Higher-order interactions between hippocampal CA1 neurons are disrupted in amnestic mice.

Across systems, higher-order interactions between components govern emergent dynamics. Here we tested whether contextual threat memory retrieval in mice relies on higher-order interactions between dorsal CA1 hippocampal neurons requiring learning-induced dendritic spine plasticity. We compared population-level Ca2+ transients as wild-type mice (with intact learning-induced spine plasticity and memory) and amnestic mice (TgCRND8 mice with high levels of amyloid-ß and deficits in learning-induced spine plasticity and memory) were tested for memory. Using machine-learning classifiers with different capacities to use input data with complex interactions, our findings indicate complex neuronal interactions in the memory representation of wild-type, but not amnestic, mice. Moreover, a peptide that partially restored learning-induced spine plasticity also restored the statistical complexity of the memory representation and memory behavior in Tg mice. These findings provide a previously missing bridge between levels of analysis in memory research, linking receptors, spines, higher-order neuronal dynamics and behavior.

Comparing behaviours induced by natural memory retrieval and optogenetic reactivation of an engram ensemble in mice.

Memories are thought to be stored within sparse collections of neurons known as engram ensembles. Neurons active during a training episode are allocated to an engram ensemble ('engram neurons'). Memory retrieval is initiated by external sensory or internal cues present at the time of training reactivating engram neurons. Interestingly, optogenetic reactivation of engram ensemble neurons alone in the absence of external sensory cues is sufficient to induce behaviour consistent with memory retrieval in mice. However, there may exist differences between the behaviours induced by natural retrieval cues or artificial engram reactivation. Here, we compared two defensive behaviours (freezing and the syllable structure of ultrasonic vocalizations, USVs) induced by sensory cues present at training (natural memory retrieval) and optogenetic engram ensemble reactivation (artificial memory retrieval) in a threat conditioning paradigm in the same mice. During natural memory recall, we observed a strong positive correlation between freezing levels and distinct USV syllable features (characterized by an unsupervised algorithm, MUPET (Mouse Ultrasonic Profile ExTraction)). Moreover, we observed strikingly similar behavioural profiles in terms of freezing and USV characteristics between natural memory recall and artificial memory recall in the absence of sensory retrieval cues. Although our analysis focused on two behavioural measures of threat memory (freezing and USV characteristics), these results underscore the similarities between threat memory recall triggered naturally and through optogenetic reactivation of engram ensembles. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Engrams.

Frankland et al. provide a history of research on engrams and their relationship to memory processes, highlighting new technologies that have allowed careful dissection of engrams and their function.

Neurogenesis-dependent remodeling of hippocampal circuits reduces PTSD-like behaviors in adult mice.

Post-traumatic stress disorder (PTSD) is a hypermnesic condition that develops in a subset of individuals following exposure to severe trauma. PTSD symptoms are debilitating, and include increased anxiety, abnormal threat generalization, and impaired extinction. In developing treatment strategies for PTSD, preclinical studies in rodents have largely focused on interventions that target post-encoding memory processes such as reconsolidation and extinction. Instead, here we focus on forgetting, another post-encoding process that regulates memory expression. Using a double trauma murine model for PTSD, we asked whether promoting neurogenesis-mediated forgetting can weaken trauma memories and associated PTSD-relevant behavioral phenotypes. In the double trauma paradigm, consecutive aversive experiences lead to a constellation of behavioral phenotypes associated with PTSD including increases in anxiety-like behavior, abnormal threat generalization, and deficient extinction. We found that post-training interventions that elevate hippocampal neurogenesis weakened the original trauma memory and decreased these PTSD-relevant phenotypes. These effects were observed using multiple methods to manipulate hippocampal neurogenesis, including interventions restricted to neural progenitor cells that selectively promoted integration of adult-generated granule cells into hippocampal circuits. The same interventions also weakened cocaine place preference memories, suggesting that promoting hippocampal neurogenesis may represent a broadly useful approach in hypermnesic conditions such as PTSD and substance abuse disorders.

Reactivation of encoding ensembles in the prelimbic cortex supports temporal associations.

Fear conditioning is encoded by strengthening synaptic connections between the neurons activated by a conditioned stimulus (CS) and those activated by an unconditioned stimulus (US), forming a memory engram, which is reactivated during memory retrieval. In temporal associations, activity within the prelimbic cortex (PL) plays a role in sustaining a short-term, transient memory of the CS, which is associated with the US after a temporal gap. However, it is unknown whether the PL has only a temporary role, transiently representing the CS, or is part of the neuronal ensembles that support the retrieval, i.e., whether PL neurons support both transient, short-term memories and stable, long-term memories. We investigated neuronal ensembles underlying temporal associations using fear conditioning with a 5-s interval between the CS and US (CFC-5s). Controls were trained in contextual fear conditioning (CFC), in which the CS-US overlaps. We used Robust Activity Marking (RAM) to selectively manipulate PL neurons activated by CFC-5s learning and Targeted Recombination in Active Populations (TRAP2) mice to label neurons activated by CFC-5s learning and reactivated by memory retrieval in the amygdala, medial prefrontal cortex, hippocampus, perirhinal cortices (PER) and subiculum. We also computed their co-reactivation to generate correlation-based networks. The optogenetic reactivation or silencing of PL encoding ensembles either promoted or impaired the retrieval of CFC-5s but not CFC. CFC-5s retrieval reactivated encoding ensembles in the PL, PER, and basolateral amygdala. The engram network of CFC-5s had higher amygdala and PER centralities and interconnectivity. The same PL neurons support learning and stable associative memories.

Excitability mediates allocation of pre-configured ensembles to a hippocampal engram supporting contextual conditioned threat in mice.

Little is understood about how engrams, sparse groups of neurons that store memories, are formed endogenously. Here, we combined calcium imaging, activity tagging, and optogenetics to examine the role of neuronal excitability and pre-existing functional connectivity on the allocation of mouse cornu ammonis area 1 (CA1) hippocampal neurons to an engram ensemble supporting a contextual threat memory. Engram neurons (high activity during recall or TRAP2-tagged during training) were more active than non-engram neurons 3 h (but not 24 h to 5 days) before training. Consistent with this, optogenetically inhibiting scFLARE2-tagged neurons active in homecage 3 h, but not 24 h, before conditioning disrupted memory retrieval, indicating that neurons with higher pre-training excitability were allocated to the engram. We also observed stable pre-configured functionally connected sub-ensembles of neurons whose activity cycled over days. Sub-ensembles that were more active before training were allocated to the engram, and their functional connectivity increased at training. Therefore, both neuronal excitability and pre-configured functional connectivity mediate allocation to an engram ensemble.

Nonsynonymous Mutations in Intellectual Disability and Autism Spectrum Disorder Gene PTCHD1 Disrupt N-Glycosylation and Reduce Protein Stability.

PTCHD1 has been implicated in Autism Spectrum Disorders (ASDs) and/or intellectual disability, where copy-number-variant losses or loss-of-function coding mutations segregate with disease in an X-linked recessive fashion. Missense variants of PTCHD1 have also been reported in patients. However, the significance of these mutations remains undetermined since the activities, subcellular localization, and regulation of the PTCHD1 protein are currently unknown. This paucity of data concerning PTCHD1 prevents the effective evaluation of sequence variants identified during diagnostic screening. Here, we characterize PTCHD1 protein binding partners, extending previously reported interactions with postsynaptic scaffolding protein, SAP102. Six rare missense variants of PTCHD1 were also identified from patients with neurodevelopmental disorders. After modelling these variants on a hypothetical three-dimensional structure of PTCHD1, based on the solved structure of NPC1, PTCHD1 variants harboring these mutations were assessed for protein stability, post-translational processing, and protein trafficking. We show here that the wild-type PTCHD1 post-translational modification includes complex N-glycosylation and that specific mutant proteins disrupt normal N-link glycosylation processing. However, regardless of their processing, these mutants still localized to PSD95-containing dendritic processes and remained competent for complexing SAP102.

Examining memory linking and generalization using scFLARE2, a temporally precise neuronal activity tagging system.

How memories are organized in the brain influences whether they are remembered discretely versus linked with other experiences or whether generalized information is applied to entirely novel situations. Here, we used scFLARE2 (single-chain fast light- and activity-regulated expression 2), a temporally precise tagging system, to manipulate mouse lateral amygdala neurons active during one of two 3 min threat experiences occurring close (3 h) or further apart (27 h) in time. Silencing scFLARE2-tagged neurons showed that two threat experiences occurring at distal times are dis-allocated to orthogonal engram ensembles and remembered discretely, whereas the same two threat experiences occurring in close temporal proximity are linked via co-allocation to overlapping engram ensembles. Moreover, we found that co-allocation mediates memory generalization applied to a completely novel stimulus. These results indicate that endogenous temporal evolution of engram ensemble neuronal excitability determines how memories are organized and remembered and that this would not be possible using conventional immediate-early gene-based tagging methods.

Neuronal transcription of autism gene PTCHD1 is regulated by a conserved downstream enhancer sequence.

Patched domain-containing 1 (PTCHD1) is a well-established susceptibility gene for autism spectrum disorder (ASD) and intellectual disability (ID). Previous studies have suggested that alterations in the dosage of PTCHD1 may contribute to the etiology of both ASD and ID. However, there has not yet been a thorough investigation regarding mechanisms that regulate PTCHD1 expression. We sought to characterize the Ptchd1 promoter in a mouse neuronal model, as well as to identify and validate cis regulatory elements. We defined specific regions of the Ptchd1 promoter essential for robust expression in P19-induced neurons. Evolutionarily-conserved putative transcription factor binding sites within these regions were subsequently identified. Using a pairwise comparison of chromatin accessibility between mouse forebrain and liver tissues, a candidate regulatory region, ~ 9.1 kbp downstream of the Ptchd1 stop codon was defined. This region harbours two ENCODE-predicted enhancer cis-regulatory elements. Further, using DNase footprint analysis, a putative YY1-binding motif was also identified. Genomic deletion of the entire 8 kbp downstream open chromatin region attenuated Ptchd1 transcription by over 60% in our neuronal model, corroborating its predicted regulatory function. This study provides mechanistic insights related to the expression of PTCHD1, and provides important context to interpret genetic and genomic variation at this locus which may influence neurodevelopment.

Time separating spatial memories does not influence their integration in humans.

Humans can navigate through similar environments-like grocery stores-by integrating across their memories to extract commonalities or by differentiating between each to find idiosyncratic locations. Here, we investigate one factor that might impact whether two related spatial memories are integrated or differentiated: Namely, the temporal delay between experiences. Rodents have been shown to integrate memories more often when they are formed within 6 hours of each other. To test if this effect influences how humans spontaneously integrate spatial memories, we had 131 participants search for rewards in two similar virtual environments. We separated these learning experiences by either 30 minutes, 3 hours, or 27 hours. Memory integration was assessed three days later. Participants were able to integrate and simultaneously differentiate related memories across experiences. However, neither memory integration nor differentiation was modulated by temporal delay, in contrast to previous work. We further showed that both the levels of initial memory reactivation during the second experience and memory generalization to novel environments were comparable across conditions. Moreover, perseveration toward the initial reward locations during the second experience was related positively to integration and negatively to differentiation-but again, these associations did not vary by delay. Our findings identify important boundary conditions on the translation of rodent memory mechanisms to humans, motivating more research to characterize how even fundamental memory mechanisms are conserved and diverge across species.

A shift in the mechanisms controlling hippocampal engram formation during brain maturation.

The ability to form precise, episodic memories develops with age, with young children only able to form gist-like memories that lack precision. The cellular and molecular events in the developing hippocampus that underlie the emergence of precise, episodic-like memory are unclear. In mice, the absence of a competitive neuronal engram allocation process in the immature hippocampus precluded the formation of sparse engrams and precise memories until the fourth postnatal week, when inhibitory circuits in the hippocampus mature. This age-dependent shift in precision of episodic-like memories involved the functional maturation of parvalbumin-expressing interneurons in subfield CA1 through assembly of extracellular perineuronal nets, which is necessary and sufficient for the onset of competitive neuronal allocation, sparse engram formation, and memory precision.

Emergence of a predictive model in the hippocampus.

The brain organizes experiences into memories that guide future behavior. Hippocampal CA1 population activity is hypothesized to reflect predictive models that contain information about future events, but little is known about how they develop. We trained mice on a series of problems with or without a common statistical structure to observe how memories are formed and updated. Mice that learned structured problems integrated their experiences into a predictive model that contained the solutions to upcoming novel problems. Retrieving the model during learning improved discrimination accuracy and facilitated learning. Using calcium imaging to track CA1 activity during learning, we found that hippocampal ensemble activity became more stable as mice formed a predictive model. The hippocampal ensemble was reactivated during training and incorporated new activity patterns from each training problem. These results show how hippocampal activity supports building predictive models by organizing new information with respect to existing memories.

Examining the engram encoding specificity hypothesis in mice.

According to the encoding specificity hypothesis, memory is best recalled by retrieval cues that overlap with training cues. Human studies generally support this hypothesis. However, memories are thought to be stored in neuronal ensembles (engrams), and retrieval cues are thought to reactivate neurons in an engram to induce memory recall. Here, we visualized engrams in mice to test whether retrieval cues that overlap with training cues produce maximal memory recall via high engram reactivation (engram encoding specificity hypothesis). Using variations of cued threat conditioning (pairing conditioned stimulus [CS] with footshock), we manipulated encoding and retrieval conditions along multiple domains, including pharmacological state, external sensory cue, and internal optogenetic cue. Maximal engram reactivation and memory recall occurred when retrieval conditions closely matched training conditions. These findings provide a biological basis for the encoding specificity hypothesis and highlight the important interaction between stored information (engram) and cues available at memory retrieval (ecphory).

Alcohol Dependence Modifies Brain Networks Activated During Withdrawal and Reaccess: A c-Fos-Based Analysis in Mice.

BACKGROUND: High-level alcohol consumption causes neuroplastic changes in the brain that promote pathological drinking behavior. Some of these changes have been characterized in defined brain circuits and cell types, but unbiased approaches are needed to explore broader patterns of adaptations. METHODS: We used whole-brain c-Fos mapping and network analysis to assess patterns of neuronal activity during alcohol withdrawal and following reaccess in a well-characterized model of alcohol dependence. Mice underwent 4 cycles of chronic intermittent ethanol to increase voluntary alcohol consumption, and a subset underwent forced swim stress to further escalate consumption. Brains were collected either 24 hours (withdrawal) or immediately following a 1-hour period of alcohol reaccess. c-fos counts were obtained for 110 brain regions using iDISCO and ClearMap. Then, we classified mice as high or low drinkers and used graph theory to identify changes in network properties associated with high-drinking behavior. RESULTS: During withdrawal, chronic intermittent ethanol mice displayed widespread increased c-Fos expression relative to air-exposed mice, independent of forced swim stress. Reaccess drinking reversed this increase. Network modularity, a measure of segregation into communities, was increased in high-drinking mice after alcohol reaccess relative to withdrawal. The cortical amygdala showed increased cross-community coactivation during withdrawal in high-drinking mice, and cortical amygdala silencing in chronic intermittent ethanol mice reduced voluntary drinking. CONCLUSIONS: Alcohol withdrawal in dependent mice causes changes in brain network organization that are attenuated by reaccess drinking. Olfactory brain regions, including the cortical amygdala, drive some of these changes and may play an important but underappreciated role in alcohol dependence.

Exercise accelerates place cell representational drift.

Stable neural ensembles are often thought to underlie stable learned behaviors and memory. Recent longitudinal experiments, however, that tracked the activity of the same neurons over days to weeks have shown that neuronal activity patterns can change over extended timescales even if behaviors remain the same - a phenomenon termed representational drift1. We have tested whether neural circuit remodeling, defined as any change in structural connectivity, contributes to representational drift. To do this, we tracked how hippocampal CA1 spatial representations of a familiar environment change with time in conventionally housed mice relative to mice housed with a running wheel. Voluntary exercise is an environmental stimulus that promotes hippocampal circuit remodeling, primarily via promoting adult neurogenesis in the dentate gyrus. Adult neurogenesis alters structural connectivity patterns, as the integration of adult-generated granule cells (abGCs) is a competitive process where new input-output synaptic connections may co-exist and/or even replace existing synaptic connections2. Comparing the spatial activity of downstream hippocampal CA1 place cells in the same familiar environment over two weeks, we found that the activity of place cells in exercise mice exhibited accelerated representational drift compared to control mice, suggesting that hippocampal circuit remodeling may indeed drive representational drift.

Formation and fate of an engram in the lateral amygdala supporting a rewarding memory in mice.

Memories allow past experiences to guide future decision making and behavior. Sparse ensembles of neurons, known as engrams, are thought to store memories in the brain. Most previous research has focused on engrams supporting threatening or fearful memories where results show that neurons involved in a particular engram ("engram neurons") are both necessary and sufficient for memory expression. Far less is understood about engrams supporting appetitive or rewarding memories. As circumstances and environments are dynamic, the fate of a previously acquired engram with changing circumstances is unknown. Here we examined how engrams supporting a rewarding cue-cocaine memory are formed and whether this original engram is important in reinstatement of memory-guided behavior following extinction. Using a variety of techniques, we show that neurons in the lateral amygdala are allocated to an engram based on relative neuronal excitability at training. Furthermore, once allocated, these neurons become both necessary and sufficient for behavior consistent with recall of that rewarding memory. Allocated neurons are also critical for cocaine-primed reinstatement of memory-guided behavior following extinction. Moreover, artificial reactivation of initially allocated neurons supports reinstatement-like behavior following extinction even in the absence of cocaine-priming. Together, these findings suggest that cocaine priming after extinction reactivates the original engram, and that memory-guided reinstatement behavior does not occur in the absence of this reactivation. Although we focused on neurons in one brain region only, our findings that manipulations of lateral amygdala engram neurons alone were sufficient to impact memory-guided behavior indicate that the lateral amygdala is a critical hub region in what may be a larger brain-wide engram.

Genetic variants in DDX53 contribute to Autism Spectrum Disorder associated with the Xp22.11 locus.

Autism Spectrum Disorder (ASD) exhibits an ~4:1 male-to-female sex bias and is characterized by early-onset impairment of social/communication skills, restricted interests, and stereotyped behaviors. Disruption of the Xp22.11 locus has been associated with ASD in males. This locus includes the three-exon PTCHD1 gene, an adjacent multi-isoform long noncoding RNA (lncRNA) named PTCHD1-AS (spanning ~1Mb), and a poorly characterized single-exon RNA helicase named DDX53 that is intronic to PTCHD1-AS. While the relationship between PTCHD1/PTCHD1-AS and ASD is being studied, the role of DDX53 has not been examined, in part because there is no apparent functional murine orthologue. Through clinical testing, here, we identified 6 males and 1 female with ASD from 6 unrelated families carrying rare, predicted-damaging or loss-of-function variants in DDX53. Then, we examined databases, including the Autism Speaks MSSNG and Simons Foundation Autism Research Initiative, as well as population controls. We identified 24 additional individuals with ASD harboring rare, damaging DDX53 variations, including the same variants detected in two families from the original clinical analysis. In this extended cohort of 31 participants with ASD (28 male, 3 female), we identified 25 mostly maternally-inherited variations in DDX53, including 18 missense changes, 2 truncating variants, 2 in-frame variants, 2 deletions in the 3' UTR and 1 copy number deletion. Our findings in humans support a direct link between DDX53 and ASD, which will be important in clinical genetic testing. These same autism-related findings, coupled with the observation that a functional orthologous gene is not found in mouse, may also influence the design and interpretation of murine-modelling of ASD.

Adult neurogenesis acts as a neural regularizer.

New neurons are continuously generated in the subgranular zone of the dentate gyrus throughout adulthood. These new neurons gradually integrate into hippocampal circuits, forming new naive synapses. Viewed from this perspective, these new neurons may represent a significant source of "wiring" noise in hippocampal networks. In machine learning, such noise injection is commonly used as a regularization technique. Regularization techniques help prevent overfitting training data and allow models to generalize learning to new, unseen data. Using a computational modeling approach, here we ask whether a neurogenesis-like process similarly acts as a regularizer, facilitating generalization in a category learning task. In a convolutional neural network (CNN) trained on the CIFAR-10 object recognition dataset, we modeled neurogenesis as a replacement/turnover mechanism, where weights for a randomly chosen small subset of hidden layer neurons were reinitialized to new values as the model learned to categorize 10 different classes of objects. We found that neurogenesis enhanced generalization on unseen test data compared to networks with no neurogenesis. Moreover, neurogenic networks either outperformed or performed similarly to networks with conventional noise injection (i.e., dropout, weight decay, and neural noise). These results suggest that neurogenesis can enhance generalization in hippocampal learning through noise injection, expanding on the roles that neurogenesis may have in cognition.

Toxoplasma infection in male mice alters dopamine-sensitive behaviors and host gene expression patterns associated with neuropsychiatric disease.

During chronic infection, the single celled parasite, Toxoplasma gondii, can migrate to the brain where it has been associated with altered dopamine function and the capacity to modulate host behavior, increasing risk of neurocognitive disorders. Here we explore alterations in dopamine-related behavior in a new mouse model based on stimulant (cocaine)-induced hyperactivity. In combination with cocaine, infection resulted in heightened sensorimotor deficits and impairment in prepulse inhibition response, which are commonly disrupted in neuropsychiatric conditions. To identify molecular pathways in the brain affected by chronic T. gondii infection, we investigated patterns of gene expression. As expected, infection was associated with an enrichment of genes associated with general immune response pathways, that otherwise limits statistical power to identify more informative pathways. To overcome this limitation and focus on pathways of neurological relevance, we developed a novel context enrichment approach that relies on a customized ontology. Applying this approach, we identified genes that exhibited unexpected patterns of expression arising from the combination of cocaine exposure and infection. These include sets of genes which exhibited dampened response to cocaine in infected mice, suggesting a possible mechanism for some observed behaviors and a neuroprotective effect that may be advantageous to parasite persistence. This model offers a powerful new approach to dissect the molecular pathways by which T. gondii infection contributes to neurocognitive disorders.