Associative Fear Memory Research Articles

3,3'-Diindolylmethane improves pathology and neurological outcome following traumatic brain injury.

3,3'-Diindolylmethane (DIM), a naturally occurring bis-indole found in cruciferous vegetables and produced in small amounts in the normal flora of the human gut, has demonstrated neuroprotective benefits in models of CNS hypoxia and stroke. In the CNS, DIM modulates the activation of the aryl hydrocarbon receptor (AhR) and inhibits its pro-inflammatory effects. Although capable of crossing the blood brain barrier, DIM's bioavailability is limited by its low solubility. Dispersed BR4044 provides a nanoscale high-solubility DIM suspension with the potential for treating traumatic brain injury (TBI). The present study aimed to determine whether BR4044 treatment could reduce pathology and improve behavioral recovery following moderate TBI. Male Sprague Dawley rats received moderate fluid percussion injury or sham surgery followed by vehicle or BR4044 treatment in the acute recovery period. TBI BR4044 animals showed significantly reduced cortical and hippocampal edema and lower levels of serum-derived extracellular vesicles compared to TBI Vehicle animals. BR4044 treatment of TBI animals preserved sensorimotor function and associative fear memory. Cortical contusion size and neuronal loss in the parietal cortex and CA3 region of the hippocampus were also significantly reduced with BR4044 treatment. BR4044 also decreased microbleeding and nuclear AhR at the contusion site. This translational study demonstrates that BR4044 ameliorates pathology and improves neurological outcomes following TBI by reducing brain edema, lowering acute extracellular vesicle release, modulating AhR, preserving cortical and hippocampal neurons, reducing red blood cell (RBC) extravasation into the injured brain, and promoting behavioral recovery.

Emotional stress increases GluA2 expression and potentiates fear memory via adenylyl cyclase 5.

Stress can alter behavior and contributes to psychiatric disorders by regulating the expression of the GluA2 AMPA receptor subunit. We have previously shown in mice that exposure to predator odor stress elevates GluA2 transcription in cerebellar molecular layer interneurons (MLIs), and MLI activity is required for fear memory consolidation. Here, we identified the critical involvement of adenylyl cyclase 5, in both the stress-induced increase in GluA2 in MLIs and the enhancement of fear memory. We found that noradrenaline release during predator odor stress activates AC5 and downstream PKA-CREB signaling. This pathway interacts synergistically with α1-adrenergic receptors to promote synaptic GluA2 expression in MLIs. At a behavioral level, predator odor stress potentiates associative fear memory, and this is abolished in AC5 knockout mice, suggesting that AC5-dependent plasticity is required for enhanced memory formation. Therefore, AC5 is a promising pharmacological target for preventing stress-enhanced fear memory.

Deconstruction of a memory engram reveals distinct ensembles recruited at learning.

How are associative memories formed? Which cells represent a memory, and when are they engaged? By visualizing and tagging cells based on their calcium influx with unparalleled temporal precision, we identified non-overlapping dorsal CA1 neuronal ensembles that are differentially active during associative fear memory acquisition. We dissected the acquisition experience into periods during which salient stimuli were presented or certain mouse behaviors occurred and found that cells associated with specific acquisition periods are sufficient alone to drive memory expression and contribute to fear engram formation. This study delineated the different identities of the cell ensembles active during learning, and revealed, for the first time, which ones form the core engram and are essential for memory formation and recall.

Time-dependent neural arbitration between cue associative and episodic fear memories.

After traumatic events, simple cue-threat associative memories strengthen while episodic memories become incoherent. However, how the brain prioritises cue associations over episodic coding of traumatic events remains unclear. Here, we developed an original episodic threat conditioning paradigm in which participants concurrently form two memory representations: cue associations and episodic cue sequence. We discovered that these two distinct memories compete for physiological fear expression, reorganising overnight from an overgeneralised cue-based to a precise sequence-based expression. With multivariate fMRI, we track inter-area communication of the memory representations to reveal that a rebalancing between hippocampal- and prefrontal control of the fear regulatory circuit governs this memory maturation. Critically, this overnight re-organisation is altered with heightened trait anxiety. Together, we show the brain prioritises generalisable associative memories under recent traumatic stress but resorts to selective episodic memories 24 h later. Time-dependent memory competition mayprovide a unifying account for memory dysfunctions in post-traumatic stress disorders.

Sex-specific role for the long noncoding RNA Pnky in mouse behavior

The aberrant expression of specific long noncoding RNAs (lncRNAs) has been associated with cognitive and psychiatric disorders. Although a growing number of lncRNAs are now known to regulate neural cell development and function, relatively few lncRNAs have been shown to underlie animal behavior. Pnky is an evolutionarily conserved, neural lncRNA that regulates brain development. Using mouse genetic strategies, we show that Pnky has sex-specific roles in mouse behavior and that this lncRNA can underlie specific behavior by functioning in trans. Male Pnky-knockout mice have decreased context generalization in a paradigm of associative fear learning and memory. In female Pnky-knockout mice, there is an increase in the acoustic startle response, a behavior that is altered in affective disorders. Remarkably, expression of Pnky from a bacterial artificial chromosome transgene decreases the acoustic startle response in female Pnky-knockout mice, demonstrating that Pnky can modulate specific animal behavior by functioning in trans. More broadly, these studies illustrate how specific lncRNAs can underlie cognitive and mood disorders.

Periaqueductal gray activates antipredatory neural responses in the amygdala of foraging rats.

Pavlovian fear conditioning research suggests that the interaction between the dorsal periaqueductal gray (dPAG) and basolateral amygdala (BLA) acts as a prediction error mechanism in the formation of associative fear memories. However, their roles in responding to naturalistic predatory threats, characterized by less explicit cues and the absence of reiterative trial-and-error learning events, remain unexplored. In this study, we conducted single-unit recordings in rats during an 'approach food-avoid predator' task, focusing on the responsiveness of dPAG and BLA neurons to a rapidly approaching robot predator. Optogenetic stimulation of the dPAG triggered fleeing behaviors and increased BLA activity in naive rats. Notably, BLA neurons activated by dPAG stimulation displayed immediate responses to the robot, demonstrating heightened synchronous activity compared to BLA neurons that did not respond to dPAG stimulation. Additionally, the use of anterograde and retrograde tracer injections into the dPAG and BLA, respectively, coupled with c-Fos activation in response to predatory threats, indicates that the midline thalamus may play an intermediary role in innate antipredatory-defensive functioning.

Periaqueductal gray activates antipredatory neural responses in the amygdala of foraging rats

Pavlovian fear conditioning research suggests that the interaction between the dorsal periaqueductal gray (dPAG) and basolateral amygdala (BLA) acts as a prediction error mechanism in the formation of associative fear memories. However, their roles in responding to naturalistic predatory threats, characterized by less explicit cues and the absence of reiterative trial-and-error learning events, remain unexplored. In this study, we conducted single-unit recordings in rats during an ‘approach food-avoid predator’ task, focusing on the responsiveness of dPAG and BLA neurons to a rapidly approaching robot predator. Optogenetic stimulation of the dPAG triggered fleeing behaviors and increased BLA activity in naive rats. Notably, BLA neurons activated by dPAG stimulation displayed immediate responses to the robot, demonstrating heightened synchronous activity compared to BLA neurons that did not respond to dPAG stimulation. Additionally, the use of anterograde and retrograde tracer injections into the dPAG and BLA, respectively, coupled with c-Fos activation in response to predatory threats, indicates that the midline thalamus may play an intermediary role in innate antipredatory-defensive functioning.

Pharmacological diacylglycerol lipase inhibition impairs contextual fear extinction in mice.

Acquisition and extinction of associative fear memories are critical for guiding adaptive behavioral responses to environmental threats, and dysregulation of these processes is thought to represent important neurobehavioral substrates of trauma and stress-related disorders including posttraumatic stress disorder (PTSD). Endogenous cannabinoid (eCB) signaling has been heavily implicated in the extinction of aversive fear memories and we have recently shown that pharmacological inhibition of 2-arachidonoylglycerol (2-AG) synthesis, a major eCB regulating synaptic suppression, impairs fear extinction in an auditory cue conditioning paradigm. Despite these data, the role of 2-AG signaling in contextual fear conditioning is not well understood. Here, we show that systemic pharmacological blockade of diacylglycerol lipase, the rate-limiting enzyme catalyzing in the synthesis of 2-AG, enhances contextual fear learning and impairs within-session extinction. In sham-conditioned mice, 2-AG synthesis inhibition causes a small increase in unconditioned freezing behavior. No effects of 2-AG synthesis inhibition were noted in the Elevated Plus Maze in micetested after fear extinction. These data provide support for 2-AG signaling in the suppression of contextual fear learning and the expression of within-session extinction of contextual fear memories.

Inhibition of fatty acid binding protein-5 in the basolateral amygdala induces anxiolytic effects and accelerates fear memory extinction.

The endocannabinoid (eCB) system critically controls anxiety and fear-related behaviours. Anandamide (AEA), a prominent eCB ligand, is a hydrophobic lipid that requires chaperone proteins such as Fatty Acid Binding Proteins (FABPs) for intracellular transport. Intracellular AEA transport is necessary for degradation, so blocking FABP activity increases AEA neurotransmission. To investigate the effects of a novel FABP5 inhibitor (SBFI-103) in the basolateral amygdala (BLA) on anxiety and fear memory. We infused SBFI-103 (0.5μg-5μg) to the BLA of adult male Sprague Dawley rats and ran various anxiety and fear memory behavioural assays, neurophysiological recordings, and localized molecular signaling analyses. We also co-infused SBFI-103 with the AEA inhibitor, LEI-401 (3μg and 10μg) to investigate the potential role of AEA in these phenomena. Acute intra-BLA administration of SBFI-103 produced strong anxiolytic effects across multiple behavioural tests. Furthermore, animals exhibited acute and long-term accelerated associative fear memory extinction following intra-BLA FABP5 inhibition. In addition, BLA FABP5 inhibition induced strong modulatory effects on putative PFC pyramidal neurons along with significantly increased gamma oscillation power. Finally, we observed local BLA changes in the phosphorylation activity of various anxiety- and fear memory-related molecular biomarkers in the PI3K/Akt and MAPK/Erk signaling pathways. At all three levels of analyses, we found the functional effects of SBFI-103 depend on availability of the AEA ligand. These findings demonstrate a novel intra-BLA FABP5 signaling mechanism regulating anxiety and fear memory behaviours, neuronal activity states, local anxiety-related molecular pathways, and functional AEA modulation.

Methylome-wide association study of anxiety disorders.

Anxiety Disorders (ANX) such as panic disorder, generalized anxiety disorder, and phobias, are highly prevalent conditions that are moderately heritable. Evidence suggests that DNA methylation may play a role, as it is involved in critical adaptations to changing environments. Applying an enrichment-based sequencing approach covering nearly 28 million autosomal CpG sites, we conducted a methylome-wide association study (MWAS) of lifetime ANX in 1132 participants (618 cases/514 controls) from the Netherlands Study of Depression and Anxiety. Using epigenomic deconvolution, we performed MWAS for the main cell types in blood: granulocytes, T-cells, B-cells and monocytes. Cell-type specific analyses identified 280 and 82 methylome-wide significant associations (q-value < 0.1) in monocytes and granulocytes, respectively. Our top finding in monocytes was located in ZNF823 on chromosome 19 (p = 1.38 × 10-10) previously associated with schizophrenia. We observed significant overlap (p < 1 × 10-06) with the same direction of effect in monocytes (210 sites), T-cells (135 sites), and B-cells (727 sites) between this Discovery MWAS signal and a comparable replication dataset from the Great Smoky Mountains Study (N = 433). Overlapping Discovery-Replication MWAS signal was enriched for findings from published GWAS of ANX, major depression, and post-traumatic stress disorder. In monocytes, two specific sites in the FZR1 gene showed significant replication after Bonferroni correction with an additional 15 nominally replicated sites in monocytes and 4 in T-cells. FZR1 regulates neurogenesis in the hippocampus, and its knockout leads to impairments in associative fear memory and long-term potentiation in mice. In the largest and most extensive methylome-wide study of ANX, we identified replicable methylation sites located in genes of potential relevance for brain mechanisms of psychiatric conditions.

Prefrontal engrams of long-term fear memory perpetuate pain perception

A painful episode can lead to a life-long increase in an individual’s experience of pain. Fearful anticipation of imminent pain could play a role in this phenomenon, but the neurobiological underpinnings are unclear because fear can both suppress and enhance pain. Here, we show in mice that long-term associative fear memory stored in neuronal engrams in the prefrontal cortex determines whether a painful episode shapes pain experience later in life. Furthermore, under conditions of inflammatory and neuropathic pain, prefrontal fear engrams expand to encompass neurons representing nociception and tactile sensation, leading to pronounced changes in prefrontal connectivity to fear-relevant brain areas. Conversely, silencing prefrontal fear engrams reverses chronically established hyperalgesia and allodynia. These results reveal that a discrete subset of prefrontal cortex neurons can account for the debilitating comorbidity of fear and chronic pain and show that attenuating the fear memory of pain can alleviate chronic pain itself.

Alcohol and Vaporized Nicotine Co-exposure During Adolescence Contribute Differentially to Sex-Specific Behavioral Effects in Adulthood.

Co-occurrence of e-cigarette use and alcohol consumption during adolescence is frequent. Here, we examined whether adolescent co-exposure to alcohol drinking and vaporized nicotine would impact reward- and cognition-related behaviors in adult male and female rats during adulthood. Four groups of male and female Sprague Dawley rats (n = 8-11/group/sex) received either nicotine (JUUL 5% nicotine pods) or vehicle vapor for 10 minutes daily between postnatal days 30-46, while having continuous voluntary access to ethanol and water during this time in a two-bottle preference design. Upon reaching adulthood, all rats underwent behavioral testing (ie, Pavlovian conditioned approach testing, fear conditioning and a two-bottle alcohol preference). A sex-dependent effect, not related to adolescent nicotine or alcohol exposure, on alcohol drinking in adulthood was found, such that females had a higher intake and preference for alcohol compared to males; both male and female adult rats also had greater alcohol preference compared to their alcohol preference as adolescents. Male rats exposed to vaporized nicotine with or without alcohol drinking during adolescence exhibited altered reward-related learning in adulthood, evidenced by enhanced levels of sign-tracking behavior. Male rats that drank alcohol with or without nicotine vapor in adolescence showed deficits in associative fear learning and memory as adults. In contrast, these effects were not seen in female rats exposed to alcohol and nicotine vapor during adolescence. The present study provides evidence that co-exposure to alcohol and vaporized nicotine during adolescence in male, but not female, rats produces long-term changes in reward- and cognition-related behaviors. These findings enhance our understanding of the effects of alcohol drinking and nicotine vapor exposure in adolescence. Moreover, they highlight potential sex differences that exist in the response to alcohol and nicotine vapor, underscoring the need for follow-up studies elucidating the neurobiological mechanisms that drive these sex differences, as well as the long-term effects of alcohol and nicotine vapor use.

Hesperetin rescues emotional memory and synaptic plasticity deficit in aged rats.

Emotional memory deficit is often accompanied by Alzheimer's disease and normal aging. It is important to do what is possible to alleviate or rescue emotional memory deficit in aging to improve the quality of older adults. Hesperetin is a flavonoid and an aglycone of hesperidin, it easily passes through the blood-brain barrier into the brain and exerts neuroprotective effects. However, little is known about its neuroprotective effect on emotional memory in aging. To address this issue, we examined the role of hesperetin in the regulation of hippocampal long-term potentiation (LTP), surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) including glutamate receptor 1 subunit-containing AMPAR (GluA1) and glutamate receptor 1 subunit-containing AMPAR (GluA2), malondialdehyde (MDA) concentration, reduced glutathione (GSH) concentration, and associative fear memory in aged rats. We found that aged rats exhibited impaired emotional memory and LTP. Furthermore, we also found oral administration of hesperetin ameliorated the impairment of emotional memory and LTP. Finally, we demonstrated hesperetin rescued impaired LTP possibly via enhancing AMPAR trafficking and oxidant-antioxidant balance in aged rats. These results imply a pivotal role for hesperetin in synaptic plasticity and associative fear memory in aged rats and suggest that hesperetin is a potential candidate for treating emotional memory deficit in aging. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Repetitive Mild Traumatic Brain Injury in Rats Impairs Cognition, Enhances Prefrontal Cortex Neuronal Activity, and Reduces Pre-synaptic Mitochondrial Function.

A major hurdle preventing effective interventions for patients with mild traumatic brain injury (mTBI) is the lack of known mechanisms for the long-term cognitive impairment that follows mTBI. The closed head impact model of repeated engineered rotational acceleration (rCHIMERA), a non-surgical animal model of repeated mTBI (rmTBI), mimics key features of rmTBI in humans. Using the rCHIMERA in rats, this study was designed to characterize rmTBI-induced behavioral disruption, underlying electrophysiological changes in the medial prefrontal cortex (mPFC), and associated mitochondrial dysfunction. Rats received 6 closed-head impacts over 2 days at 2 Joules of energy. Behavioral testing included automated analysis of behavior in open field and home-cage environments, rotarod test for motor skills, novel object recognition, and fear conditioning. Following rmTBI, rats spent less time grooming and less time in the center of the open field arena. Rats in their home cage had reduced inactivity time 1 week after mTBI and increased exploration time 1 month after injury. Impaired associative fear learning and memory in fear conditioning test, and reduced short-term memory in novel object recognition test were found 4 weeks after rmTBI. Single-unit in vivo recordings showed increased neuronal activity in the mPFC after rmTBI, partially attributable to neuronal disinhibition from reduced inhibitory synaptic transmission, possibly secondary to impaired mitochondrial function. These findings help validate this rat rmTBI model as replicating clinical features, and point to impaired mitochondrial functions after injury as causing imbalanced synaptic transmission and consequent impaired long-term cognitive dysfunction.

Synaptic correlates of associative fear memory in the lateral amygdala

Successful adaptation to the environment requires an accurate response to external threats by recalling specific memories. Memory formation and recall require engram cell activity and synaptic strengthening among activated neuronal ensembles. However, elucidation of the underlying neural substrates of associative fear memory has remained limited without a direct interrogation of extinction-induced changes of specific synapses that encode a specific auditory fear memory. Using dual-eGRASP (enhanced green fluorescent protein reconstitution across synaptic partners), we found that synapses among activated neuronal ensembles or activated synaptic ensembles showed a significantly larger spine morphology at auditory cortex (AC)-to-lateral amygdala (LA) projections after auditory fear conditioning in mice. Fear extinction reversed these enhanced synaptic ensemble spines, whereas re-conditioning with the same tone and shock restored the spine size of the synaptic ensemble. We suggest that synaptic ensembles encode and represent different fear memory states.

Imaging of C-fos Activity in Neurons of the Mouse Parietal Association Cortex during Acquisition and Retrieval of Associative Fear Memory.

The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.

Oxytocin receptor activation does not mediate associative fear deficits in a Williams Syndrome model.

Williams Syndrome results in distinct behavioral phenotypes, which include learning deficits, anxiety, increased phobias and hypersociability. While the underlying mechanisms driving this subset of phenotypes is unknown, oxytocin (OT) dysregulation is hypothesized to be involved as some studies have shown elevated blood OT and altered OT receptor expression in patients. A "Complete Deletion" (CD) mouse, modeling the hemizygous deletion in Williams Syndrome, recapitulates many of the phenotypes present in humans. These CD mice also exhibit impaired fear responses in the conditioned fear task. Here, we address whether OT dysregulation is responsible for this impaired associative fear memory response. We show direct delivery of an OT receptor antagonist to the central nervous system did not rescue the attenuated contextual or cued fear memory responses in CD mice. Thus, increased OT signaling is not acutely responsible for this phenotype. We also evaluated OT receptor and serotonin transporter availability in regions related to fear learning, memory and sociability using autoradiography in wild type and CD mice. While no differences withstood correction, we identified regions that may warrant further investigation. There was a nonsignificant decrease in OT receptor expression in the lateral septal nucleus and nonsignificant lowered serotonin transporter availability in the striatum and orbitofrontal cortex. Together, these data suggest the fear conditioning anomalies in the Williams Syndrome mouse model are independent of any alterations in the oxytocinergic system caused by deletion of the Williams locus.

GluN2D NMDA Receptors Gate Fear Extinction Learning and Interneuron Plasticity.

The cerebellum is critically involved in the formation of associative fear memory and in subsequent extinction learning. Fear conditioning is associated with a long-term potentiation at both excitatory and inhibitory synapses onto Purkinje cells. We therefore tested whether fear conditioning unmasks novel forms of synaptic plasticity, which enable subsequent extinction learning to reset cerebellar circuitry. We found that fear learning enhanced GABA release from molecular layer interneurons and this was reversed after fear extinction learning. Importantly an extinction-like stimulation of parallel fibers after fear learning is sufficient to induce a lasting decrease in inhibitory transmission (I-LTDstim) in the cerebellar cortex, a form of plasticity that is absent in naïve animals. While NMDA (N-methyl-D-aspartate) receptors are required for the formation and extinction of associative memory, the role of GluN2D, one of the four major NMDA receptor subunits, in learning and memory has not been determined. We found that fear conditioning elevates spontaneous GABA release in GluN2D KO as shown in WT mice. Deletion of GluN2D, however, abolished the I-LTDstim induced by parallel fiber stimulation after learning. At the behavioral level, genetic deletion of GluN2D subunits did not affect associative learning and memory retention, but impaired subsequent fear extinction learning. D-cycloserine, a partial NMDA receptor (NMDAR) agonist, failed to rescue extinction learning in mutant mice. Our results identify GluN2D as a critical NMDAR subunit for extinction learning and reveal a form of GluN2D-dependent metaplasticity that is associated with extinction in the cerebellum.

Traumatic memory: Molecular and cellular mechanisms of post‐traumatic stress disorder

The mechanisms of post-traumatic stress disorder (PTSD) have attracted increasing attention in recent years. However, despite the great success in the study of PTSD at the behavioral, psychological and physiological level in humans, as well as large number animal models of PTSD, the phenomenon of PTSD remains largely incomprehensible in theoretical terms. The aim of this project is to establish the neural basis for the pathological stability of traumatic memory that formed when encountering a stressful situation leading to the development of PTSD and to study the differences between the mechanisms of normal and traumatic memory formation at the neural level. First, we subjected mice to single traumatic experience and studied the dependence of PTSD development on protein synthesis during the consolidation and reconsolidation of traumatic memory. Mice injected with protein synthesis inhibitor cycloheximide (90 mg/kg, i.p.) 30 min before PTSD induction retained contextual associative memory but did not show a pronounced contextual fear. The results of elevated plus maze test suggests that the protein synthesis inhibition during the consolidation of traumatic memory can not only prevent the development of PTSD, but also disrupt the natural defensive behavior of animals, making them less anxious than control mice who had no previous negative experience. Than we showed that protein synthesis inhibition during the reconsolidation of traumatic memory almost completely abolish non-specific symptoms of trauma in mice, such as increased sensitization, fear generalization and anxiety. At the same time, however, cycloheximide injection during reminder did not affect the associative fear memory. These results show a dissociation between associative and nonassociative consequences of a traumatic experience in terms of their sensitivity to impairment during memory retrieval. We also studied the dependence of PTSD development on de novo methylation processes. Methyltransferase inhibitors 5-AZA and RG108 injection (0.5 mg/kg, i.p.) 30 min before traumatic episode reduced PTSD symptoms in mice: it decreased the level of conditioned fear and affected the level of behavioral sensitization and anxiety, but not the generalization. The inhibition of methylation processes during the reconsolidation of traumatic memory did not affect the symptoms of PTSD according to our data. We analyzed the c-Fos activity of the brain in the formation of normal contextual fear memory (FC) and traumatic memory in order to answer the second question of this project. The formation of traumatic memory was accompanied by activation of a pattern of brain areas similar to that activated during formation of FC memory. However, in PTSD the activity of amygdala, prelimbic cortex and dentate gyrus was considerably more pronounced. These results show important differences between normal and traumatic forms of memory at the time of their formation. In addition we hypothesize that increased activation of the amygdala and prefrontal cortex in PTSD may be associated with pathological persistence of traumatic memory.

The BAD-BAX-Caspase-3 Cascade Modulates Synaptic Vesicle Pools via Autophagy.

The BAD-BAX-caspase-3 cascade is a canonical apoptosis pathway. Macroautophagy ("autophagy" hereinafter) is a process by which organelles and aggregated proteins are delivered to lysosomes for degradation. Here, we report a new function of the BAD-BAX-caspase-3 cascade and autophagy in the control of synaptic vesicle pools. We found that, in hippocampal neurons of male mice, the BAD-BAX-caspase-3 pathway regulates autophagy, which in turn limits the size of synaptic vesicle pools and influences the kinetics of activity-induced depletion and recovery of synaptic vesicle pools. Moreover, the caspase-autophagy pathway is engaged by fear conditioning to facilitate associative fear learning and memory. This work identifies a new mechanism for controlling synaptic vesicle pools, and a novel, nonapoptotic, presynaptic function of the BAD-BAX-caspase-3 cascade.SIGNIFICANCE STATEMENT Despite the importance of synaptic vesicles for neurons, little is known about how the size of synaptic vesicle pools is maintained under basal conditions and regulated by neural activity. This study identifies a new mechanism for the control of synaptic vesicle pools, and a new, nonapoptotic function of the BAD-BAX-caspase-3 pathway in presynaptic terminals. Additionally, it indicates that autophagy is not only a homeostatic mechanism to maintain the integrity of cells and tissues, but also a process engaged by neural activity to regulate synaptic vesicle pools for optimal synaptic responses, learning, and memory.