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.

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.

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.

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.

An inhibitory hippocampal-thalamic pathway modulates remote memory retrieval.

Memories are supported by distributed hippocampal-thalamic-cortical networks, but the brain regions that contribute to network activity may vary with memory age. This process of reorganization is referred to as systems consolidation, and previous studies have examined the relationship between the activation of different hippocampal, thalamic, and cortical brain regions and memory age at the time of recall. While the activation of some brain regions increases with memory age, other regions become less active. In mice, here we show that the active disengagement of one such brain region, the anterodorsal thalamic nucleus, is necessary for recall at remote time-points and, in addition, which projection(s) mediate such inhibition. Specifically, we identified a sparse inhibitory projection from CA3 to the anterodorsal thalamic nucleus that becomes more active during systems consolidation, such that it is necessary for contextual fear memory retrieval at remote, but not recent, time-points post-learning.

Automated Curation of CNMF-E-Extracted ROI Spatial Footprints and Calcium Traces Using Open-Source AutoML Tools.

In vivo 1-photon (1p) calcium imaging is an increasingly prevalent method in behavioral neuroscience. Numerous analysis pipelines have been developed to improve the reliability and scalability of pre-processing and ROI extraction for these large calcium imaging datasets. Despite these advancements in pre-processing methods, manual curation of the extracted spatial footprints and calcium traces of neurons remains important for quality control. Here, we propose an additional semi-automated curation step for sorting spatial footprints and calcium traces from putative neurons extracted using the popular constrained non-negative matrixfactorization for microendoscopic data (CNMF-E) algorithm. We used the automated machine learning (AutoML) tools TPOT and AutoSklearn to generate classifiers to curate the extracted ROIs trained on a subset of human-labeled data. AutoSklearn produced the best performing classifier, achieving an F1 score >92% on the ground truth test dataset. This automated approach is a useful strategy for filtering ROIs with relatively few labeled data points and can be easily added to pre-existing pipelines currently using CNMF-E for ROI extraction.

The ontogeny of memory persistence and specificity.

Interest in the ontogeny of memory blossomed in the twentieth century following the initial observations that memories from infancy and early childhood are rapidly forgotten. The intense exploration of infantile amnesia in subsequent years has led to a thorough characterization of its psychological determinants, although the neurobiology of memory persistence has long remained elusive. By contrast, other phenomena in the ontogeny of memory like infantile generalization have received relatively less attention. Despite strong evidence for reduced memory specificity during ontogeny, infantile generalization is poorly understood from psychological and neurobiological perspectives. In this review, we examine the ontogeny of memory persistence and specificity in humans and nonhuman animals at the levels of behavior and the brain. To this end, we first describe the behavioral phenotypes associated with each phenomenon. Looking into the brain, we then discuss neurobiological mechanisms in the hippocampus that contribute to the ontogeny of memory. Hippocampal neurogenesis and critical period mechanisms have recently been discovered to underlie amnesia during early development, and at the same time, we speculate that similar processes may contribute to the early bias towards memory generalization.

A Compact Head-Mounted Endoscope for In Vivo Calcium Imaging in Freely Behaving Mice.

Miniaturized fluorescence microscopes for imaging calcium transients are a promising tool for investigating the relationship between behavior and population-level neuronal activity in rodents. However, commercially available miniature microscopes may be costly and, because they are closed source, may not be easily modified based on particular experimental requirements. Here, we describe how to build and use a low-cost compact head-mounted endoscope (CHEndoscope) system for in vivo calcium imaging. The CHEndoscope uses an implanted gradient index lens along with the genetically encoded calcium indicator GCaMP6 to image calcium transients from hundreds of neurons simultaneously in awake behaving mice. This system is affordable, open source, and flexible, permitting modification depending on the particular experiment. This article describes in detail the assembly, surgical implantation, data collection, and processing of calcium signals using the CHEndoscope system. The aim of this open framework is to provide an accessible set of miniaturized calcium imaging tools for the neuroscience research community. © 2018 by John Wiley & Sons, Inc.

Elevation of Hippocampal Neurogenesis Induces a Temporally Graded Pattern of Forgetting of Contextual Fear Memories.

Throughout life neurons are continuously generated in the subgranular zone of the hippocampus. The subsequent integration of newly generated neurons alters patterns of dentate gyrus input and output connectivity, potentially rendering memories already stored in those circuits harder to access. Consistent with this prediction, we previously showed that increasing hippocampal neurogenesis after training induces forgetting of hippocampus-dependent memories, including contextual fear memory. However, the brain regions supporting contextual fear memories change with time, and this time-dependent memory reorganization might regulate the sensitivity of contextual fear memories to fluctuations in hippocampal neurogenesis. By virally expressing the inhibitory designer receptor exclusively activated by designer drugs, hM4Di, we first confirmed that chemogenetic inhibition of dorsal hippocampal neurons impairs retrieval of recent (day-old) but not remote (month-old) contextual fear memories in male mice. We then contrasted the effects of increasing hippocampal neurogenesis at recent versus remote time points after contextual fear conditioning in male and female mice. Increasing hippocampal neurogenesis immediately following training reduced conditioned freezing when mice were replaced in the context 1 month later. In contrast, when hippocampal neurogenesis was increased time points remote to training, conditioned freezing levels were unaltered when mice were subsequently tested. These temporally graded forgetting effects were observed using both environmental and genetic interventions to increase hippocampal neurogenesis. Our experiments identify memory age as a boundary condition for neurogenesis-mediated forgetting and suggest that, as contextual fear memories mature, they become less sensitive to changes in hippocampal neurogenesis levels because they no longer depend on the hippocampus for their expression.SIGNIFICANCE STATEMENT New neurons are generated in the hippocampus throughout life. As they integrate into the hippocampus, they remodel neural circuitry, potentially making information stored in those circuits harder to access. Consistent with this, increasing hippocampal neurogenesis after learning induces forgetting of the learnt information. The current study in mice asks whether these forgetting effects depend on the age of the memory. We found that post-training increases in hippocampal neurogenesis only impacted recently acquired, and not remotely acquired, hippocampal memories. These experiments identify memory age as a boundary condition for neurogenesis-mediated forgetting, and suggest remote memories are less sensitive to changes in hippocampal neurogenesis levels because they no longer depend critically on the hippocampus for their expression.

Determinants of object-in-context and object-place-context recognition in the developing rat.

Since the seminal report on novel object recognition in the rat (Ennaceur & Delacour, 1988), novelty recognition paradigms have become increasingly prevalent in learning and memory research. Novelty recognition tasks do not require extensive training or complex behaviors, and thus are especially suitable for studying the ontogeny of various forms of memory (e.g., object, spatial, and contextual memory). However, relatively little is known about the determinants of recognition memory during development. The present study extends our recent research on the development of recognition memory by further characterizing the ontogeny of contextual recognition (Ramsaran, Westbrook, & Stanton, 2016). We report that long-term retention of object-in-context (OiC) memory emerges during early development in the rat (Experiment 1), and that performance of object-place-context (OPC), a spatial variant of the OiC task, also displays protracted development until early adolescence (Experiment 2). In addition, we examined the role of NMDA receptors (NMDARs) in contextual recognition and found that OiC memory is not dependent on NMDAR-mediated plasticity whereas performance of spatial task variants including the distal cue OiC (Ramsaran et al., 2016) and OPC tasks are NMDAR-dependent (Experiments 3 and 4). The ontogeny of contextual recognition is influenced by memory retention and spatial processing demands, which may also determine the neurobiological mechanisms supporting task performance.

The Young and the Promiscuous.

It is now well accepted that new neurons continue to be generated in the adult hippocampus. By visualizing and manipulating new neurons in behaving mice, Danielson et al. (2016) begin to define how new neurons contribute to hippocampal function.

Ontogeny of object-in-context recognition in the rat.

The object-in-context recognition (OiC) task [19] is a spontaneous exploration task that serves as an index of incidental contextual learning and memory. During the test phase, rats prefer to explore the object mismatched to the testing context based on previous object-context pairings experienced during training. The mechanisms of OiC memory have been explored in adult rats [12,35]; however, little is known about its determinants during development. Thus, the present study examined the ontogeny of the OiC task in preweanling through adolescent rats. We demonstrate that postnatal day (PD) 17, 21, 26, and 31 rats can perform the OiC task (Experiment 1) and that preference for the novel target is eliminated when rats are tested in an alternate context not encountered during training (Experiment 2). Lastly, we show that PD26 but not PD17 rats can perform the OiC task when the training contexts only differed by distal spatial cues (Experiment 3). These data demonstrate for the first time that PD17 rats can acquire and retain short-term OiC memory, which involves associative learning of object and context information. However, we also provide evidence that preweanling rats' ability to utilize certain aspects of a context (i.e., distal spatial cues) in the OiC task is not equivalent to that of their older counterparts. Implications for the development of contextual memory and its related neural substrates are discussed.