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INTRODUCTION: Medicinal cannabis has proliferated around the world and there is increasing interest in the therapeutic potential of individual plant-derived cannabinoids (phytocannabinoids). Preclinical evidence suggests the phytocannabinoid cannabigerol (CBG) could be useful in treating brain disorders, including stress and anxiety-related disorders. In this study, we aimed to explore whether CBG disrupts various contextually conditioned fear memory processes and trauma-induced anxiety-related behavior in a mouse model of post-traumatic stress disorder (PTSD). MATERIALS AND METHODS: All mice underwent contextual fear conditioning. CBG was administered between 1 and 60 mg/kg intraperitoneally (i.p.). We first assessed the effects of repeated CBG exposure on long-term fear memories. We also examined whether acute CBG affected various fear memory processes, namely expression, acquisition, consolidation, and reconsolidation of conditioned fear. Finally, the effect of acute CBG administration on stress-induced anxiety in the light/dark test was assessed. RESULTS: Repeated CBG exposure did not affect long-term conditioned fear that was observed 24 days after the conditioning session. Moreover, acute CBG administration did not influence the acquisition, consolidation, reconsolidation, or expression of contextually conditioned fear. Acute CBG treatment also did not affect stress-induced anxiety-related behaviors in the light/dark test. CONCLUSIONS: CBG was ineffective in disrupting long-term fear memories, various conditioned fear memory processes, or stress-induced anxiety-related behavior in mice. These preclinical data suggest CBG may have limited scope in the treatment of PTSD and stress-related anxiety.

Neuron-astrocyte interactions are now recognized as essential to brain functioning and information processing. They occur at multiple levels, at different cellular locations, and upon different physiological states. Hence, the general aim of this thesis was to unravel structural and molecular interactions of astrocytes with neurons that contribute to synaptic function and memory processing. To that end: In Chapter 2, I aimed to describe the implementation of a recently developed new technique, NAPA (neuron-astrocyte proximity assay), that measures and characterizes astrocyte leaflets proximity to synapses in living tissue. Hence, I present a stepwise protocol on how to perform and measure sensitized emission Förster resonance energy transfer (SE-FRET) in cell lines and acute brain slices. After in vitro testing in HEK293T cells, we used the approach to visualize and track the static and dynamic proximity of striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons (MSNs) in living tissue. In Chapter 3, I aimed to examine astrocyte leaflet-synapse spatial interaction in the hippocampus during the formation and consolidation of fear memories, and to determine whether this proximity is critical for synaptic transmission and fear memory expression. I manipulated astrocyte leaflets in vivo by depleting Ezrin, a leaflet-structural protein, using CRISPR-saCas9 gene editing. Next, I used multidisciplinary approaches, including FRET analysis, electron microscopy, genetically encoded glutamate sensors, whole-cell patch-clamp recordings and contextual fear conditioning to show that the apposition of astrocyte leaflets to the synaptic cleft influences contextual fear memory expression and gates neuronal activation in a context- and time-dependent manner. In Chapter 4, I aimed to characterize hippocampal high-affinity glutamate transporter (GLT-1) protein complexes by combining interactive proteomics approaches. We showed that GLT-1 form at least four stable distinct protein complexes that differ in size and protein composition. Next, we revealed that GLT-1 protein complexes are dynamically assembled during contextual fear conditioning, and differently regulated in the early stages of AD. In Chapter 5, I aimed to develop an automated high-throughput analysis method to measure reactive astrogliosis in vitro, and to determine the effect of nutritional supplementation (FC) on the induction of astrocyte reactivity by pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We found that extracellular factors, such as extracellular matrix proteins, and culture media have a great influence on the development of the reactive phenotype. In addition, we showed that treatment with FC has an acute beneficial effect on cytokine-induced reactive astrogliosis. Taken together, with this research I deepened our understanding on the structural and molecular interactions of hippocampal astrocytes with neurons, and how this dynamic interplay contribute to synaptic function and mnemonic processing. Furthermore, this data sheds the light on the role of astrocytes in maladaptive/dysfunctional neuronal networks and strengthens the importance to therapeutically target astrocytes to tackle neurological disorders.

Neuron-astrocyte interactions are now recognized as essential to brain functioning and information processing. They occur at multiple levels, at different cellular locations, and upon different physiological states. Hence, the general aim of this thesis was to unravel structural and molecular interactions of astrocytes with neurons that contribute to synaptic function and memory processing. To that end: In Chapter 2, I aimed to describe the implementation of a recently developed new technique, NAPA (neuron-astrocyte proximity assay), that measures and characterizes astrocyte leaflets proximity to synapses in living tissue. Hence, I present a stepwise protocol on how to perform and measure sensitized emission Förster resonance energy transfer (SE-FRET) in cell lines and acute brain slices. After in vitro testing in HEK293T cells, we used the approach to visualize and track the static and dynamic proximity of striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons (MSNs) in living tissue. In Chapter 3, I aimed to examine astrocyte leaflet-synapse spatial interaction in the hippocampus during the formation and consolidation of fear memories, and to determine whether this proximity is critical for synaptic transmission and fear memory expression. I manipulated astrocyte leaflets in vivo by depleting Ezrin, a leaflet-structural protein, using CRISPR-saCas9 gene editing. Next, I used multidisciplinary approaches, including FRET analysis, electron microscopy, genetically encoded glutamate sensors, whole-cell patch-clamp recordings and contextual fear conditioning to show that the apposition of astrocyte leaflets to the synaptic cleft influences contextual fear memory expression and gates neuronal activation in a context- and time-dependent manner. In Chapter 4, I aimed to characterize hippocampal high-affinity glutamate transporter (GLT-1) protein complexes by combining interactive proteomics approaches. We showed that GLT-1 form at least four stable distinct protein complexes that differ in size and protein composition. Next, we revealed that GLT-1 protein complexes are dynamically assembled during contextual fear conditioning, and differently regulated in the early stages of AD. In Chapter 5, I aimed to develop an automated high-throughput analysis method to measure reactive astrogliosis in vitro, and to determine the effect of nutritional supplementation (FC) on the induction of astrocyte reactivity by pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We found that extracellular factors, such as extracellular matrix proteins, and culture media have a great influence on the development of the reactive phenotype. In addition, we showed that treatment with FC has an acute beneficial effect on cytokine-induced reactive astrogliosis. Taken together, with this research I deepened our understanding on the structural and molecular interactions of hippocampal astrocytes with neurons, and how this dynamic interplay contribute to synaptic function and mnemonic processing. Furthermore, this data sheds the light on the role of astrocytes in maladaptive/dysfunctional neuronal networks and strengthens the importance to therapeutically target astrocytes to tackle neurological disorders.

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.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.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.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.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.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.

The prospect of exploiting memory reconsolidation to treat mental health disorders has received great research interest, particularly following demonstrations that the β-adrenergic receptor antagonist propranolol, which is safe for use in humans, can disrupt the reconsolidation of pavlovian conditioned fear memories. However, recent studies have failed to replicate the effects of propranolol on fear memory reconsolidation, and have questioned whether treatments based upon reconsolidation blockade would be robust enough for clinical translation. It remains possible, though, that studies reporting no effect of propranolol on memory reconsolidation could be due to a failure to engage the memory destabilisation process, which is necessary for the memory to become susceptible to disruption with amnestic agents. Demonstrating that memory destabilisation has not been engaged is challenging when only using behavioural measures, but there are molecular correlates of memory destabilisation that can be used to determine whether memory lability has been induced. Here, we attempted to replicate the classic finding that systemic administration of propranolol disrupts the reconsolidation of a pavlovian auditory fear memory. Following a failure to replicate, we manipulated the parameters of the memory reactivation session to enhance prediction error in an attempt to overcome the boundary conditions of reconsolidation. On finding no disruption of memory despite these manipulations, we examined the expression of the post-synaptic density protein Shank in the basolateral amygdala. Degradation of Shank has been shown to correlate with the induction of memory lability, but we found no effect on Shank expression, consistent with the lack of observed behavioural effects.

During fear learning, defensive behaviors like freezing need to be finely balanced in the presence or absence of threat-predicting cues (conditioned stimulus, CS). Nevertheless, the circuits underlying such balancing are largely unknown. Here, we investigate the role of the ventral tail striatum (vTS) in auditory-cued fear learning of male mice.In-vivoCa2+imaging showed that robust sub- populations of direct (D1R+) and indirect pathway neurons (Adora+) in the vTS responded to footshocks, and to the initiation of movements after freezing; moreover, a sub-population of D1R+ neurons increased its responsiveness to an auditory CS during fear learning.In-vivooptogenetic silencing shows that footshock-driven activity of D1R+ neurons contributes to fear memory formation, whereas Adora+ neurons modulate freezing in the absence of a learned CS. Circuit tracing identified the posterior insular cortex (pInsCx) as an important cortical input to the vTS, and recording of optogenetically-evoked EPSCs revealed long-term plasticity with opposite outcomes at the cortical synapses onto D1R+ - and Adora+ neurons. Thus, direct- and indirect pathways neurons of the vTS show differential signs of plasticity after fear learning, and balance defensive behaviors in the presence and absence of learned sensory cues.

This page contains the links to the preregistration and clinical trial registration of the human study that is part of https://osf.io/ydwba.

The claustrum is a small subcortical nucleus present in reptiles, birds and mammals that has extensive reciprocal connections with the whole cortex and with subcortical areas. Thus far the claustrum has been speculated to be involved in a variety of brain mechanisms, but its precise function remains unknown. We used in vivo electrophysiological recording during freely moving behavior and fear conditioning in combination with chemogenetic tools to investigate the role of the claustrum in cortico-cortical communication. We showed that inhibition of the claustrum induced a modification in the flow of information, as well as in the co-occurrence of brain oscillations associated with memory events, across distant cortical areas. These changes were associated with alterations in the consolidation and the retrieval of fear memory. Our findings indicate that the claustrum acts as a conductor of cortico-cortical communication, and that this communication is necessary for the correct expression of fear memory.

Preregistrations and data related to Luyten et al., 2023.

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 STATEMENTCoordinated 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.

Clustered protocadherins (Pcdhs) are a large family of cadherin-like cell adhesion proteins that are central for neurite self-avoidance and neuronal connectivity in the brain. Their downstream nonreceptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2, also known as Ptk2b, Cakb, Raftk, Fak2, and Cadtk) is predominantly expressed in the hippocampus. We constructed Pyk2-null mouse lines and found that these mutant mice showed enhancement in contextual fear memory, without significant change in auditory-cued and spatial-referenced learning and memory. In addition, by preparing Y402F mutant mice, we observed that Pyk2 suppressed contextual fear memory in an autophosphorylation-independent manner. Moreover, using high-throughput RNA sequencing, we found that immediate early genes, such as Npas4, cFos, Zif268/Egr1, Arc, and Nr4a1, were enhanced in Pyk2-null mice. We further showed that Pyk2 disruption affected pyramidal neuronal complexity and spine dynamics. Thus, we demonstrated that Pyk2 is a novel fear memory suppressor molecule and Pyk2-null mice provide a model for understanding fear-related disorders. These findings have interesting implications regarding dysregulation of the Pcdh‒Pyk2 axis in neuropsychiatric disorders.

Fear extinction is not permanent but is instead more vulnerable than the original fear memory, as traditionally shown by the return of fear phenomena. Because of this, techniques to mitigate the return of fear are needed in the clinical treatment of related psychological conditions. One promising strategy is the occasional reinforced extinction treatment, introducing a gradual and sparse number of conditioned stimulus–unconditioned stimulus (CS-US) pairings within the extinction treatment. We present the results of three experiments in which we used a threat conditioning procedure in humans. Our main aim was to evaluate whether occasional reinforced extinction could reduce two different forms of relapse: spontaneous recovery (Experiments 1 and 2) and reinstatement (Experiment 3). Contrary to our predictions and previous literature, the results indicate that an occasional reinforcement treatment did not mitigate relapse compared with standard extinction. From a theoretical standpoint, these results are more consistent with the idea that extinction entails the acquisition of new knowledge than with the idea that there are conditions in which extinction leads to a weakening of the original fear memory. These findings also question the generality of the potential benefits of using occasional reinforced extinction in clinical settings.

Mild traumatic brain injury (mTBI) increases the risk of posttraumatic stress disorder (PTSD) in military populations. Utilizing translationally relevant animal models is imperative for establishing a platform to delineate neurobehavioral deficits common to clinical PTSD that emerge in the months to years following mTBI. Such platforms are required to facilitate preclinical development of novel therapeutics. First, this mini review provides an overview of the incidence of PTSD following mTBI in military service members. Secondly, the translational relevance of fear conditioning paradigms used in conjunction with mTBI in preclinical studies is evaluated. Next, this review addresses an important gap in the current preclinical literature; while incubation of fear has been studied in other areas of research, there are relatively few studies pertaining to the enhancement of cued and contextual fear memory over time following mTBI. Incubation of fear paradigms in conjunction with mTBI are proposed as a novel behavioral approach to advance this critical area of research. Lastly, this review discusses potential neurobiological substrates implicated in altered fear memory post mTBI.

BACKGROUND: The formation and retrieval of fear memories depends on orchestrated synaptic activity of neuronal ensembles within the hippocampus, and it is becoming increasingly evident that astrocytes residing in the environment of these synapses play a central role in shaping cellular memory representations. Astrocyte distal processes, known as leaflets, fine-tune synaptic activity by clearing neurotransmitters and limiting glutamate diffusion. However, how astroglial synaptic coverage contributes to mnemonic processing of fearful experiences remains largely unknown.METHODS: We used electron microscopy to observe changes in astroglial coverage of hippocampal synapses during consolidation of fear memory in mice. To manipulate astroglial synaptic coverage, we depleted ezrin, an integral leaflet-structural protein, from hippocampal astrocytes using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing. Next, a combination of Föster resonance energy transfer analysis, genetically encoded glutamate sensors, and whole-cell patch-clamp recordings was used to determine whether the proximity of astrocyte leaflets to the synapse is critical for synaptic integrity and function.RESULTS: We found that consolidation of a recent fear memory is accompanied by a transient retraction of astrocyte leaflets from hippocampal synapses and increased activation of NMDA receptors. Accordingly, astrocyte-specific depletion of ezrin resulted in shorter astrocyte leaflets and reduced astrocyte contact with the synaptic cleft, which consequently boosted extrasynaptic glutamate diffusion and NMDA receptor activation. Importantly, after fear conditioning, these cellular phenotypes translated to increased retrieval-evoked activation of CA1 pyramidal neurons and enhanced fear memory expression.CONCLUSIONS: Together, our data show that withdrawal of astrocyte leaflets from the synaptic cleft is an experience-induced, temporally regulated process that gates the strength of fear memories.

The aim of the current study is to gauge the presence of publication bias in a well-delineated part of the reconsolidation literature. To this end, we will perform a systematic review of the literature on reactivation-dependent amnesia for contextual fear memories in rodents, followed by a statistical assessment of publication bias in this sample. In addition, relevant researchers will be contacted for unpublished results, which will be included in the current manuscript.

Endocannabinoids are involved in various physiological functions, including synaptic plasticity and memory, and some psychiatric disorders, such as posttraumatic stress disorder (PTSD), through the activation of cannabinoid (CB) receptors. Patients with PTSD often show excessive fear memory and impairment of fear extinction (FE). It has been reported that the stability of acquired fear memory is altered through multiple memory stages, such as consolidation and reconsolidation. FE also affects the stability of fear memory. Each stage of fear memory formation and FE are regulated by different molecular mechanisms, including the CB system. However, to the best of our knowledge, no review summarizes the role of the CB system during each stage of fear memory formation and FE. In this review, we summarize the roles of endocannabinoids in fear memory formation and FE. Moreover, based on the summary, we propose a new hypothesis for the role of endocannabinoids in fear regulation, and discuss treatment for PTSD using CB system-related drugs.

Post-traumatic stress disorder (PTSD) is characterized by the co-existence of a persistent strong memory of the traumatic experience and amnesia for the peritraumatic context. Most animal models, however, fail to account for the contextual amnesia which is considered to play a critical role in the etiology of PTSD intrusive memories. It is also unclear how aging affects PTSD-like memory. Glucocorticoids alter the formation and retention of fear-associated memory. Here, we investigated whether a deficiency of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) (an intracellular glucocorticoid generating enzyme) and aging modulates fear conditioning and PTSD-like memory in mice. We first measured memory in 6 months and 24 months old 11β-HSD1 deficient (HSD1 KO) and wildtype (WT) mice following paired tone-shock fear conditioning. Then, separate groups of mice were exposed to restraint stress immediately after unpaired tone-shock contextual fear conditioning. Compared with young controls, aged WT mice exhibited enhanced auditory cued fear memory, but contextual fear memory was not different. Contextual fear memory retention was attenuated in both young and aged HSD1 KO mice. In contrast, auditory cued fear memory was reduced 24 h after training only in aged HSD1 KO mice. When fear conditioned with stress, WT mice displayed PTSD-like memory (i.e., increased fear to tone not predictive of shock and reduced fear to ‘aversive’ conditioning context); this was unchanged with aging. In contrast, young HSD1 KO mice fear conditioned with stress showed normal fear memory (i.e., increased fear response to conditioning context), as observed in WT mice fear conditioned alone. While aged HSD1 KO mice fear conditioned with stress also displayed normal contextual fear memory, the fear response to the ‘safe’ tone remained. Thus, a deficiency of 11β-HSD1 protects against both amnesia for the conditioning context and hypermnesia for a salient tone in young adult mice but only contextual amnesia is prevented in aged mice. These results suggest that brain 11β-HSD1 generated glucocorticoids make a significant contribution to fear conditioning and PTSD-like memory. 11β-HSD1 inhibition may be useful in prevention and/or treatment of PTSD.

Background A core symptom of posttraumatic stress disorder is persistent fear memory, which can be defined as fear memory that is resistant to updating, inhibition, or extinction. posttraumatic stress disorder emerges after traumatic stress exposure, but neurobiological mechanisms via which traumatic stress leads to persistent fear memory are not well defined. Akt signaling within the amygdala (Amy) is enhanced with traumatic stress, and phosphatidylinositol kinase 3 (PI3K) activation of Akt within the basolateral Amy (BLA) has been implicated as critical to fear memory formation. These findings raise the possibility that traumatic stress enhances PI3K→Akt signaling in the BLA, which leads to persistent fear memory. Methods To test this hypothesis, rats were exposed to traumatic stress using the single prolonged stress model, and changes in Akt phosphorylation were assayed in the Amy at 0 and 30 minutes after fear conditioning (FC). In a separate experiment, we inhibited PI3K→Akt signaling in the BLA prior to FC and observed the effect this had on acquisition, expression, and extinction of FC in stressed and control rats. Results Enhanced Akt phosphorylation in the Amy at both time points was observed in stressed rats, but not in control rats. PI3K→Akt inhibition in the BLA had no effect on freezing in control rats but decreased freezing during extinction training and testing in stressed rats. Conclusion These findings suggest that PI3K→Akt signaling in the BLA could be a mechanism via which traumatic stress leads to fear memory that is resistant to extinction.

Central glucocorticoid receptor (GR) activity is enhanced following traumatic events, playing a key role in the stress-related cognitive abnormalities of posttraumatic stress disorder (PTSD). GR antagonists are expected to have potential as pharmacological agents to treat PTSD-related symptoms such as anxiety and fear memory disruption. However, an incubation period is usually required and stress-induced abnormalities do not develop immediately following the trauma; thus, the optimal intervention timing should be considered. Single prolonged stress (SPS) was employed as a rodent PTSD model to examine the effects of early or late (1–7 versus 8–14 days after the SPS) sub-chronic RU486 (a GR antagonist) administration. Behaviorally, fear conditioning and anxiety behavior were assessed using the fear-conditioning test and elevated T-maze (ETM), respectively. Neurochemically, the expressions of GR, FK506-binding proteins 4 and 5 (FKBP4 and FKBP5), and early growth response-1 (Egr-1) were assessed in the hippocampus, medial prefrontal cortex (mPFC), amygdala, and hypothalamus, together with the level of plasma corticosterone. Early RU486 administration could inhibit SPS-induced behavioral abnormalities and glucocorticoid system dysregulation by reversing the SPS-induced fear extinction deficit, and preventing SPS-reduced plasma corticosterone levels and SPS-induced Egr-1 overexpression in the hippocampus. Early RU486 administration following SPS also increased the FKBP5 level in the hippocampus and hypothalamus. Finally, both early and late RU486 administration inhibited the elevated hippocampal FKBP4 level and hypothalamus GR level in the SPS rats. Early intervention with a GR antagonist aids in the correction of traumatic stress-induced fear and anxiety dysregulation.

Although women and men are equally likely to receive ketamine following traumatic injury, little is known regarding sex-related differences in the impact of ketamine on traumatic memory. We previously reported that subanesthetic doses of an intravenous (IV) ketamine infusion following fear conditioning impaired fear extinction and altered regional brain glucose metabolism (BGluM) in male rats. Here, we investigated the effects of IV ketamine infusion on fear memory, stress hormone levels, and BGluM in female rats. Adult female Sprague–Dawley rats received a single IV ketamine infusion (0, 2, 10, or 20 mg/kg, over a 2-h period) following auditory fear conditioning (three pairings of tone and footshock). Levels of plasma stress hormones, corticosterone (CORT) and progesterone, were measured after the ketamine infusion. Two days after ketamine infusion, fear memory retrieval, extinction, and renewal were tested over a three-day period. The effects of IV ketamine infusion on BGluM were determined using 18F-fluoro-deoxyglucose positron emission tomography (18F-FDG-PET) and computed tomography (CT). The 2 and 10 mg/kg ketamine infusions reduced locomotor activity, while 20 mg/kg infusion produced reduction (first hour) followed by stimulation (second hour) of activity. The 10 and 20 mg/kg ketamine infusions significantly elevated plasma CORT and progesterone levels. All three doses enhanced fear memory retrieval, impaired fear extinction, and enhanced cued fear renewal in female rats. Ketamine infusion produced dose-dependent effects on BGluM in fear- and stress-sensitive brain regions of female rats. The current findings indicate that subanesthetic doses of IV ketamine produce robust effects on the hypothalamic–pituitary–adrenal (HPA) axis and brain energy utilization that may contribute to enhanced fear memory observed in female rats.

Exposure to a catastrophic event or intense stimulation can trigger fear memories, and the threatening memories persist even over a lifetime. Exposure therapy is based on extinction learning and is widely used to treat fear-related disorders, but its effect on remote fear memory is modest. Berberine, an isoquinoline alkaloid derived from Coptis chinensis or Berberis spp., has been recently reported to exert a diversity of pharmacological effects on the central nervous system, such as facilitating extinction of drug memory. Here, we explored the effect of berberine on extinction of fear memory using a classical contextual fear conditioning (CFC) paradigm, which is Pavlovian conditioning, can rapidly create fear memories related to contexts. Twenty-four hours or 30 days after CFC training, mice were subjected to context extinction (10 days) to extinguish their behaviors and treated with 12.5 or 25 mg/kg berberine intragastrically 1 or 6 h after each extinction session, followed by reinstatement and spontaneous recovery tests. The results showed that intragastric administration of 25 mg/kg berberine 1 h after extinction significantly promoted the extinction of recent and remote fear memories and prevented reinstatement and spontaneous recovery of extinguished fear in mice. These findings indicate that berberine combined with extinction training could serve as a promising novel avenue for the treatment of fear-related disorders.

Fear memories can be reactivated by a fear-associated conditioned stimulus (CS) or unconditioned stimulus (US) and then undergo reconsolidation. Propranolol administration during CS retrieval-induced reconsolidation can impair fear memory that is specific to the reactivated CS. However, from a practical perspective, the US is often associated with multiple CSs, and each CS can induce a fear response. The present study sought to develop and test a US-based memory retrieval interference procedure with propranolol to disrupt the original fear memory and eliminate all CS-associated fear responses in humans. We recruited 127 young healthy volunteers and conducted three experiments. All of the subjects acquired fear conditioning, after which they received the β-adrenergic receptor antagonist propranolol (40 mg) or placebo (vitamin C) and were exposed to the US or CS to reactivate the original fear memory. Fear responses were measured. Oral propranolol administration 1 h before US retrieval significantly decreased subsequent fear responses and disrupted associations between all CSs and the US. However, propranolol administration before CS retrieval only inhibited the fear memory that was related to the reactivated CS. Moreover, the propranolol-induced inhibition of fear memory reconsolidation that was retrieved by the US had a relatively long-lasting effect (at least 2 weeks) and was also effective for remote fear memory. These findings indicate that the US-based memory retrieval interference procedure with propranolol can permanently decrease the fear response and prevent the return of fear for all CSs in humans. This procedure may open new avenues for treating fear-related disorders.

Background Research on catastrophic misinterpretations of bodily sensations in patients with a diagnosis of panic disorder has yielded inconsistent findings concerning the question of how typical these misinterpretations are and how this compares with other anxiety disorders. Limitations of assessment strategies concerning catastrophic misinterpretations have been discussed. We assessed catastrophic misinterpretations by activating participants’ fear memory, as has been suggested. Methods Participants in the experimental group (EG) were shown a suspenseful film clip to induce physiological arousal before completing a measure of catastrophic misinterpretation (BSIQ-FR). Skin conductance level (SCL) was used as marker for physiological arousal. Results As expected, the film manipulation led to a significant increase in physiological arousal in the EG compared to the control group (CG) across all disorder groups. ANOVAs did not show significant interactions between factors Group (Panic Disorder, Other Anxiety Disorder, and Healthy Controls) and Condition (EG, CG). However, comparison of means indicated that participants with panic disorder showed more catastrophic misinterpretations of bodily sensations than patients with other anxiety disorders in the EG, but not in the CG. Conclusions The findings indicate that the activation of fear memory via induction of physiological arousal facilitated the measurement of catastrophic misinterpretations, and provide further evidence that catastrophic misinterpretations of bodily sensations are typical for panic disorder.

BackgroundFear memory is a fundamental capability for animals and humans to survive. Its impairment results in the disability to avoid danger. When memory is reactivated, a reconsolidation process, which can be disrupted by various stimuli, including inflammation, is required to become permanent. Nicotine has been shown to improve cognitive deficits induced by inflammation and other stimuli. Therefore, in the present study, we investigated the effect of nicotine on lipopolysaccharide (LPS)-induced impairment of fear memory reconsolidation and the underlying mechanism.MethodsStep-through inhibitory avoidance task was recruited to study fear memory of rat, i.p. LPS (0.5 mg/kg) treatment was used to induce inflammation, and western blot and immunostaining were applied to detect protein expression and distribution in medial prefrontal cortex and hippocampus.ResultsOur data showed that LPS induced fear memory reconsolidation impairment without affecting retrieval. In addition, LPS significantly increased inflammation factors tumor necrosis factor-α and interleukin-1 beta and decreased CREB-regulated transcription coactivator 1 (CRTC1) expression and adenosine monophosphate-activated protein kinase (AMPK) activation in hippocampus. More importantly, LPS significantly decreased CRTC1 expression and AMPK activation in neurons by activating microglia cells. Of note, either nicotine treatment or activation of AMPK by intracerebroventricular infusion of metformin reduced LPS-induced impairment of fear memory reconsolidation and ameliorated inflammation factor tumor necrosis factor-α and interleukin-1 beta as well as the expression of CRTC1.ConclusionsIn conclusion, our results showed that acute nicotine treatment alleviates LPS-induced impairment of fear memory reconsolidation through activation of AMPK and upregulation of CRTC1 in hippocampus.

Several studies suggest that the perirhinal cortex (PER) may function to unitize stimulus components across time or modalities. While the PER has been shown to be critical for fear acquisition to discontinuous stimuli, the role of the PER in fear extinction memory has not been evaluated. The current study assessed the involvement of the PER during fear extinction training to a continuous or discontinuous conditioned stimulus (CS). Rats were randomly assigned to 1 of 4 groups based on 2 factors: the CS type (a continuous or discontinuous light) and a pretesting PER manipulation (muscimol inactivation or saline). Results showed that PER inactivation impaired fear memory to both CS types; however, PER inactivation had only impaired extinction memory to the discontinuous light. These results suggest the role of the PER in stimulus unitization extends to supporting the acquisition of fear extinction memory. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Fear extinction is an active learning process whereby previously established conditioned responses to a conditioned stimulus are suppressed. Paradoxically, when extinction training is performed immediately following fear acquisition, the extinction memory is weakened. Here, we demonstrate that corticotrophin-releasing factor (CRF)-expressing neurons in the central amygdala (CeA) antagonize the extinction memory following immediate extinction training. CeA-CRF neurons transition from responding to the unconditioned stimulus to the conditioned stimulus during the acquisition of a fear memory that persists during immediate extinction training, but diminishes during delayed extinction training. Inhibition of CeA-CRF neurons during immediate extinction training is sufficient to promote enhanced extinction memories, and activation of these neurons following delay extinction training is sufficient to reinstate a previously extinguished fear memory. These results demonstrate CeA-CRF neurons are an important substrate for the persistence of fear and have broad implications for the neural basis of persistent negative affective behavioral states., Learned conditioned fear associations can be weakened (extinction learning), but extinction is less effective if performed too soon after the original fear conditioning. Here, the authors show that persistent activation of CRF-expressing neurons in the central amygdala is involved in the early fear extinction deficit.

PTSD is a common consequence of individuals' exposure to life-threatening traumatic events, such as traffic accidents. It is closely related to fear memories, leading to trauma recurrence, avoidance behavior, impaired function, and emotional distress. In healthy people, memories of terror fade automatically after they are formed, but people with PTSD are less able to fade memories of terror by themselves. Experiments have shown that humans can eliminate the horrible memories formed in the hippocampus and amygdala by eliminating memory training, and TMS can enhance the learning and consolidation of fading memories. The purpose of this study was to test whether TMS could eliminate the horrible memories of traffic accidents. The results show that According to the correlation degree analysis of traffic accident related pictures, the first, second, seventh and eighth pictures with significant differences were excluded. Rule out pairs 4 and 5 according to the neutral rule. Rule out the sixth pair of pictures according to the same property principle. According to the experimental data, the subjects successfully combined traffic-related pictures with fear in the acquisition stage, changed the fear memory association in the extinction stage, and showed no significant difference in CS+ and CS- in the arousal stage. It is proved that TMS can be used to fade the memory of traffic fear, and TMS stimulation of F3 and F4 can both produce the effect of fading the memory of traffic fear. TMS can promote the treatment of PTSD patients caused by traffic accidents. This study can provide references for the treatment of PTSD patients caused by other types of horrible memories and the application of TMS technology. get date from 2 groups analyze data from NFE group in acquisation check if result is proi__ned analyze data from NFE group in extinction phase analyze resulted trend analyze data from recall phase fiom 2 goups compare and draw conclusion Figure 1 First Secord day day acquisition 20CS+(P)+US 10min extinction recall 20CS+(P) 40CS+(T+P) 40CS+P 40CS-(P) 40CS-(T +P) 40CS-P acquisition 20CS+(P)+US 10min extinction recall 20CS+(P) 40CS+P 40CS+P 40CS-(P) 40CS-P 40CS-P Figure 2

BackgroundIn both rodents and humans, the basolateral amygdala (BLA) is essential for encoding and retrieving conditioned fear memories. Although the BLA is a putative storage site for these memories, recent evidence suggests that they become independent of the BLA with the passage of time.MethodsWe systematically examined the role for the BLA in the retrieval of recent (1 day) and remote (2 weeks) fear memory using optogenetic, electrophysiological, and calcium imaging methods in male and female Long-Evans rats. Critically, we used a behavioral design that permits within-subjects comparison of recent and remote memory at the same time point; freezing behavior served as the index of learned fear.ResultsWe found that BLA c-Fos expression was similar after the retrieval of recent or remote fear memories. Extracellular single-unit recordings in awake, behaving animals revealed that single BLA neurons exhibit robust increases in spike firing to both recent and remote conditional stimuli (CSs). Fiber photometry recordings revealed that these patterns of activity emerge from principal neurons. Consistent with these results, optogenetic inhibition of BLA principal neurons impaired conditioned freezing to both recent and remote CSs. There were no sex differences in any of the measures or manipulations.ConclusionsThese data reveal that BLA neurons encode both recent and remote fear memories, suggesting substantial overlap in the allocation of temporally distinct events. This may underlie the broad generalization of fear memories across both space and time. Ultimately, these results provide evidence that the BLA is a long-term storage site for emotional memories.

Transient Reseptör Potansiyel Vanilloid 1 (TRPV1) kanalları hücre membranı yerleşimli, içinde bir katyon kanalı bulunduran ve açıldığı zaman hücre içine kalsiyum girişine neden olan bir kanaldır. Bu çalışmada TRPV1 agonisti kapsaisin ve antagonisti kapsazepin’in korku koşullanma üzerine etkisinin araştırılması amaçlanmaktadır. Çalışmada her grupta 10 hayvan olacak şekilde Balb/c türü toplam 300 adet fare kullanıldı. Davranış deneylerinde deneklere gruplarına uygun olarak TRPV1 agonisti kapsaisin (1 mg/kg, i.p.) veya TRPV1 antagonisti kapsazepin (1 mg/kg, i.p.) veya çözücü (1:1:8) uygulandı. Tüm gruplara rota-rod performans testi bağlamsal korku koşullanma testi ve ipuçlu korku koşullanma testi yapıldı. Kapsaisin ve kapsazepin gerek bağlamsal gerek ipuçlu korku koşullanma testinde öğrenme ve bellek sürecinin hiçbir aşamasında (edinim, konsolidasyon, geri çağırma, sönme ve rekonsolidasyon) istatistiksel anlamlı bir değişiklik oluşturmadı. Elde ettiğimiz sonuçlar kapsaisin ve kapsazepin’in ve dolayısı ile TRPV1 reseptörlerinin farelerde bağlamsal ve ipuçlu korku koşullanma testi ile incelenen belleğin bazal durumu üzerine herhangi bir etkisi olmadığını göstermektedir. Bununla birlikte, yapılan testlerin sağlıklı genç erişkin farelerde gerçekleştirildiğinin mutlaka göz önünde bulundurulması gereklidir. Elde ettiğimiz sonuçlar kapsaisin ve kapsazepin’in ve dolayısı ile TRPV1 reseptörlerinin farelerde bağlamsal ve ipuçlu korku koşullanma testi ile incelenen belleğin bazal durumu üzerine herhangi bir etkisi olmadığını göstermektedir. In total 300 Balb/c mice were divided into 30 groups (n= 10 animals for each group). In the behavioral experiments, the TRPV1 antagonist capsazepine (1 mg/kg, i.p.) or TRPV1 agonist capsaicin (1 mg/kg, i.p.) or solvent (1:1:8) were administered to subjects according to their groups. Rota-rod performance test, contextual and cued fear conditioning test were performed in all groups. Capsaicin and capsazepine were not statistically significant at any stage (acquisition, consolidation, retrieval, extinction, and reconsolidation) of the learning and memory process in the contextual and cued fear conditioning test. Our results show that capsaicin and capsazepine, and thus TRPV1 receptors, have no effect on the basal state of memory examined by contextual and cued fear conditioning testing in mice. However, it must be considered that the tests were performed in healthy young adult mice. The results show that capsaicin and capsazepine, and thus TRPV1 receptors, have no effect on the basal state of memory examined by the contextual and cue fear conditioning test in mice.

Zebrafish (Danio rerio) are widely used as a translational model for human neuropsychiatric conditions. Many studies have not considered sex differences in their analyses. Here, we studied sex differences of adult zebrafish in two behavioral domains: Anxiety and Memory. To assess whether sex influences anxiety‐like responses, we used two different behavioral protocols, the novel tank diving task and the light‐dark test. To assess sex differences in learning and memory tasks, we explored two memory domains, short‐term spatial memory (free movement pattern Y‐maze task) and short‐term fear memory (Pavlovian fear conditioning task). Although we did not find any significant difference in learning and memory tasks, female zebrafish showed robust increases in anxiety‐like behavioral endpoints in both anxiety tests. Overall, our data suggest that zebrafish is a sensitive model to work with sex differences when modeling anxiety‐related disorders and this should be an important factor to consider in different experimental designs.

Abnormality of fear memory is one of the important pathogenic factors leading to post-traumatic stress disorder (PTSD), anxiety disorder, and other mental disorders. Clinically, although exposure therapy, which is based on the principle of fear memory extinction, has a certain effect on these diseases, it still relapses frequently in some cases. These troubles can be effectively solved by retrieving the memory in a certain time window before the extinction of fear memory. Therefore, it is generally believed that the extinction of fear memory is the result of forming new safe memory to competitively inhibit the original fear memory, while the retrieval-extinction operation is the updating or erasure of the original fear memory, thus, which has greater clinical therapeutic potential. However, what are the detailed molecular networks, specifically the circular RNAs (circRNAs), involved in fear memory updating, and the differences with fear extinction, are still unknown. In this study, we systematically observed the expression of mRNAs, microRNAs (miRNA), long non-coding RNAs (lncRNAs), and circRNAs in the basolateral amygdala of mice after fear memory formation, extinction, and updating by whole-transcriptional sequencing, then a variety of inter-group comparison and bioinformatics analysis were used to find the differential expressed RNAs, enrich the function of them, and construct the molecular interaction networks. Moreover, competing endogenous RNA (ceRNA) molecular networks and transcriptional regulatory networks for the candidate circRNAs were constructed. Through these analyses, we found that about 10% of molecules were both involved in the fear memory extinction and formation, but the molecules and their signaling pathways were almost completely different between fear memory extinction and updating. This study describes a relatively detailed molecular network for fear memory updating, which might provide some novel directions for further mechanism research, and help to develop a specific physical method for fear memory intervention, based on the regulation of these key molecules.

The basolateral amygdala (BLA) mediates the effects of stress and fear on rapid eye movement sleep (REM) and on REM-related theta (θ) oscillatory activity in the electroencephalograph (EEG), which is implicated in fear memory consolidation. We used optogenetics to assess the potential role of BLA glutamate neurons (BLAGlu) in regulating behavioral, stress and sleep indices of fear memory, and their relationship to altered θ. An excitatory optogenetic construct targeting glutamatergic cells (AAV-CaMKIIα-hChR2-eYFP) was injected into the BLA of mice. Telemetry was used for real-time monitoring of EEG, activity, and body temperature to determine sleep states and stress-induced hyperthermia (SIH). For 3 h following shock training (ST: 20 footshocks, 0.5 mA, 0.5 s, 1 min interval), BLA was optogenetically stimulated only during REM (REM + L) or NREM (NREM + L). Mice were then re-exposed to the fear context at 24 h, 48 h, and 1 week after ST and assessed for behavior, SIH, sleep and θ activity. Control mice were infected with a construct without ChR2 (eYFP) and studied under the same conditions. REM + L significantly reduced freezing and facilitated immediate recovery of REM tested at 24 h and 48 h post-ST during contextual re-exposures, whereas NREM + L had no significant effect. REM + L significantly reduced post-ST REM-θ, but attenuated REM-θ reductions at 24 h compared to those found in NREM + L and control mice. Fear-conditioned SIH persisted regardless of treatment. The results demonstrate that BLAGlu activity during post-ST REM mediates the integration of behavioral and sleep indices of fear memory by processes that are associated with θ oscillations within the amygdalo-hippocampal pathway. They also demonstrate that fear memories can remain stressful (as indicated by SIH) even when fear conditioned behavior (freezing) and changes in sleep are attenuated.

Although fear memory formation is essential for survival and fear-related mental disorders, the neural circuitry and mechanism are incompletely understood. Here, we utilized trace fear conditioning to study the formation of trace fear memory in mice. We identified the entorhinal cortex (EC) as a critical component of sensory signaling to the amygdala. We adopted both loss-of-function and gain-of-function experiments to demonstrate that release of the cholecystokinin (CCK) from the EC is required for trace fear memory formation. We discovered that CCK-positive neurons project from the EC to the lateral nuclei of the amygdala (LA), and inhibition of CCK-dependent signaling in the EC prevented long-term potentiation of the auditory response in the LA and formation of trace fear memory. In summary, high-frequency activation of EC neurons triggers the release of CCK in their projection terminals in the LA, potentiating auditory response in LA neurons. The neural plasticity in the LA leads to trace fear memory formation.

SummaryDisinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory and excitatory neurons suggests that each circuit motif is controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a newly-developed genetically-encoded GRP sensor and trans-synaptic tracing we reveal that GRP regulates VIP cells via extrasynaptic diffusion from several putative local and long-range sources. In vivo photometry and CRISPR/Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and that GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

A growing body of evidence suggests that memories of fearful events may be altered after initial acquisition or learning. Although much of this work has been done in rodents using Pavlovian fear conditioning, it may have important implications for fear memories in humans such as in post-traumatic stress disorder (PTSD). A recent study suggested that cued fear memories, made labile by memory retrieval, were made additionally labile and thus more vulnerable to subsequent modification when mice inhaled 10% carbon dioxide (CO2) during retrieval. In light of this finding, we hypothesized that 10% CO2 inhalation soon after fear acquisition might affect memory recall 24 h later. We found that both cue and context fear memory were increased by CO2 exposure after fear acquisition. The effect of CO2 was time-dependent, as CO2 inhalation administered 1 or 4 h after cued fear acquisition increased fear memory, whereas CO2 inhalation 4 h before or 24 h after cued fear acquisition did not increase fear memory. The ability of CO2 exposure following acquisition to enhance fear memory was not a general consequence of stress, as restraining mice after acquisition did not alter cued fear memory. The memory-enhancing action of CO2 may be relatively specific to fear conditioning as novel object recognition was impaired by post-training CO2 inhalation. To explore the molecular underpinnings of these effects, we tested if they depended on the acid-sensing ion channel-1a (ASIC1A), a proton-gated cation channel that mediates other effects of CO2, likely via its ability to sense acidosis induced during CO2 inhalation. We found that CO2 inhalation did not alter cued or context fear memory in Asic1a–/– mice, suggesting that this phenomenon critically depends on ASIC1A. These results suggest that brain acidosis around the time of a traumatic event may enhance memory of the trauma, and may thus constitute an important risk factor for developing PTSD. Moreover, preventing peritraumatic acidosis might reduce risk of PTSD.

Memories are stored in the brain as cellular ensembles activated during learning and reactivated during retrieval. Using the Tet-tag system, we labeled dorsal dentate gyrus (dDG) neurons activated by positive, neutral or negative experiences with channelrhodopsin-2. Following fear-conditioning, these cells were artificially reactivated during fear memory recall. Optical stimulation of a competing positive memory was sufficient to disrupt reconsolidation, thereby reducing conditioned fear acutely and enduringly. Moreover, mice demonstrated operant responding for reactivation of a positive memory, confirming its rewarding properties. These results show that interference from a rewarding experience can counteract negative affective states. While interference induced by memory reactivation involved a relatively small set of neurons, we also found that activating a large population of randomly labeled dDG neurons was effective at disrupting reconsolidation. Importantly, reconsolidation-interference was specific to the fear memory. These findings implicate the dDG as a potential therapeutic node for modulating memories to suppress fear.

Introduction: Single housing of laboratory mice is a common practice to meet experimental needs, or to avoid intermale aggression. However, single housing is considered to negatively affect animal welfare and may compromise the scientific validity of experiments. The aim of this study was to investigate whether the use of a cage with a cage divider, which avoids physical contact between mice while maintaining sensory contact, may be a potential refinement strategy for experiments in which group housing of mice is not possible.Methods: Eight-week-old male C57BL/6JRj mice were single housed, pair housed or pair housed with a cage divider for four (experiment 1) or ten (experiment 2) weeks, after which we performed an open field test, Y-maze spontaneous alternation test, elevated plus maze test, an auditory fear conditioning task, and assessed responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis.Results: Housing conditions did not affect body weight, exploratory activity, anxiety, working memory, fear memory processing or markers for HPA-axis functioning in either experiment 1 or experiment 2. There was an increased distance traveled in mice housed with a cage divider compared to pair housed mice after 4 weeks, and after 10 weeks mice housed with a cage divider made significantly more arm entries in the Y-maze spontaneous alternation test.Conclusion: Taken together, our study did not provide evidence for robust differences in exploratory activity, anxiety, working memory and fear memory processing in male C57BL/6JRj mice that were single housed, pair housed or pair housed with a cage divider.

Reconsolidation enables the activity-dependent modification of memory traces and has been used to reverse addiction, fear memory, and pain hypersensitivity in animal models. We demonstrate that non-ionotropic NMDA receptor signalling in the spinal dorsal horn is sufficient to reverse pain hypersensitivity and necessary for pain modulation by spinal reconsolidation. These findings reveal a key process by which reconsolidation disrupts memory traces that may be exploited in the treatment of pain and other disorders.

Stress can elicit glucocorticoid release to promote coping mechanisms and influence learning and memory performance. Individual memory performance varies in response to stress, and the underlying mechanism is not clear yet. Peroxiredoxin 6 (PRDX6) is a multifunctional enzyme participating in both physiological and pathological conditions. Several studies have demonstrated the correlation between PRDX6 expression level and stress-related disorders. Our recent finding indicates that lack of the Prdx6 gene leads to enhanced fear memory. However, it is unknown whether PRDX6 is involved in changes in anxiety response and memory performance upon stress. The present study reveals that hippocampal PRDX6 level is downregulated 30 min after acute immobilization stress (AIS) and trace fear conditioning (TFC). In human retinal pigment epithelium (ARPE-19) cells, the PRDX6 expression level decreases after being treated with stress hormone corticosterone. Lack of PRDX6 caused elevated basal H2O2 levels in the hippocampus, basolateral amygdala, and medial prefrontal cortex, brain regions involved in anxiety response and fear memory formation. Additionally, this H2O2 level was still high in the medial prefrontal cortex of the knockout mice under AIS. Anxiety behavior of Prdx6−/− mice was enhanced after immobilization for 30 min. After exposure to AIS before a contextual test, Prdx6−/− mice displayed a contextual fear memory deficit. Our results showed that the memory performance of Prdx6−/− mice was impaired when responding to AIS, accompanied by dysregulated H2O2 levels. The present study helps better understand the function of PRDX6 in memory performance after acute stress.

Emotionally arousing experiences are retained very well as seen in posttraumatic stress disorder (PTSD). Various lines of evidence indicate that reactivation of these memories renders them labile which offers a potential time-window for intervention. We tested in non-human primates whether ketamine, administered during fear memory reactivation, affected passive (inhibitory) avoidance learning. For the consolidation of contextual emotional memory, the unescapable foot-shock paradigm in a passive avoidance task with two compartments (dark vs illuminated) was used. After entering the dark compartment, marmoset monkeys received four random foot-shocks (1 mA, 4 s) within 15-min. This stressful exposure increased the saliva cortisol and heart rate and impaired REM-sleep ( p < 0.05). One week later the monkeys were re-exposed to the stressful situation for the reconsolidation of the fearful experience. During the re-exposure the monkeys were treated with ketamine (0.5 mg/kg) or saline. In week 3, the monkeys were placed in the experimental setting to test their memory for the fearful experience. In contrast to the vehicle-treated monkeys, who avoided the dark compartment, the ketamine-treated monkeys entered the dark compartment that was previously associated with the fearful experience ( p < 0.05). Post-mortem analysis of the hippocampus showed that ketamine-treated animals exhibited less doublecortin positive neurons and BrdU-labeled cells in the dentate gyrus. This study reveals that a single low dose of ketamine, administered upon fear retrieval in monkeys, reduce contextual fear memory and attenuate neurogenesis in the hippocampus. These are important findings for considering ketamine as a potential candidate to target traumatic memories in PTSD. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

Fear learning and memory are crucial for animal survival. Abnormal fear memory is a hallmark of many neuropsychiatric disorders. Appropriate neuronal activation and excitability in the basolateral amygdala (BLA) are necessary for the formation of fear memory. The gene cylindromatosis (Cyld), which encodes a lysine-63 deubiquitinase, is expressed in several brain regions including the amygdala. The functions of the cylindromatosis protein (CYLD) in the regulation of the neuronal activity, neural circuits and fear memory, remain largely unknown, however. Here, we report that Cyld knockout impairs amygdala-dependent tone-cued fear memory. The number of c-Fos+ neurons responding to the tone-cued fear test was reduced in the BLA of Cyld–/– mice, suggesting that the absence of CYLD causes aberrant neuronal activation. We found that this aberrant neuronal activation in the BLA of Cyld–/– mice may relate to the decreased excitability of principal neurons. Another possibility of aberrant neuronal activation could be the impaired excitatory synaptic transmission in the BLA of Cyld–/– mice. Specifically, both the frequency of spontaneous excitatory postsynaptic currents and the amplitude of miniature excitatory postsynaptic currents in BLA principal neurons were decreased. In addition, Cyld mutation caused an increase in both the frequency of miniature inhibitory postsynaptic currents in principal neurons and the number of parvalbumin+ interneurons, consistent with excessive local circuit inhibition in the BLA of Cyld–/– mice. Taken together, these results suggest that CYLD deficiency disrupts the neuronal activity and synaptic transmission in the BLA of mice which may contribute to the impaired fear memory observed in Cyld–/– mice.

O envelhecimento ? um processo que acarreta mudan?as fisiol?gicas, psicol?gicas e sociais, incluindo a vulnerabilidade a doen?as neurodegenerativas e d?ficits de mem?ria. As vias de sinaliza??o da Wnt est?o envolvidas na forma??o de mem?rias no hipocampo, mas sua fun??o em outras regi?es associadas ? mem?ria, como o c?rtex pr?-frontal medial (CPFm), ainda ? pouco conhecida. Assim, neste trabalho investigamos o envolvimento das vias de sinaliza??o da Wnt do c?rtex pr?-frontal medial nos processos de aquisi??o, consolida??o e evoca??o associados a mem?ria de medo. Para isto, ratos Wistar machos foram submetidos ao protocolo de medo condicionado ao contexto (MCC) e receberam infus?es de DKK1 e SFRP1, antagonistas das vias can?nica Wnt/?-catenina e n?o-can?nica Wnt/Ca2+, respectivamente, na subdivis?o pr?-l?mbica (PrL) do CPFm em diferentes momentos e, se avaliou a aquisi??o, consolida??o e evoca??o da mem?ria de curta e longa dura??o. Observamos que o bloqueio das vias de sinaliza??o can?nica Wnt/?-catenina e n?o can?nica Wnt/Ca2+, 15 minutos antes ou imediatamente ap?s o treino da tarefa de MCC n?o teve efeito sobre a mem?ria de curta dura??o (MCD) e de longa dura??o (MLD), enquanto que seu bloqueio 15 minutos antes do teste de reten??o prejudicou a evoca??o da MCD e da MLD. Esses resultados destacam a import?ncia do CPFm na evoca??o da mem?ria de medo, demonstrando que ambas as vias de sinaliza??o can?nica Wnt/?-catenina e n?o can?nica Wnt/Ca2+ s?o necess?rias neste processo. A compreens?o de como os sistemas cerebrais agem nas mem?rias de medo pode fornecer um novo alvo para o tratamento de transtornos relacionados ao medo, como transtorno de estresse p?s-traum?tico e outros transtornos de ansiedade. Aging is a process which incurs physiological, psychological and social changes, including vulnerability to neurodegenerative disorders and memory deficits. The Wnt signaling pathways are involved in memory formation in the hippocampus, albeit its role in other memory-related regions, such as the medial prefrontal cortex (mPFC) is still to be elucidated. In this paper, we investigate the involvement of Wnt?s signaling pathways in the medial prefrontal cortex, in the processes of acquisition, consolidation and retrieval, associated with fear memory. Towards that goal, male Wistar rats were subjected to a contextual fear conditioning protocol (CFC) and were injected with DKK1 and SFRP1, which are Wnt antagonists of the canonical Wnt/?-catenin and non-canonical Wnt/Ca2 pathways, respectively, in the prelimbic (PrL) subdivision of the mPFC, in different moments, and acquisition, consolidation and retrieval of short- and long-term memory were assessed. We observed that the blockade of the canonical Wnt/?-catenin, and non-canonical Wnt/Ca2, signaling pathways 15 minutes before, or immediately after, the CFC task training, did not affect short- (STM) and long-term memory (LTM); however, when the blockade occurred 15 minutes before the retention test, it impaired the retrieval of short and long-term, memory. These findings highlight the importance of the mPFC in the retrieval of fear memory, showing that both the canonical Wnt/?-catenin and the non-canonical Wnt/Ca2+ pathways are necessary for this process. The understanding of cerebral systems act on fear memories might provide a new target for the treatment of disorders related to fear memories, such as post-traumatic stress disorder and other anxiety disorders. Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES

Serial section electron microscopy (ssEM) can provide comprehensive 3D ultrastructural information of the brain with exceptional computational cost. Targeted reconstruction of subcellular structures from ssEM datasets is less computationally demanding but still highly informative. We thus developed a Region-CNN-based deep learning method to identify, segment, and reconstruct synapses and mitochondria to explore the structural plasticity of synapses and mitochondria in the auditory cortex of mice subjected to fear conditioning. Upon reconstructing over 135,000 mitochondria and 160,000 synapses, we found that fear conditioning significantly increases the number of mitochondria but decreases their size, and promotes the formation of multi-contact synapses comprising a single axonal bouton and multiple postsynaptic sites from different dendrites. Modeling indicates that such multi-contact configuration increases the information storage capacity of new synapses by over 50%. With high accuracy and speed in reconstruction, our method yields structural and functional insight into cellular plasticity associated with fear learning.

Adenosine A2A receptors (A2AR) control fear memory and the underlying processes of synaptic plasticity in the amygdala. In other brain regions, A2AR activation is ensured by ATP-derived extracellular adenosine formed by ecto-5′-nucleotidase or CD73. We now tested whether CD73 is also responsible to provide for the activation of A2AR in controlling fear memory and amygdala long-term potentiation (LTP). The bilateral intracerebroventricular injection of the CD73 inhibitor αβ-methylene ADP (AOPCP, 1 nmol/ventricle/day) phenocopied the effect of the A2AR blockade by decreasing the expression of fear memory, an effect disappearing in CD73-knockout (KO) mice and in forebrain neuronal A2AR-KO mice. In the presence of PPADS (20 μM) to eliminate any modification of ATP/ADP-mediated P2 receptor effects, both AOPCP (100 μM) and the A2AR antagonist, SCH58261 (50 nM), decreased LTP magnitude in synapses of projection from the external capsula into the lateral amygdala, an effect eliminated in slices from both forebrain neuronal A2AR-KO mice and CD73-KO mice. These data indicate a key role of CD73 in the process of A2AR-mediated control of fear memory and underlying synaptic plasticity processes in the amygdala, paving the way to envisage CD73 as a new therapeutic target to interfere with abnormal fear-like emotional processing.

Stress induces neuroinflammation and disrupts sleep, which together can promote a number of stress-related disorders. Fear memories associated with stress can resurface and reproduce symptoms. Our previous studies have demonstrated sleep outcomes can be modified by stressor controllability following stress and fear memory recall. However, it is unknown how stressor controllability alters neuroinflammatory signaling and its association with sleep following fear memory recall. Mice were implanted with telemetry transmitters and experienced escapable or inescapable footshock and then were re-exposed to the shuttlebox context one week later. Gene expression was assessed with Nanostring® panels using RNA extracted from the basolateral amygdala and hippocampus. Freezing and temperature were examined as behavioral measures of fear. Increased sleep after escapable stress was associated with a down-regulation in neuro-inflammatory and neuro-degenerative related genes, while decreased sleep after inescapable stress was associated with an up-regulation in these genes. Behavioral measures of fear were virtually identical. Sleep and neuroimmune responses appear to be integrated during fear conditioning and reproduced by fear memory recall. The established roles of disrupted sleep and neuroinflammation in stress-related disorders indicate that these differences may serve as informative indices of how fear memory can lead to psychopathology.