNF-α administered directly into the brain would elicit behavioral effects related to learning and memory impairments. Rats subjected to single-dose intra-amygdala TNF-α infusion showed a significant delay in the acquisition and extinction of auditory fear conditioning. Accordingly, the glutamate level of the tissue samples from amygdala was elevated after the TNF-α treatment. Furthermore, pharmacological blockade of NMDAR before the TNF-α treatment reversed the TNF-α induced impairments in fear learning. Our findings suggest that TNF-α can impair the learning and memory functions through glutamate–NMDAR neurotoxicity, and present the possibility to develop therapeutic modalities directing at glutamate transmission for the treatment of neuro-inflammative dysfunctions.

Mineralocorticoid receptors in the ventral hippocampus are involved in extinction memory in rats.

Fear extinction decreases conditioned fear responses that normally occur when a conditioned stimulus (CS) is repeatedly presented in the absence of an aversive unconditioned stimulus (US), which is the behavioral basis of exposure therapy for posttraumatic stress disorder (PTSD). However, knowledge about the neurobiology of extinction is insufficient. The present study investigated changes in the protein expression of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in the dorsal hippocampus (DH)and ventral hippocampus (VH), induced by extinction and return of conditioned fear responses to an auditory signal. Western blot analysis indicated that shock stress enhanced the expression of MRs in only the DH, whereas extinction selectively increased the expression of MRs in the VH. The infusion of MRs antagonist spironolactone in the VH indicated that MRs in the VH did not affect the retrieval of fear and extinction memories, but facilitated the formation of extinction memory. However, no changes in GRs in either the DH or VH were observed in each phase of auditory fear conditioning. These results suggest that MRs in the DH and VH have differential functions in the extinction of auditory fear conditioning. MRs in the DH appear to be related to only stressful experiences, whereas MRs in the VH are involved in extinction formation. The enhancement of MRs in the VH might be necessary to improve PTSD.

Persistence of amygdala gamma oscillations during extinction learning predicts spontaneous fear recovery

Extinction of auditory fear conditioning induces a temporary inhibition of conditioned fear responses that can spontaneously reappear with the passage of time. Several lines of evidence indicate that extinction learning relies on the recruitment of specific neuronal populations within the basolateral amygdala. In contrast, post-extinction spontaneous fear recovery is thought to result from deficits in the consolidation of extinction memory within prefrontal neuronal circuits. Interestingly, recent data indicates that the strength of gamma oscillations in the basolateral amygdala during auditory fear conditioning correlates with retrieval of conditioned fear responses. In the present manuscript we evaluated the hypothesis that post-extinction spontaneous fear recovery might depend on the maintenance of gamma oscillations within the basolateral amygdala by using single unit and local field potential recordings in behaving mice. Our results indicate that gamma oscillations in the basolateral amygdala were enhanced following fear conditioning, whereas during extinction learning gamma profiles were more heterogeneous despite similar extinction learning rates. Remarkably, variations in the strength of gamma power within the basolateral amygdala between early and late stages of extinction linearly predicted the level of post-extinction spontaneous fear recovery. These data suggest that maintenance of gamma oscillations in the basolateral amygdala during extinction learning is a strong predictive factor of long term spontaneous fear recovery.

Microinfusion of the D1 receptor antagonist, SCH23390 into the IL but not the BLA impairs consolidation of extinction of auditory fear conditioning

In auditory fear conditioning, repeated presentation of the tone in the absence of the shock leads to extinction of the acquired fear response. Both the medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA) are involved in extinction. Here we examined this involvement by antagonizing D1 receptors in both regions, in the rat. We microinfused the D1 receptor antagonist, SCH23390, into the infra-limbic part of the mPFC (IL) or BLA at different time points. SCH23390 mircoinfused into the IL either before extinction acquisition or following short extinction training resulted in impairment of extinction consolidation. Microinfusion of SCH23390 into the BLA, prior to acquisition of extinction caused impairment in acquisition of extinction without affecting extinction consolidation. This is supported by the results showing that microinfusion of SCH23390 into the BLA following a short-training session did not affect consolidation. These results further strengthen the role of mPFC in consolidation of extinction while highlighting the role of the D1 receptors in this process.

Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Whereas the neuronal substrates underlying the acquisition of auditory fear conditioning have been widely studied, the substrates and mechanisms mediating the acquisition of fear extinction remain largely elusive. Previous reports indicate that consolidation of fear extinction depends on the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) signalling pathway and on protein synthesis in the medial prefrontal cortex (mPFC). Based on experiments using the fear-potentiated startle paradigm suggesting a role for neuronal plasticity in the basolateral amygdala (BLA) during fear extinction, we directly addressed whether MAPK/ERK signalling in the basolateral amygdala is necessary for the acquisition of fear extinction using conditioned freezing as a read-out. First, we investigated the regional and temporal pattern of MAPK/ERK activation in the BLA following extinction learning in C57Bl/6J mice. Our results indicate that acquisition of extinction is associated with an increase of phosphorylated MAPK/ERK in the BLA. Moreover, we found that inhibition of the MAPK/ERK signalling pathway by intrabasolateral amygdala infusion of the MEK inhibitor, U0126, completely blocks acquisition of extinction. Thus, our results indicate that the MAPK/ERK signalling pathway is required for extinction of auditory fear conditioning in the BLA, and support a role for neuronal plasticity in the BLA during the acquisition of fear extinction.

Electrical stimulation of medial prefrontal cortex reduces conditioned fear in a temporally specific manner.

The authors recently showed that extinction of auditory fear conditioning leads to potentiation of tone-evoked activity of neurons in the infralimbic (IL) subregion of the medial prefrontal cortex, suggesting that IL inhibits fear after extinction (M. R. Milad, & G. J. Quirk, 2002). In support of this finding, pairing conditioned tones with brief (300-ms) electrical stimulation of IL reduces conditioned freezing. The present study showed that IL stimulation inhibits freezing if given 0.1 s after tone onset (the latency of tone-evoked responses) but has no effect if given either 1 s before or 1 s after tone onset. This suggests that IL gates the response of downstream structures such as the amygdala to fear stimuli.

Stimulation of Medial Prefrontal Cortex Decreases the Responsiveness of Central Amygdala Output Neurons

In extinction of auditory fear conditioning, rats learn that a tone no longer predicts the occurrence of a footshock. Recent lesion and unit recording studies suggest that the medial prefrontal cortex (mPFC) plays an essential role in the inhibition of conditioned fear following extinction. mPFC has robust projections to the amygdala, a structure that is known to mediate the acquisition and expression of conditioned fear. Fear conditioning potentiates the tone responses of neurons in the basolateral amygdala (BLA), which excite neurons in the central nucleus (Ce) of the amygdala. In turn, the Ce projects to the brainstem and hypothalamic areas that mediate fear responses. The present study was undertaken to test the hypothesis that the mPFC inhibits conditioned fear via feedforward inhibition of Ce output neurons. Recording extracellularly from physiologically identified brainstem-projecting Ce neurons, we tested the effect of mPFC prestimulation on Ce responsiveness to synaptic input. In support of our hypothesis, mPFC prestimulation dramatically reduced the responsiveness of Ce output neurons to inputs from the insular cortex and BLA. Thus, our findings support the idea that mPFC gates impulse transmission from the BLA to Ce, perhaps through GABAergic intercalated cells, thereby gating the expression of conditioned fear.