Behavioral characterization of Capn15 conditional knockout mice.

Calpain 15 (CAPN15) is an intracellular cysteine protease belonging to the non-classical small optic lobe (SOL) family of calpains, which has an important role in development. Loss of Capn15 in mice leads to developmental eye anomalies and volumetric changes in the brain. Human individuals with biallelic variants in CAPN15 have developmental delay, neurodevelopmental disorders, as well as congenital malformations. In Aplysia, a reductionist model to study learning and memory, SOL calpain is important for non-associative long-term facilitation, the cellular analog of sensitization behavior. However, how CAPN15 is involved in adult behavior or learning and memory in vertebrates is unknown. Here, using Capn15 conditional knockout mice, we show that loss of the CAPN15 protein in excitatory forebrain neurons reduces self-grooming and marble burying, decreases performance in the accelerated roto-rod and reduces pre-tone freezing after strong fear conditioning. Thus, CAPN15 plays a role in regulating behavior in the adult mouse.

A novel stress-based intervention reduces cigarette use in non-treatment seeking smokers.

Tobacco use is the leading cause of preventable mortality worldwide. Since current smoking cessation aids show only modest efficacy, new interventions are needed. Given the evidence that stress is a potent trigger for smoking, the present randomized clinical trial tested whether stress could augment the effects of a memory updating (retrieval-extinction) intervention. Non-treatment seeking smokers (n = 76) were assigned to one of four conditions composed of either a stressful or non-stressful psychosocial challenge followed by either smoking or neutral cues. Ten minutes after this manipulation, all underwent a 60-minute extinction procedure during which they viewed smoking-related videos and images and manipulated smoking paraphernalia. Compared to participants who were not exposed to the laboratory stressor, the stressor-exposed groups exhibited greater psychophysiological responses during their intervention and greater decreases in cigarette use at two- and six-weeks follow-up independent of smoking cue exposure. Together, these findings suggest that the ability of stress to activate cigarette seeking processes can be exploited to decrease cigarette use. With replication, the stress-based intervention could become a novel strategy for decreasing cigarette use in non-treatment seeking smokers.Clinicaltrials.gov identifier: NCT04843969.

Memory Destabilization and Reconsolidation Dynamically Regulate the PKMζ Maintenance Mechanism.

Useful memory must balance between stability and malleability. This puts effective memory storage at odds with plasticity processes, such as reconsolidation. What becomes of memory maintenance processes during synaptic plasticity is unknown. Here we examined the fate of the memory maintenance protein PKMζ during memory destabilization and reconsolidation in male rats. We found that NMDAR activation and proteasome activity induced a transient reduction in PKMζ protein following retrieval. During reconsolidation, new PKMζ was synthesized to re-store the memory. Failure to synthesize new PKMζ during reconsolidation impaired memory but uninterrupted PKMζ translation was not necessary for maintenance itself. Finally, NMDAR activation was necessary to render memories vulnerable to the amnesic effect of PKMζ-antisense. These findings outline a transient disruption and renewal of the PKMζ memory maintenance mechanism during plasticity. We argue that dynamic changes in PKMζ protein levels can serve as an exemplary model of the molecular changes underlying memory destabilization and reconsolidation.SIGNIFICANCE STATEMENT Maintenance of long-term memory relies on the persistent activity of PKMζ. However, after retrieval, memories can become transiently destabilized and must be reconsolidated within a few hours to persist. During this period of plasticity, what happens to maintenance processes, such as those involving PKMζ, is unknown. Here we describe dynamic changes to PKMζ expression during both destabilization and reconsolidation of auditory fear memory in the amygdala. We show that destabilization induces a NMDAR- and proteasome-dependent loss of synaptic PKMζ and that reconsolidation requires synthesis of new PKMζ. This work provides clear evidence that memory destabilization disrupts ongoing synaptic maintenance processes which are restored during reconsolidation.

Inhibitory interneurons mediate autism-associated behaviors via 4E-BP2.

Translational control plays a key role in regulation of neuronal activity and behavior. Deletion of the translational repressor 4E-BP2 in mice alters excitatory and inhibitory synaptic functions, engendering autistic-like behaviors. The contribution of 4E-BP2-dependent translational control in excitatory and inhibitory neurons and astrocytic cells to these behaviors remains unknown. To investigate this, we generated cell-type-specific conditional 4E-BP2 knockout mice and tested them for the salient features of autism, including repetitive stereotyped behaviors (self-grooming and marble burying), sociability (3-chamber social and direct social interaction tests), and communication (ultrasonic vocalizations in pups). We found that deletion of 4E-BP2 in GABAergic inhibitory neurons, defined by Gad2, resulted in impairments in social interaction and vocal communication. In contrast, deletion of 4E-BP2 in forebrain glutamatergic excitatory neurons, defined by Camk2a, or in astrocytes, defined by Gfap, failed to cause autistic-like behavioral abnormalities. Taken together, we provide evidence for an inhibitory-cell-specific role of 4E-BP2 in engendering autism-related behaviors.

Cortico-hippocampal Schemas Enable NMDAR-Independent Fear Conditioning in Rats.

The neurobiology of memory formation has been studied primarily in experimentally naive animals, but the majority of learning unfolds on a background of prior experience. Considerable evidence now indicates that the brain processes initial and subsequent learning differently. In rodents, a first instance of contextual fear conditioning requires NMDA receptor (NMDAR) activation in the dorsal hippocampus, but subsequent conditioning to another context does not. This shift may result from a change in molecular plasticity mechanisms or in the information required to learn the second task. To clarify how related events are encoded, it is critical to identify which aspect of a first task engages NMDAR-independent learning and the brain regions that maintain this state. Here, we show in rats that the requirement for NMDARs in hippocampus depends neither on prior exposure to context nor footshock alone but rather on the procedural similarity between two conditioning tasks. Importantly, NMDAR-independent learning requires the memory of the first task to remain hippocampus dependent. Furthermore, disrupting memory maintenance in the anterior cingulate cortex after the first task also reinstates NMDAR dependency. These results reveal cortico-hippocampal interactions supporting experience-dependent learning.

Differential role of the anterior and intralaminar/lateral thalamic nuclei in systems consolidation and reconsolidation.

The anterior thalamic nuclei (ATN) and the intralaminar/lateral thalamic nuclei (ILN/LT) play different roles in memory processes. The ATN are believed to be part of an extended hippocampal system, and the ILN/LT have strong connections with the medial prefrontal cortex. It was shown that the ILN/LT are involved in systems consolidation. However, whether they are necessary for memory retrieval as well remains unclear. We, therefore, used c-Fos immunohistochemistry and reversible inactivations to investigate the role of the ATN and ILN/LT in recent and remote contextual fear memory retrieval in rats. The results confirm a differential role of the ATN and ILN/LT in systems consolidation, showing the involvement of the ATN in recent but not remote memory retrieval. This study also pinpoints which specific nuclei are involved in retrieval: the anterodorsal nucleus for recent memories, and the lateral mediodorsal nucleus for remote memories. Lastly, we also show that the ATN are not involved in reconsolidation. Together, the results suggest that these nuclei provide critical feedback for successful memory retrieval and systems consolidation.

Metformin ameliorates core deficits in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is the leading monogenic cause of autism spectrum disorders (ASD). Trinucleotide repeat expansions in FMR1 abolish FMRP expression, leading to hyperactivation of ERK and mTOR signaling upstream of mRNA translation. Here we show that metformin, the most widely used drug for type 2 diabetes, rescues core phenotypes in Fmr1-/y mice and selectively normalizes ERK signaling, eIF4E phosphorylation and the expression of MMP-9. Thus, metformin is a potential FXS therapeutic.

The X-linked inhibitor of apoptosis regulates long-term depression and learning rate.

Hippocampal long-term depression (LTD) is an active form of synaptic plasticity that is necessary for consolidation of spatial memory, contextual fear memory, and novelty acquisition. Recent studies have shown that caspases (CASPs) play an important role in NMDA receptor-dependent LTD and are involved in postsynaptic remodeling and synaptic maturation. In the present study, we examined the role of X-linked inhibitor of apoptosis (XIAP), a putative endogenous CASP inhibitor, in synaptic plasticity in the hippocampus. Analysis in acute brain slices and in cultured hippocampal neurons revealed that XIAP deletion increases CASP-3 activity, enhances α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization, sharply increases LTD, and significantly reduces synapse density. In vivo behaviors related to memory were also altered in XIAP(-/-) mice, with faster acquisition of spatial object location and increased fear memory observed. Together, these results indicate that XIAP plays an important physiologic role in regulating sublethal CASP-3 activity within central neurons and thereby facilitates synaptic plasticity and memory acquisition.-Gibon, J., Unsain, N., Gamache, K., Thomas, R. A., De Leon, A., Johnstone, A., Nader, K., Séguéla, P., Barker, P. A. The X-linked inhibitor of apoptosis regulates long-term depression and learning rate.

Memory retrieval requires ongoing protein synthesis and NMDA receptor activity-mediated AMPA receptor trafficking.

Whereas consolidation and reconsolidation are considered dynamic processes requiring protein synthesis, memory retrieval has long been considered a passive readout of previously established plasticity. However, previous findings suggest that memory retrieval may be more dynamic than previously thought. This study therefore aimed at investigating the molecular mechanisms underlying memory retrieval in the rat. Infusion of protein synthesis inhibitors (rapamycin or anisomycin) in the amygdala 10 min before memory retrieval transiently impaired auditory fear memory expression, suggesting ongoing protein synthesis is required to enable memory retrieval. We then investigated the role of protein synthesis in NMDA receptor activity-mediated AMPA receptor trafficking. Coinfusion of an NMDA receptor antagonist (ifenprodil) or infusion of an AMPA receptor endocytosis inhibitor (GluA23Y) before rapamycin prevented this memory impairment. Furthermore, rapamycin transiently decreased GluA1 levels at the postsynaptic density (PSD), but did not affect extrasynaptic sites. This effect at the PSD was prevented by an infusion of GluA23Y before rapamycin. Together, these data show that ongoing protein synthesis is required before memory retrieval is engaged, and suggest that this protein synthesis may be involved in the NMDAR activity-mediated trafficking of AMPA receptors that takes place during memory retrieval.

Pharmacological brake-release of mRNA translation enhances cognitive memory.

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the 'integrated stress response' (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. DOI:http://dx.doi.org/10.7554/eLife.00498.001.

Control of synaptic plasticity and memory via suppression of poly(A)-binding protein.

Control of protein synthesis is critical for synaptic plasticity and memory formation. However, the molecular mechanisms linking neuronal activity to activation of mRNA translation are not fully understood. Here, we report that the translational repressor poly(A)-binding protein (PABP)-interacting protein 2A (PAIP2A), an inhibitor of PABP, is rapidly proteolyzed by calpains in stimulated neurons and following training for contextual memory. Paip2a knockout mice exhibit a lowered threshold for the induction of sustained long-term potentiation and an enhancement of long-term memory after weak training. Translation of CaMKIIα mRNA is enhanced in Paip2a⁻/⁻ slices upon tetanic stimulation and in the hippocampus of Paip2a⁻/⁻ mice following contextual fear learning. We demonstrate that activity-dependent degradation of PAIP2A relieves translational inhibition of memory-related genes through PABP reactivation and conclude that PAIP2A is a pivotal translational regulator of synaptic plasticity and memory.

Preclinical evaluation of reconsolidation blockade by clonidine as a potential novel treatment for posttraumatic stress disorder.

Exposure to traumatic events can lead to posttraumatic stress disorder (PTSD). Current PTSD treatments typically only produce partial improvement. Hence, there is a need for preclinical research to identify new candidate drugs and to develop novel therapeutic approaches. Animal studies have indicated that fear memories can be weakened by blocking restabilization after retrieval, a process known as reconsolidation. Furthermore, evidence suggests that there are important alterations of the noradrenergic system in PTSD, and hence it may be of interest to study drugs that target this pathway. Here, we investigated the efficacy of clonidine, an α2-adrenoreceptor agonist, to block reconsolidation in an animal model of persistent traumatic memories. Using an auditory fear conditioning paradigm in rats, we tested the efficacy of clonidine to weaken fear memory retention when administered systemically after retrieval. We evaluated dosage, number of treatments, and specificity in reconsolidation blockade. We found that postretrieval administration of clonidine disrupts fear-related memories in a dose-dependent manner and that two treatments are sufficient for maximal memory impairment. Furthermore, we determined that this effect is long lasting and specific to reconsolidation processes as shown by the selectivity to affect reactivated memories and the absence of spontaneous recovery and of postreactivation short-term memory impairment. Our results demonstrate the efficacy of systemic administration of clonidine following retrieval to persistently disrupt fear memory retention through reconsolidation blockade. This study provides important preclinical parameters for future therapeutic strategies involving clonidine to block reconsolidation as a novel treatment for PTSD symptoms.

Systemic mifepristone blocks reconsolidation of cue-conditioned fear; propranolol prevents this effect.

Reducing reconsolidation of reactivated traumatic memories may offer a novel pharmacological treatment for posttraumatic stress disorder (PTSD). Preclinical research is needed to identify candidate drugs. We evaluated the ability of postreactivation mifepristone (RU38486, a glucocorticoid antagonist), alone and in combination with propranolol (a beta-adrenergic blocker), both given systemically, to reduce cue-conditioned fear in rats. On Day 1, a 30-s tone conditioned stimulus (CS) was paired with an electric shock unconditioned stimulus (US). On Day 2, the CS was presented without the US (reactivation), and the freezing conditioned response (CR) was measured. This was immediately followed by subcutaneous injection of vehicle, mifepristone 30 mg/kg, propranolol 10 mg/kg, or both. On Day 3, the CR was again measured as a test of postreactivation long-term memory (PR-LTM). On Day 10, the CR was again measured to evaluate spontaneous recovery. On Day 11, the US was presented alone (reinstatement). On Day 12, the CR was again measured. A fifth group received mifepristone without the CS presentation (nonreactivation) on Day 2. A sixth group was tested four hours after the Day 2 mifepristone injection to measure postreactivation short-term memory. Postreactivation, but not nonreactivation, mifepristone produced a decrement in the CR that did not undergo spontaneous recovery and underwent only modest reinstatement. Mifepristone did not exert its effect when administered concurrently with propranolol. Postreactivation mifepristone did not impair short-term memory. Systemic mifepristone blocks the reconsolidation of cue-conditioned fear in rats. Concurrent administration of propranolol prevents this effect. Postreactivation mifepristone may be a promising treatment for PTSD, but not necessarily in combination with propranolol.

PKMzeta maintains memories by regulating GluR2-dependent AMPA receptor trafficking.

The maintenance of long-term memory in hippocampus, neocortex and amygdala requires the persistent action of the atypical protein kinase C isoform, protein kinase Mzeta (PKMzeta). We found that inactivating PKMzeta in the amygdala impaired fear memory in rats and that the extent of the impairment was positively correlated with a decrease in postsynaptic GluR2. Blocking the GluR2-dependent removal of postsynaptic AMPA receptors abolished the behavioral impairment caused by PKMzeta inhibition and the associated decrease in postsynaptic GluR2 expression, which correlated with performance. Similarly, blocking this pathway for removal of GluR2-containing receptors from postsynaptic sites in amygdala slices prevented the reversal of long-term potentiation caused by inactivating PKMzeta. Similar behavioral results were obtained in the hippocampus for unreinforced recognition memory of object location. Together, these findings indicate that PKMzeta maintains long-term memory by regulating the trafficking of GluR2-containing AMPA receptors, the postsynaptic expression of which directly predicts memory retention.

eIF2alpha phosphorylation bidirectionally regulates the switch from short- to long-term synaptic plasticity and memory.

The late phase of long-term potentiation (LTP) and memory (LTM) requires new gene expression, but the molecular mechanisms that underlie these processes are not fully understood. Phosphorylation of eIF2alpha inhibits general translation but selectively stimulates translation of ATF4, a repressor of CREB-mediated late-LTP (L-LTP) and LTM. We used a pharmacogenetic bidirectional approach to examine the role of eIF2alpha phosphorylation in synaptic plasticity and behavioral learning. We show that in eIF2alpha(+/S51A) mice, in which eIF2alpha phosphorylation is reduced, the threshold for eliciting L-LTP in hippocampal slices is lowered, and memory is enhanced. In contrast, only early-LTP is evoked by repeated tetanic stimulation and LTM is impaired, when eIF2alpha phosphorylation is increased by injecting into the hippocampus a small molecule, Sal003, which prevents the dephosphorylation of eIF2alpha. These findings highlight the importance of a single phosphorylation site in eIF2alpha as a key regulator of L-LTP and LTM formation.

NMDA receptors are critical for unleashing consolidated auditory fear memories.

Memories are dynamic and can change when recalled. The process that returns memories to a labile state during remembering is unclear. We found that the presence of NMDA, but not AMPA, receptor antagonists in the amygdala prior to recall prevented the consolidated fear memory from returning to a labile state. These findings suggest that NMDA receptors in the amygdala are critical for transforming a memory from a fixed to a labile state.