Systemic corticosterone enhances fear memory extinction in rats: Involvement of the infralimbic medial prefrontal cortex GABAA and GABAB receptors.

PURPOSE: The infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) regulates the extinction of conditioned fear memory. Glucocorticoid and gamma-aminobutyric acid (GABA) receptors are expressed in the mPFC and are also critical in fear extinction. This study investigated the possible interactive effects of the glucocorticoids and GABAergic system in the IL on the regulation of fear extinction. METHOD: The rats were trained using an auditory fear conditioning task during which they received three conditioned stimuli (tones, 30 s, 4 kHz, 80 dB), co-terminated with the three unconditioned stimuli (footshock, 0.8 mA, 1 s). Extinction testing was conducted over 3 days (Ext 1-3). Thirty minutes before the first extinction trial (Ext 1), the rats received bicuculline (BIC, 1 mg/kg/2 mL, intraperitoneal [i.p.]) as a GABAA receptor antagonist or CGP55845 (CGP, 0.1 mg/kg/2 ML, i.p.) as a GABAB receptor antagonist followed by systemic injection of corticosterone (CORT, 3 mg/kg/2 ML, i.p.). Furthermore, separate groups of rats received a bilateral intra-IL injection of BIC (100 ng/0.3 µL/side) or CGP (10 ng/0.3 µL/side) followed by a systemic injection of CORT (3 mg/kg/2 ML, i.p.) before the first extinction trial (Ext 1). The extracellular signal-regulated kinase (ERK1) and cAMP response element-binding (CREB) activity in the IL was examined by Western blot analysis after Ext 1. FINDING: The results indicated that systemic CORT injection facilitated fear extinction and increased the expression of ERK1 but not CREB in the IL. Both systemic and intra-IL co-injection of BIC or CGP blocked the effects of CORT on fear extinction and ERK1 expression. CONCLUSION: These findings suggest that glucocorticoids and the GABAergic system may modulate fear extinction through the ERK pathway in the IL.

Minimizing Variability in Developmental Fear Studies in Mice: Toward Improved Replicability in the Field.

In rodents, the first weeks of postnatal life feature remarkable changes in fear memory acquisition, retention, extinction, and discrimination. Early development is also marked by profound changes in brain circuits underlying fear memory processing, with heightened sensitivity to environmental influences and stress, providing a powerful model to study the intersection between brain structure, function, and the impacts of stress. Nevertheless, difficulties related to breeding and housing young rodents, preweaning manipulations, and potential increased variability within that population pose considerable challenges to developmental fear research. Here we discuss several factors that may promote variability in studies examining fear conditioning in young rodents and provide recommendations to increase replicability. We focus primarily on experimental conditions, design, and analysis of rodent fear data, with an emphasis on mouse studies. The convergence of anatomical, synaptic, physiological, and behavioral changes during early life may increase variability, but careful practice and transparency in reporting may improve rigor and consensus in the field. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Hippocampus-to-amygdala pathway drives the separation of remote memories of related events.

The mammalian brain can store and retrieve memories of related events as distinct memories and remember common features of those experiences. How it computes this function remains elusive. Here, we show in rats that recent memories of two closely timed auditory fear events share overlapping neuronal ensembles in the basolateral amygdala (BLA) and are functionally linked. However, remote memories have reduced neuronal overlap and are functionally independent. The activity of parvalbumin (PV)-expressing neurons in the BLA plays a crucial role in forming separate remote memories. Chemogenetic blockade of PV preserves individual remote memories but prevents their segregation, resulting in reciprocal associations. The hippocampus drives this process through specific excitatory connections with BLA GABAergic interneurons. These findings provide insights into the neuronal mechanisms that minimize the overlap between distinct remote memories and enable the retrieval of related memories separately.

The sexually divergent cFos activation map of fear extinction.

Objective: Post-traumatic stress disorder (PTSD) is a neuropsychiatric disorder that can develop after experiencing or witnessing a traumatic event. Exposure therapy is a common treatment for PTSD, but it has varying levels of efficacy depending on sex. In this study, we aimed to compare the sexual dimorphism in brain activation during the extinction of fear conditioning in male and female rats by detecting the c-fos levels in the whole brain. Methods: Thirty-two rats (Male: n = 16; Female: n = 16) were randomly separated into the extinction group as well as the non-extinction group, and fear conditioning was followed by extinction and non-extinction, respectively. Subsequently, brain sections from the sacrificed animal were performed immunofluorescence and the collected data were analyzed by repeated two-way ANOVAs as well as Pearson Correlation Coefficient. Results: Our findings showed that most brain areas activated during extinction were similar in both male and female rats, except for the reuniens thalamic nucleus and ventral hippocampi. Furthermore, we found differences in the correlation between c-fos activation levels and freezing behavior during extinction between male and female rats. Specifically, in male rats, c-fos activation in the anterior cingulate cortex was negatively correlated with the freezing level, while c-fos activation in the retrosplenial granular cortex was positively correlated with the freezing level; but in female rats did not exhibit any correlation between c-fos activation and freezing level. Finally, the functional connectivity analysis revealed differences in the neural networks involved in extinction learning between male and female rats. In male rats, the infralimbic cortex and insular cortex, anterior cingulate cortex and retrosplenial granular cortex, and dorsal dentate gyrus and dCA3 were strongly correlated after extinction. In female rats, prelimbic cortex and basolateral amygdala, insular cortex and dCA3, and anterior cingulate cortex and dCA1 were significantly correlated. Conclusion: These results suggest divergent neural networks involved in extinction learning in male and female rats and provide a clue for improving the clinical treatment of exposure therapy based on the sexual difference.

Cerebellar nuclei neurons projecting to the lateral parabrachial nucleus modulate classical fear conditioning.

Multiple brain regions are engaged in classical fear conditioning. Despite evidence for cerebellar involvement in fear conditioning, the mechanisms by which cerebellar outputs modulate fear learning and memory remain unclear. We identify a population of deep cerebellar nucleus (DCN) neurons with monosynaptic glutamatergic projections to the lateral parabrachial nucleus (lPBN) (DCN→lPBN neurons) in mice. While optogenetic suppression of DCN→lPBN neurons impairs auditory fear memory, activation of DCN→lPBN neurons elicits freezing behavior only after auditory fear conditioning. Moreover, auditory fear conditioning potentiates DCN-lPBN synapses, and subsequently, auditory cue activates lPBN neurons after fear conditioning. Furthermore, DCN→lPBN neuron activation can replace the auditory cue but not footshock in fear conditioning. These findings demonstrate that cerebellar nuclei modulate auditory fear conditioning via transmitting conditioned stimuli signals to the lPBN. Collectively, our findings suggest that the DCN-lPBN circuit is a part of neuronal substrates within interconnected brain regions underscoring auditory fear memory.

Involvement of canonical Wnt/ß-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/ß-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-GSK3ß and nuclear ß-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/ß-catenin pathway is involved in AFC extinction. To determine how Dkk1 affects canonical Wnt/ß-catenin signaling in AFC extinction, the protein levels of p-GSK3ß and ß-catenin were measured. We found that DKK1 produces a decrease in p-GSK3ß and ß-catenin. Moreover, we found that upregulating the Wnt/ß-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/ß-catenin signaling pathway might be a suitable way of therapeutically treating psychiatric disorders.

Corticosterone injection into the infralimbic prefrontal cortex enhances fear memory extinction: Involvement of GABA receptors and the extracellular signal-regulated kinase.

This study investigated the interactive effect of glucocorticoid and Gamma-aminobutyric acid (GABA) receptors in the Infralimbic (IL) cortex on fear extinction in rats' auditory fear conditioning task (AFC). Animals received 3 conditioning trial tones (conditioned stimulus, 30 s, 4 kHz, 80 dB) co-terminated with a footshock (unconditioned stimulus, 0.8 mA, 1 s). Extinction testing was conducted over 3 days (Ext 1-3) after conditioning. Intra-IL injection of corticosterone (CORT, 20 ng/0.3 µl/side) was performed 15 min before the first extinction trial (Ext 1) which attenuated auditory fear expression in subsequent extinction trials (Ext 1-3), demonstrating fear memory extinction enhancement. Co-injection of the GABAA agonist muscimol (250 ng/0.3 µl/side) or the GABAB agonist baclofen (250 ng/0.3 µl/side) 15 min before corticosterone, did not significantly affect the facilitative effects of corticosterone on fear extinction. However, co-injection of the GABAA antagonist bicuculline (BIC, 100 ng/0.3 µl/side) or the GABAB antagonist CGP35348 (CGP, 100 ng/0.3 µl/side) 15 min before corticosterone, blocked the facilitative effects of corticosterone on fear extinction. Moreover, extracellular signal-regulated kinase (ERK) and cAMP response element-binding (CREB) in the IL were examined by Western blotting analysis after the first extinction trial (Ext 1) in some groups. Intra-IL injection of corticosterone increased the ERK activity but not CREB. Co-injection of the bicuculline or CGP35348 blocked the enhancing effect of corticosterone on ERK expression in the IL. Glucocorticoid receptors (GRs) activation in the IL cortex by corticosterone increased ERK activity and facilitated fear extinction. GABAA or GABAB antagonists decreased ERK activity and inhibited corticosterone's effect. GRs and GABA receptors in the IL cortex jointly modulate the fear extinction processes via the ERK pathway. This pre-clinical animal study may highlight GRs and GABA interactions in the IL cortex modulating fear memory processes in fear-related disorders such as post-traumatic stress disorder (PTSD).

When Artificial Intelligence Voices Human Concerns: The Paradoxical Effects of AI Voice on Climate Risk Perception and Pro-Environmental Behavioral Intention.

Artificial intelligence (AI)-enabled text-to-speech transformation has been widely employed to deliver online information in various fields. However, few studies have investigated the effect of the AI voice in environmental risk communication, especially in the field of climate change, an issue that poses a severe threat to global public health. To address this gap, the current study examines how the AI voice impacts the persuasive outcome of climate-related information and the potential mechanism that underlies this process. Based on the social and affect heuristics of voice, we propose a serial mediation model to test the effect of climate-related information delivered by different voice types (AI voice vs. human voice) in eliciting risk perception and motivating pro-environmental behavioral intention. Through an online auditory experiment (N = 397), we found the following. First, the AI voice was as effective as the human voice in eliciting risk perception and motivating pro-environmental behavioral intention. Second, compared with human voice, the AI voice yielded a listener's lower level of perceived identity oneness with the speaker, which decreased risk perception and subsequently inhibited pro-environmental behavioral intention. Third, compared with human voice, the AI voice produced a higher level of auditory fear, which increased risk perception and thereby led to stronger pro-environmental behavioral intention. The paradoxical role of the AI voice and its wise use in environmental risk communication for promoting global public health are discussed.

Local memory allocation recruits memory ensembles across brain regions.

Memories are thought to be stored in ensembles of neurons across multiple brain regions. However, whether and how these ensembles are coordinated at the time of learning remains largely unknown. Here, we combined CREB-mediated memory allocation with transsynaptic retrograde tracing to demonstrate that the allocation of aversive memories to a group of neurons in one brain region directly affects the allocation of interconnected neurons in upstream brain regions in a behavioral- and brain region-specific manner in mice. Our analysis suggests that this cross-regional recruitment of presynaptic neurons is initiated by downstream memory neurons through a retrograde mechanism. Together with statistical modeling, our results indicate that in addition to the anterograde flow of information between brain regions, the establishment of interconnected, brain-wide memory traces relies on a retrograde mechanism that coordinates memory ensembles at the time of learning.

A stable sensory map emerges from a dynamic equilibrium of neurons with unstable tuning properties.

Recent long-term measurements of neuronal activity have revealed that, despite stability in large-scale topographic maps, the tuning properties of individual cortical neurons can undergo substantial reformatting over days. To shed light on this apparent contradiction, we captured the sound response dynamics of auditory cortical neurons using repeated 2-photon calcium imaging in awake mice. We measured sound-evoked responses to a set of pure tone and complex sound stimuli in more than 20,000 auditory cortex neurons over several days. We found that a substantial fraction of neurons dropped in and out of the population response. We modeled these dynamics as a simple discrete-time Markov chain, capturing the continuous changes in responsiveness observed during stable behavioral and environmental conditions. Although only a minority of neurons were driven by the sound stimuli at a given time point, the model predicts that most cells would at least transiently become responsive within 100 days. We observe that, despite single-neuron volatility, the population-level representation of sound frequency was stably maintained, demonstrating the dynamic equilibrium underlying the tonotopic map. Our results show that sensory maps are maintained by shifting subpopulations of neurons "sharing" the job of creating a sensory representation.

Role of Amygdala-Infralimbic Cortex Circuitry in Glucocorticoid-induced Facilitation of Auditory Fear Memory Extinction.

Introduction: The basolateral amygdala (BLA) and infralimbic area (IL) of the medial prefrontal cortex (mPFC) are two interconnected brain structures that mediate both fear memory expression and extinction. Besides the well-known role of the BLA in the acquisition and expression of fear memory, projections from IL to BLA inhibit fear expression and have a critical role in fear extinction. However, the details of IL-BLA interaction have remained unclear. Here, we investigated the role of functional reciprocal interactions between BLA and IL in mediating fear memory extinction. Methods: Using lidocaine (LID), male rats underwent unilateral or bilateral inactivation of the BLA and then unilateral intra-IL infusion of corticosterone (CORT) prior to extinction training of the auditory fear conditioning paradigm. Freezing behavior was reported as an index for conditioned fear. Infusions were performed before the extinction training, allowing us to examine the effects on fear expression and further extinction memory. Experiments 1-3 investigated the effects of left or right infusion of CORT into IL and LID unilaterally into BLA on fear memory extinction. Results: Intra-IL infusion of CORT in the right hemisphere reduced freezing behavior when administrated before the extinction training. Auditory fear memory extinction was impaired by asymmetric inactivation of BLA and CORT infusion in the right IL; however, the same effect was not observed with symmetric inactivation of BLA. Conclusion: IL-BLA neural circuit may provide additional evidence for the contribution of this circuit to auditory fear extinction. This study demonstrates dissociable roles for right or left BLA in subserving the auditory fear extinction. Our finding also raises the possibility that left BLA-IL circuitry may mediate auditory fear memory extinction via underlying mechanisms. However, further research is required in this area. Highlights: Corticosterone infusion in the right (but not the left) infralimbic area facilitates auditory fear memory extinction.Corticosterone infusion in the right infralimbic area following symmetric basolateral amygdala inactivation has no effect on auditory fear memory extinction.Asymmetric basolateral amygdala inactivation prior to corticosterone infusion into the right infralimbic area impairs auditory fear memory extinction. Plain Language Summary: Previous studies have established that glucocorticoids, which are released in stressful conditions, enhance fear memory extinction. In this study, we found that corticosterone infusion into the right infralimbic area, but not the left one, facilitates auditory fear memory extinction. The effect of corticosterone infusion in the infralimbic area was not blocked by the intra-basolateral amygdala injections of lidocaine when administrated in the ipsilateral hemisphere. However, asymmetric basolateral amygdala inactivation and corticosterone infusion into the right infralimbic area impairs auditory fear memory extinction.

Frequency-Dependent Plasticity in the Temporal Association Cortex Originates from the Primary Auditory Cortex, and Is Modified by the Secondary Auditory Cortex and the Medial Geniculate Body.

The brain areas that mediate the formation of auditory threat memory and perceptual decisions remain uncertain to date. Candidates include the primary (A1) and secondary (A2) auditory cortex, the medial division of the medial geniculate body (MGm), amygdala, and the temporal association cortex. We used chemogenetic and optogenetic manipulations with in vivo and in vitro patch-clamp recordings to assess the roles of these brain regions in threat memory learning in female mice. We found that conditioned sound (CS) frequency-dependent plasticity resulted in the formation of auditory threat memory in the temporal association cortex. This neural correlated auditory threat memory depended on CS frequency information from A1 glutamatergic subthreshold monosynaptic inputs, CS lateral inhibition from A2 glutamatergic disynaptic inputs, and non-frequency-specific facilitation from MGm glutamatergic monosynaptic inputs. These results indicate that the A2 and MGm work together in an inhibitory-facilitative role.SIGNIFICANCE STATEMENT: The ability to recognize specific sounds to avoid predators or seek prey is a useful survival tool. Improving this ability through experiential learning is an added advantage requiring neural plasticity. As an example, humans must learn to distinguish the sound of a car horn, and thus avoid oncoming traffic. Our research discovered that the temporal association cortex can encode this kind of auditory information through tonal receptive field plasticity. In addition, the results revealed the underlying synaptic mechanisms of this process. These results extended our understanding of how meaningful auditory information is processed in an animal's brain.

Oxytocin in dorsal hippocampus facilitates auditory fear memory extinction in rats.

Fear extinction is impaired in some psychiatric disorders. Any treatment that facilitates the extinction of fear is a way to advance the treatment of related psychiatric disorders. Recent studies have highlighted the role of oxytocin (OT) in fear extinction, but the endogenous release of OT during fear extinction in the dorsal hippocampal (dHPC) is not clear. We investigated the release of OT during fear extinction and the role of the HPC - medial prefrontal cortex (mPFC) circuit and BDNF in the effects of exogenous OT on auditory fear conditioning in male rats. We found that the release of endogenous OT in the dHPC is significantly increased during the fear extinction process as measured by the microdialysis method. Increased freezing response in the OT-treated rats compared to saline-treated rats showed that exogenous OT in the dHPC enhanced the fear extinction. Injection of BDNF antagonist (ANA-12) into the infralimbic (IL) blocked the effect of exogenous OT on the dHPC. Following OT injection, BDNF levels increased in the dHPC, ventral HPC, and IL cortex; but decreased in the prelimbic cortex (PL). Finally, OT microinjected into the dHPC significantly increased neural activity of pyramidal neurons of the CA1-vHPC and IL but decreased the neural activity in the PL cortex. Our findings strongly support that the dHPC endogenous OT plays a crucial role in enhancing fear extinction. It seems that the activation of the HPC-mPFC pathway, and consequently, the release of BDNF in the IL cortex mediates the enhancing effects of OT on fear extinction.

Chronic light deprivation induces different effects on spatial and fear memory and hippocampal BDNF/TRKB expression during light and dark phases of rat diurnal rhythm.

Disruptions in light/dark cycle have been associated with an altered ability to form and retrieve memory in human and animals. Animal studies have shown that chronic light deprivation disrupts the light/dark cycle and alters the neural connections that mediate hippocampal memory formation. In order to better understand how light deprivation affects the formation and retrieval of memory in adult rats, we examined the effect of total darkness on spatial and auditory fear learning and memory formation and BDNF/TRKB protein levels during the light and dark phases of the rat circadian cycle. Male Wistar rats (n = 60), were randomly divided into two main groups: normal rearing (NR, 12 h light/dark cycle for 3 weeks) and dark rearing (DR, kept in constant darkness for 3 weeks); and each of these groups had a "light (day)" and "dark (night)" sub-group. After 3 weeks, the Morris Water maze and auditory fear conditioning were used to assess spatial and fear memory acquisition and retrieval, respectively. BDNF and TRKB protein levels in the hippocampus of rats from the four sub-groups were measured by Western blot, at the completion of the 3 week constant darkness exposure and after the behavioral experiments. These studies revealed that DR for 3 weeks impaired spatial memory retrieval and enhanced extinction of auditory fear memory specifically during the light (day) phase. DR also eliminated the normal fluctuations in BDNF/TRKB levels observed in the hippocampus across the light/dark cycle.

Auditory fear conditioning facilitates neurotransmitter release at lateral amygdala to basal amygdala synapses.

The lateral amygdala (LA) is a main sensory input site from the cortical and thalamic regions. In turn, LA glutamatergic pyramidal neurons strongly project to the basal amygdala (BA). Although it is well known that auditory fear conditioning involves synaptic potentiation in the LA, it is not clear whether the LA-BA synaptic transmission is modified upon auditory fear conditioning. Here we found that high-frequency stimulation ex vivo resulted in long-term potentiation (LTP) with a concomitant enhancement of neurotransmitter release at LA-BA synapses. Auditory fear conditioning also led to the presynaptic facilitation at LA-BA synapses. Meanwhile, AMPA/NMDA current ratio was not changed upon fear conditioning, excluding the involvement of postsynaptic mechanism. Notably, fear conditioning occluded electrically induced ex vivo LTP in the LA-BA pathway, indicating that the conditioning and electrically induced LTP share common mechanisms. Our findings suggest that the presynaptic potentiation of LA-BA synapses may be involved in fear conditioning.

Dissociated Role of Thalamic and Cortical Input to the Lateral Amygdala for Consolidation of Long-Term Fear Memory.

Post-encoding coordinated reactivation of memory traces distributed throughout interconnected brain regions is thought to be critical for consolidation of memories. However, little is known about the role of neural circuit pathways during post-learning periods for consolidation of memories. To investigate this question, we optogenetically silenced the inputs from both auditory cortex and thalamus in the lateral amygdala (LA) for 15 min immediately following auditory fear conditioning (FC) and examined its effect on fear memory formation in mice of both sexes. Optogenetic inhibition of both inputs disrupted long-term fear memory formation tested 24 h after FC. This effect was specific such that the same inhibition did not affect short-term memory and context-dependent memory. Moreover, long-term memory was intact if the inputs were inhibited at much later time points after FC (3 h or 1 d after FC), indicating that optical inhibition for 15 min itself does not produce any nonspecific deleterious effect on fear memory retrieval. Selective inhibition of thalamic input was sufficient to impair consolidation of auditory fear memory. In contrast, selective inhibition of cortical input disrupted remote fear memory without affecting recent memory. These results reveal a dissociated role of thalamic and cortical input to the LA during early post-learning periods for consolidation of long-term fear memory.SIGNIFICANCE STATEMENT Coordinated communications between brain regions are thought to be essential during post-learning periods for consolidation of memories. However, the role of specific neural circuit pathways in this process has been scarcely explored. Using a precise optogenetic inhibition of auditory input pathways, either thalamic or cortical or both, to the LA during post-training periods, we here show that thalamic input is required for consolidation of both recent and remote fear memory, whereas cortical input is crucial for consolidation of remote fear memory. These results reveal a dissociated role of auditory input pathways to the LA for consolidation of long-term fear memory.

Microglial synaptic pruning on axon initial segment spines of dentate granule cells: Sexually dimorphic effects of early-life stress and consequences for adult fear response.

Axon initial segments (AIS) of dentate granule cells in the hippocampus exhibit prominent spines (AISS) during early development that are associated with microglial contacts. In the present study, we investigated whether developmental changes in AISS could be modified by early-life stress (ELS), specifically neonatal maternal separation (MS), through stress hormones and microglial activation and examined the potential behavioural consequences. We examined AISS at postnatal day (PND)5, 15 and 50, using Golgi-Cox staining and anatomical analysis. Neurone-microglial interaction was assessed using antibodies against ankyrin-G, PSD-95 and Iba1, for AIS, AISS and microglia visualisation, respectively, in normally reared and neonatal maternally separated male and female rats. We observed a higher density of AISS in ELS rats at both PND15 and PND50 compared to controls. Effects were more pronounced in females than males. AIS-associated microglia in ELS rats showed a hyper-ramified morphology and less co-localisation with PSD-95 compared to controls at PND15. ELS-associated alteration in microglial morphology and synaptic pruning was mimicked by treatment of acute hippocampal slices of normally reared rats with vasopressin. ELS rats exhibited increased freezing behaviour during auditory fear memory testing, which was more pronounced in female subjects and corresponded with increased Fos expression in dorsal and ventral dentate granule cells. Thus, microglial synaptic pruning in dentate AIS of hippocampus is influenced by ELS, with demonstrable sex bias regarding its anatomical characteristics and subsequent fear-induced defensive behaviours.

Temporary inactivation of the infralimbic cortex impairs while the blockade of its dopamine D2 receptors enhances auditory fear extinction in rats.

Fear extinction is defined as decline in conditioned fear responses that occurs with repeated and non-reinforced exposure to a feared conditioned stimulus. Experimental evidence suggests that the extinction of fear memory requires the integration of the medial prefrontal cortex (mPFC); nevertheless, the role of its sub-regions in regulating the expression and extinction of auditory fear has been rarely addressed in literature. The present study examined the roles of the infra-limbic (IL) and pre-limbic (PL) regions of the mPFC in the expression and extinction of auditory fear by temporally deactivating these regions using lidocaine (10 µg/0.5 µl) before training male Wistar rats in auditory fear-conditioning tasks. The results showed increased freezing levels and impaired extinction through deactivating the IL rather than the PL cortex. Given the role of the dopaminergic pathways in regulating fear memory, this study also investigated the role of D2 receptors located in the IL cortex in fear extinction. Fear extinction was improved in an inverted U-shape pattern through the intra-IL infusion of 15.125, 31.25, 62.5, 125, 250 and 500 ng/0.5 µl of the D2 receptor antagonist sulpiride. In other words, the moderate doses, i.e. 31.25, 62.5, 125, 250 ng/0.5 µl, enhanced auditory fear extinction, whereas the lowest and highest doses, i.e. 15.125 and 500 ng/0.5 µl, were ineffective. These findings demonstrated the key roles of the IL cortex and its dopamine D2 receptors in regulating auditory fear in rats.

Lack of drug-induced post-retrieval amnesia for auditory fear memories in rats.

BACKGROUND: Long-term memory formation is generally assumed to involve the permanent storage of recently acquired memories, making them relatively insensitive to disruption, a process referred to as memory consolidation. However, when retrieved under specific circumstances, consolidated fear memories are thought to return to a labile state, thereby opening a window for modification (e.g., attenuation) of the memory. Several interventions during a critical time frame after this destabilization seem to be able to alter the retrieved memory, for example by pharmacologically interfering with the restabilization process, either by direct protein synthesis inhibition or indirectly, using drugs that can be safely administered in patients (e.g., propranolol). Here, we find that, contrary to expectations, systemic pharmacological manipulations in auditory fear-conditioned rats do not lead to drug-induced post-retrieval amnesia. RESULTS: In a series of well-powered auditory fear conditioning experiments (four with propranolol, 10 mg/kg, two with rapamycin, 20-40 mg/kg, one with anisomycin, 150 mg/kg and cycloheximide, 1.5 mg/kg), we found no evidence for reduced cued fear memory expression during a drug-free test in adult male Sprague-Dawley rats that had previously received a systemic drug injection upon retrieval of the tone fear memory. All experiments used standard fear conditioning and reactivation procedures with freezing as the behavioral read-out (conceptual or exact replications of published reports) and common pharmacological agents. Additional tests confirmed that the applied drug doses and administration routes were effective in inducing their conventional effects on expression of fear (propranolol, acutely), body weight (rapamycin, anisomycin, cycloheximide), and consolidation of extinction memories (cycloheximide). CONCLUSIONS: In contrast with previously published studies, we did not find evidence for drug-induced post-retrieval amnesia, underlining that this effect, as well as its clinical applicability, may be considerably more constrained and less readily reproduced than what the current literature would suggest.

The role of p21-activated kinase in maintaining the fear learning-induced modulation of excitation/inhibition ratio in lateral amygdala.

We study the relations between different learning paradigms and enduring changes in excitatory synaptic transmission. Here we show that auditory fear conditioning (AFC), but not olfactory fear conditioning (OFC) training, led to enduring enhancement in AMPA-mediated miniature EPSCs (mEPSCs). Moreover, olfactory unpaired training led to a stable significant reduction in excitatory synaptic transmission. However, olfactory discrimination learning (OD) did not modulate postsynaptic AMPA-mediated mEPSCs in LA. The p21-activated kinase (PAK) activity, previously shown to have a key role in maintaining persistent long-lasting enhancement in synaptic inhibition after OFC, has an opposing effect on excitatory synaptic transmission. PAK maintained the level of excitatory synaptic transmission in the amygdala in all experimental groups, except in neurons in the OFC trained rats. PAK also maintained excitatory synaptic transmission in all neurons of auditory fear conditioning and naïve training groups except in neurons of the auditory safety learning. Safety learning was previously shown in our study to enhance synaptic inhibition. We thus suggest that PAK maintains inhibitory synaptic transmission in a learning-dependent manner and on the other hand affects excitatory synaptic transmission only in groups where learning has not affected inhibitory transmission. Thus, PAK controls learning-induced changes in the excitation/inhibition balance.