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.

Specific behaviors during auditory fear conditioning and postsynaptic expression of AMPA receptors in the basolateral amygdala predict interindividual differences in fear generalization in male rats.

Auditory fear conditioning in rats is a widely used method to study learning, memory, and emotional responding. Despite procedural standardizations and optimizations, there is substantial interindividual variability in fear expression during test, notably in terms of fear expressed toward the testing context alone. To better understand which factors could explain this variation between subjects, we here explored whether behavior during training and expression of AMPA receptors (AMPARs) after long-term memory formation in the amygdala could predict freezing during test. We studied outbred male rats and found strong variation in fear generalization to a different context. Hierarchical clustering of these data identified two distinct groups of subjects that independently correlated with a specific pattern of behaviors expressed during initial training (i.e., rearing and freezing). The extent of fear generalization correlated positively with postsynaptic expression of GluA1-containing AMPA receptors in the basolateral nucleus of the amygdala. Our data thus identify candidate behavioral and molecular predictors of fear generalization that may inform our understanding of some anxiety-related disorders, such as posttraumatic stress disorder (PTSD), that are characterized by overgeneralized fear.

Infusing zeta inhibitory peptide into the perirhinal cortex of rats abolishes long-term object recognition memory without affecting novel object location recognition.

Infusing the amnesic agent zeta inhibitory peptide (ZIP) into the dorsal hippocampus disrupts established long-term object location recognition memory without affecting object identity recognition, which likely depends on the perirhinal cortex. Here, we tested whether infusing ZIP into the perirhinal cortex can abolish long-term memory supporting object identity recognition, leaving long-term object location recognition memory intact. We infused ZIP into the perirhinal cortex of rats either 1 day or 6 days after exposing them to two identical objects in an open field arena. One day after ZIP infusion, that is, 2 or 7 days after object exposure, we either assessed whether the animals recognized that now one of the two objects was novel or whether they recognized that one of the two familiar objects was at a new location. Our results show for both retention intervals, infusions of ZIP into the perirhinal cortex impaired novel object recognition but spared novel object location recognition. Rats that received a scrambled version of ZIP had no deficit in either test at both retention intervals and expressed stronger novel object recognition compared to rats infused with ZIP. These findings support the view that object recognition depends on dissociable memory representations distributed across different brain areas, with perirhinal cortex maintaining long-term memory for what objects had been encountered, and hippocampus supporting memory for where these objects had been placed.

Imbalance of flight-freeze responses and their cellular correlates in the Nlgn3-/y rat model of autism.

BACKGROUND: Mutations in the postsynaptic transmembrane protein neuroligin-3 are highly correlative with autism spectrum disorders (ASDs) and intellectual disabilities (IDs). Fear learning is well studied in models of these disorders, however differences in fear response behaviours are often overlooked. We aim to examine fear behaviour and its cellular underpinnings in a rat model of ASD/ID lacking Nlgn3. METHODS: This study uses a range of behavioural tests to understand differences in fear response behaviour in Nlgn3-/y rats. Following this, we examined the physiological underpinnings of this in neurons of the periaqueductal grey (PAG), a midbrain area involved in flight-or-freeze responses. We used whole-cell patch-clamp recordings from ex vivo PAG slices, in addition to in vivo local-field potential recordings and electrical stimulation of the PAG in wildtype and Nlgn3-/y rats. We analysed behavioural data with two- and three-way ANOVAS and electrophysiological data with generalised linear mixed modelling (GLMM). RESULTS: We observed that, unlike the wildtype, Nlgn3-/y rats are more likely to response with flight rather than freezing in threatening situations. Electrophysiological findings were in agreement with these behavioural outcomes. We found in ex vivo slices from Nlgn3-/y rats that neurons in dorsal PAG (dPAG) showed intrinsic hyperexcitability compared to wildtype. Similarly, stimulating dPAG in vivo revealed that lower magnitudes sufficed to evoke flight behaviour in Nlgn3-/y than wildtype rats, indicating the functional impact of the increased cellular excitability. LIMITATIONS: Our findings do not examine what specific cell type in the PAG is likely responsible for these phenotypes. Furthermore, we have focussed on phenotypes in young adult animals, whilst the human condition associated with NLGN3 mutations appears during the first few years of life. CONCLUSIONS: We describe altered fear responses in Nlgn3-/y rats and provide evidence that this is the result of a circuit bias that predisposes flight over freeze responses. Additionally, we demonstrate the first link between PAG dysfunction and ASD/ID. This study provides new insight into potential pathophysiologies leading to anxiety disorders and changes to fear responses in individuals with ASD.

The Black Box effect: sensory stimulation after learning interferes with the retention of long-term object location memory in rats.

Reducing sensory experiences during the period that immediately follows learning improves long-term memory retention in healthy humans, and even preserves memory in patients with amnesia. To date, it is entirely unclear why this is the case, and identifying the neurobiological mechanisms underpinning this effect requires suitable animal models, which are currently lacking. Here, we describe a straightforward experimental procedure in rats that future studies can use to directly address this issue. Using this method, we replicated the central findings on quiet wakefulness obtained in humans: We show that rats that spent 1 h alone in a familiar dark and quiet chamber (the Black Box) after exploring two objects in an open field expressed long-term memory for the object locations 6 h later, while rats that instead directly went back into their home cage with their cage mates did not. We discovered that both visual stimulation and being together with conspecifics contributed to the memory loss in the home cage, as exposing rats either to light or to a cage mate in the Black Box was sufficient to disrupt memory for object locations. Our results suggest that in both rats and humans, everyday sensory experiences that normally follow learning in natural settings can interfere with processes that promote long-term memory retention, thereby causing forgetting in form of retroactive interference. The processes involved in this effect are not sleep-dependent because we prevented sleep in periods of reduced sensory experience. Our findings, which also have implications for research practices, describe a potentially useful method to study the neurobiological mechanisms that might explain why normal sensory processing after learning impairs memory both in healthy humans and in patients suffering from amnesia.

Maintained memory and long-term potentiation in a mouse model of Alzheimer's disease with both amyloid pathology and human tau.

One of the key knowledge gaps in the field of Alzheimer's disease research is the lack of understanding of how amyloid beta and tau cooperate to cause neurodegeneration. We recently generated a mouse model (APP/PS1 + Tau) that develops amyloid plaque pathology and expresses human tau in the absence of endogenous murine tau. These mice exhibit an age-related behavioural hyperactivity phenotype and transcriptional deficits which are ameliorated by tau transgene suppression. We hypothesized that these mice would also display memory and hippocampal synaptic plasticity deficits as has been reported for many plaque bearing mouse models which express endogenous mouse tau. We observed that our APP/PS1 + Tau model does not exhibit novel object memory or robust long-term potentiation deficits with age, whereas the parent APP/PS1 line with mouse tau did develop the expected deficits. These data are important as they highlight potential functional differences between mouse and human tau and the need to use multiple models to fully understand Alzheimer's disease pathogenesis and develop effective therapeutic strategies.

Recapitulation of the EEF1A2 D252H neurodevelopmental disorder-causing missense mutation in mice reveals a toxic gain of function.

Heterozygous de novo mutations in EEF1A2, encoding the tissue-specific translation elongation factor eEF1A2, have been shown to cause neurodevelopmental disorders including often severe epilepsy and intellectual disability. The mutational profile is unusual; ~50 different missense mutations have been identified but no obvious loss of function mutations, though large heterozygous deletions are known to be compatible with life. A key question is whether the heterozygous missense mutations operate through haploinsufficiency or a gain of function mechanism, an important prerequisite for design of therapeutic strategies. In order both to address this question and to provide a novel model for neurodevelopmental disorders resulting from mutations in EEF1A2, we created a new mouse model of the D252H mutation. This mutation causes the eEF1A2 protein to be expressed at lower levels in brain but higher in muscle in the mice. We compared both heterozygous and homozygous D252H and null mutant mice using behavioural and motor phenotyping alongside molecular modelling and analysis of binding partners. Although the proteomic analysis pointed to a loss of function for the D252H mutant protein, the D252H homozygous mice were more severely affected than null homozygotes on the same genetic background. Mice that are heterozygous for the missense mutation show no behavioural abnormalities but do have sex-specific deficits in body mass and motor function. The phenotyping of our novel mouse lines, together with analysis of molecular modelling and interacting proteins, suggest that the D252H mutation results in a gain of function.

Amyloid Beta and Tau Cooperate to Cause Reversible Behavioral and Transcriptional Deficits in a Model of Alzheimer's Disease.

A key knowledge gap blocking development of effective therapeutics for Alzheimer's disease (AD) is the lack of understanding of how amyloid beta (Aß) peptide and pathological forms of the tau protein cooperate in causing disease phenotypes. Within a mouse tau-deficient background, we probed the molecular, cellular, and behavioral disruption triggered by the influence of wild-type human tau on human Aß-induced pathology. We find that Aß and tau work cooperatively to cause a hyperactivity behavioral phenotype and to cause downregulation of transcription of genes involved in synaptic function. In both our mouse model and human postmortem tissue, we observe accumulation of pathological tau in synapses, supporting the potential importance of synaptic tau. Importantly, tau reduction in the mice initiated after behavioral deficits emerge corrects behavioral deficits, reduces synaptic tau levels, and substantially reverses transcriptional perturbations, suggesting that lowering synaptic tau levels may be beneficial in AD.

NMDA receptor activity bidirectionally controls active decay of long-term spatial memory in the dorsal hippocampus.

The time-dependent forgetting of long-term spatial memories involves activation of NMDA receptors (NMDARs) in the hippocampus. Here, we tested whether NMDARs regulate memory persistence bidirectionally, decreasing or increasing the rate of forgetting. We found that blocking NMDAR activation with AP5 or the GluN2B-selective antagonist Ro25-6981 in the dorsal hippocampus (dHPC) prevented the natural forgetting of long-term memory for the locations of objects in an open field arena. In contrast, while enhancing NMDAR function with the partial agonist D-Cycloserine did not affect the speed of forgetting for these types of memories, infusing the NMDAR co-agonist D-Serine significantly shortened their persistence. These results suggest that NMDAR activity can modulate the speed of constitutive long-term memory decay in the dHPC and that regulating NMDAR expression and D-Serine availability could provide a mechanism to control the duration of long-term memory.

Terminological and Epistemological Issues in Current Memory Research.

A number of observations in recent years demonstrates that across all levels of organization, memory is inherently fluid. On the cognitive-behavioral level, the innocent act of remembering can irrevocably alter the contents of established long-term memories, while the content of dormant long-term memories that is deemed irrelevant, superfluous, or limiting may be pragmatically erased or suppressed. On the cellular level, the proteins implementing the molecular alterations underpinning memories are in a constant state of flux, with proteins being turned over, translocated, reconfigured, substituted, and replaced. Yet, the general perception of memory, and the words used to describe it, suggest a static system characterized by the goal of preserving records of past experiences with high fidelity, in contrast to the reality of an inherently adaptive system purposed to enable survival in a changing world with a pragmatic disregard for the fate of acquired memories. Here, we examine present memory terminology and how it corresponds to our actual understanding of the molecules, cells, and systems underlying memory. We will identify where terms lead us astray and line out possible ways to reform memory nomenclature to better fit the true nature of memory as we begin to know it.

Systems consolidation revisited, but not revised: The promise and limits of optogenetics in the study of memory.

Episodic memories (in humans) and event-like memories (in non-human animals) require the hippocampus for some time after acquisition, but at remote points seem to depend more on cortical areas instead. Systems consolidation refers to the process that promotes this reorganization of memory. Various theoretical frameworks accounting for this process have been proposed, but clear evidence favoring one or another of these positions has been lacking. Addressing this issue, a recent study deployed some of the most advanced neurobiological technologies - optogenetics and calcium imaging - and provided high resolution, precise observations regarding brain systems involved in recent and remote contextual fear memories. We critically review these findings within their historical context and conclude that they do not resolve the debate concerning systems consolidation. This is because the relevant question concerning the quality of memory at recent and remote time points has not been answered: Does the memory reorganization taking place during systems consolidation result in changes to the content of memory?

Social propinquity in rodents as measured by tube cooccupancy differs between inbred and outbred genotypes.

Existing assays of social interaction are suboptimal, and none measures propinquity, the tendency of rodents to maintain close physical proximity. These assays are ubiquitously performed using inbred mouse strains and mutations placed on inbred genetic backgrounds. We developed the automatable tube cooccupancy test (TCOT) based on propinquity, the tendency of freely mobile rodents to maintain close physical proximity, and assessed TCOT behavior on a variety of genotypes and social and environmental conditions. In outbred mice and rats, familiarity determined willingness to cooccupy the tube, with siblings and/or cagemates of both sexes exhibiting higher cooccupancy behavior than strangers. Subsequent testing using multiple genotypes revealed that inbred strain siblings do not cooccupy at higher rates than strangers, in marked contrast to both outbred and rederived wild mice. Mutant mouse strains with "autistic-like" phenotypes (Fmr1-/y and Eif4e Ser209Ala) displayed significantly decreased cooccupancy.

Forgetting of long-term memory requires activation of NMDA receptors, L-type voltage-dependent Ca2+ channels, and calcineurin.

In the past decades, the cellular and molecular mechanisms underlying memory consolidation, reconsolidation, and extinction have been well characterized. However, the neurobiological underpinnings of forgetting processes remain to be elucidated. Here we used behavioral, pharmacological and electrophysiological approaches to explore mechanisms controlling forgetting. We found that post-acquisition chronic inhibition of the N-methyl-D-aspartate receptor (NMDAR), L-type voltage-dependent Ca(2+) channel (LVDCC), and protein phosphatase calcineurin (CaN), maintains long-term object location memory that otherwise would have been forgotten. We further show that NMDAR activation is necessary to induce forgetting of object recognition memory. Studying the role of NMDAR activation in the decay of the early phase of long-term potentiation (E-LTP) in the hippocampus, we found that ifenprodil infused 30 min after LTP induction in vivo blocks the decay of CA1-evoked postsynaptic plasticity, suggesting that GluN2B-containing NMDARs activation are critical to promote LTP decay. Taken together, these findings indicate that a well-regulated forgetting process, initiated by Ca(2+) influx through LVDCCs and GluN2B-NMDARs followed by CaN activation, controls the maintenance of hippocampal LTP and long-term memories over time.

Blocking Synaptic Removal of GluA2-Containing AMPA Receptors Prevents the Natural Forgetting of Long-Term Memories.

The neurobiological processes underpinning the natural forgetting of long-term memories are poorly understood. Based on the critical role of GluA2-containing AMPA receptors (GluA2/AMPARs) in long-term memory persistence, we tested in rats whether their synaptic removal underpins time-dependent memory loss. We found that blocking GluA2/AMPAR removal with the interference peptides GluA23Y or G2CT in the dorsal hippocampus during a memory retention interval prevented the normal forgetting of established, long-term object location memories, but did not affect their acquisition. The same intervention also preserved associative memories of food-reward conditioned place preference that would otherwise be lost over time. We then explored whether this forgetting process could play a part in behavioral phenomena involving time-dependent memory change. We found that infusing GluA23Y into the dorsal hippocampus during a 2 week retention interval blocked generalization of contextual fear expression, whereas infusing it into the infralimbic cortex after extinction of auditory fear prevented spontaneous recovery of the conditioned response. Exploring possible physiological mechanisms that could be involved in this form of memory decay, we found that bath application of GluA23Y prevented depotentiation, but not induction of long-term potentiation, in a hippocampal slice preparation. Together, these findings suggest that a decay-like forgetting process that involves the synaptic removal of GluA2/AMPARs erases consolidated long-term memories in the hippocampus and other brain structures over time. This well regulated forgetting process may critically contribute to establishing adaptive behavior, whereas its dysregulation could promote the decline of memory and cognition in neuropathological disorders. SIGNIFICANCE STATEMENT: The neurobiological mechanisms involved in the natural forgetting of long-term memory and its possible functions are not fully understood. Based on our previous work describing the role of GluA2-containing AMPA receptors in memory maintenance, here, we tested their role in forgetting of long-term memory. We found that blocking their synaptic removal after long-term memory formation extended the natural lifetime of several forms of memory. In the hippocampus, it preserved spatial memories and inhibited contextual fear generalization; in the infralimbic cortex, it blocked the spontaneous recovery of extinguished fear. These findings suggest that a constitutive decay-like forgetting process erases long-term memories over time, which, depending on the memory removed, may critically contribute to developing adaptive behavioral responses.

The maintenance of long-term memory in the hippocampus depends on the interaction between N-ethylmaleimide-sensitive factor and GluA2.

The maintenance of established memories has recently been shown to involve the stabilization of GluA2-containing AMPA receptors (GluA2/AMPARs) at postsynaptic membranes. Previous studies have suggested that N-ethylmaleimide-sensitive factor (NSF) regulates the stabilization of AMPARs at the synaptic membrane. We therefore disrupted the interaction between GluA2 and NSF in the dorsal hippocampus and examined its effect on the maintenance of object location and contextual fear memory. We used two interference peptides, pep2m and pepR845A, that have been shown to block the binding of NSF to GluA2 and reduce GluA2 synaptic content. Either peptide disrupted consolidated memory, and these effects persisted for at least 5 or 28 days after peptide administration. Following peptide administration and long-term memory disruption, rats were able to acquire new memories. Memory acquisition or consolidation was not impaired when pepR845A was given immediately before the training sessions. Blocking GluA2 endocytosis with the peptide GluA23Y prevented the memory impairment effect of pepR845A. Taken together, our results indicate that the persistence of long-term memory depends on the maintenance of a steady-state level of synaptic GluA2/AMPARs, which requires the interaction of NSF with GluA2.

GluA2-dependent AMPA receptor endocytosis and the decay of early and late long-term potentiation: possible mechanisms for forgetting of short- and long-term memories.

The molecular processes involved in establishing long-term potentiation (LTP) have been characterized well, but the decay of early and late LTP (E-LTP and L-LTP) is poorly understood. We review recent advances in describing the mechanisms involved in maintaining LTP and homeostatic plasticity. We discuss how these phenomena could relate to processes that might underpin the loss of synaptic potentiation over time, and how they might contribute to the forgetting of short-term and long-term memories. We propose that homeostatic downscaling mediates the loss of E-LTP, and that metaplastic parameters determine the decay rate of L-LTP, while both processes require the activity-dependent removal of postsynaptic GluA2-containing AMPA receptors.

Decay happens: the role of active forgetting in memory.

Although the biological bases of forgetting remain obscure, the consensus among cognitive psychologists emphasizes interference processes, rejecting decay in accounting for memory loss. In contrast to this view, recent advances in understanding the neurobiology of long-term memory maintenance lead us to propose that a brain-wide well-regulated decay process, occurring mostly during sleep, systematically removes selected memories. Down-regulation of this decay process can increase the life expectancy of a memory and may eventually prevent its loss. Memory interference usually occurs during certain active processing phases, such as encoding and retrieval, and will be stronger in brain areas with minimal sensory integration and less pattern separation. In areas with efficient pattern separation, such as the hippocampus, interference-driven forgetting will be minimal, and, consequently, decay will cause most forgetting.

Memory as a new therapeutic target.

This review aims to demonstrate how an understanding of the brain mechanisms involved in memory provides a basis for; (i) reconceptualizing some mental disorders; (ii) refining existing therapeutic tools; and (iii) designing new ones for targeting processes that maintain these disorders. First, some of the stages which a memory undergoes are defined, and the clinical relevance of an understanding of memory processing by the brain is discussed. This is followed by a brief review of some of the clinical studies that have targeted memory processes. Finally, some new insights provided by the field of neuroscience with implications for conceptualizing mental disorders are presented.

Esta revisión busca demostrar cómo una comprensión de los mecanismos cerebrales involucrados en la memoria aportan las bases para: 1) reconceptualizar algunos trastornos mentales, 2) perfeccionar las herramientas terapéuticas existentes y 3) diseñar nuevas terapias para los procesos clave que sustentan estos trastornos. Primero se definen algunas de las fases que están a la base de la memoria y se discute la relevancia clinica de la comprensión de los procesos de memoria por el cerebro. Luego se revisan brevemente algunos estudios clínicos que se han enfocado en procesos de memoria y finalmente se presentan algunas nuevas perspectivas provenientes de las neurociencias que tienen repercusiones para la conceptualización de los trastornos mentales.

Cet article démontre comment la compréhension des mécanismes cérébraux impliqués dans la mémoire sert de base à: a) reconceptualiser certains troubles mentaux; b) améliorer les outils thérapeutiques existants; et c) élaborer de nouveaux outils pour cibler les processus qui entretiennent ces troubles. Nous définissons tout d'abord certains des stades par lesquels passe la mémoire, et nous analysons la pertinence clinique du traitement de la mémoire par le cerveau. Nous poursuivons par une brève mise au point de quelques études cliniques sur les processus mnésiques. Nous présentons enfin les nouveautés dans le domaine des neurosciences et leurs conséquences dans la conceptualisation des troubles mentaux.

Dorsal hippocampus is necessary for novel learning but sufficient for subsequent similar learning.

Our current understanding of brain mechanisms involved in learning and memory has been derived largely from studies using experimentally naïve animals. However, it is becoming increasingly clear that not all identified mechanisms may generalize to subsequent learning. For example, N-methyl-D-aspartate glutamate (NMDA) receptors in the dorsal hippocampus are required for contextual fear conditioning in naïve animals but not in animals previously trained in a similar task. Here we investigated how animals learn contextual fear conditioning for a second time-a response which is not due to habituation or generalization. We found that dorsal hippocampus infusions of voltage-dependent calcium channel blockers or the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) agonist impaired the first, not the second contextual learning. Only manipulations of the entire hippocampus led to an impairment in second learning. Specifically, inactivation of either the dorsal or ventral hippocampus caused the remaining portion of the hippocampus to acquire and consolidate the second learning. Thus, dorsal hippocampus seems necessary for initial contextual fear conditioning, but either the dorsal or ventral hippocampus is sufficient for subsequent conditioning in a different context. Together, these findings suggest that prior training experiences can change how the hippocampus processes subsequent similar learning.

Update on memory systems and processes.

Ideas about how the brain organizes learning and memory have been evolving in recent years, with potentially important ramifications. We review traditional thinking about learning and memory and consider more closely emerging trends from both human and animal research that could lead to profound shifts in how we understand the neural basis of memory.