Event-based memories may be encoded with high (e.g., episodic) or low (e.g., gist) mnemonic resolution. While the CA1 region of the hippocampus encodes events at both scales, it is unclear how such dual coding emerges. Consistent with theoretical predictions1,2, here we show that trisynaptic (from CA3) and monosynaptic (from the medial entorhinal cortex) projections to CA1 encode high- and low-resolution event-based memories, respectively. Recruitment of the trisynaptic pathway during contextual fear conditioning promotes feedforward inhibition of CA1 via activation of parvalbumin-positive (PV+) interneurons. This allows sparse encoding of event-based memories in high-dimensional neural states that support high-resolution contextual fear memories, where mice exhibit conditioned fear only in the training context and not similar contexts. Inhibiting this input during training reduces activation of CA1 PV+ interneurons and feedforward inhibition, shifting neural dynamics to low-dimensional states that support low-resolution contextual fear memories where mice freeze in the training context and similar contexts. These experiments identify a circuit-based mechanism that causally links the dimensionality of CA1 neural dynamics to mnemonic resolution.

Activity tagging of neuronal ensembles has become an important tool in neuroscience. Here, we present a protocol for visualizing separate neuronal ensembles active during three distinct phases of a memory in transgenic mice. We describe steps to label active neurons using viral microinjection, inducing GFP expression under the robust activity marker (RAM) promoter, and transgenic mice, inducing tdTomato (TdT) expression, and immunohistochemical (IHC) visualization of endogenous cFos expression. We then detail procedures for preparation of tissue, imaging, and quantification of memory events. For complete details on the use and execution of this protocol, please refer to Lesuis et al.1.

Memories for events (i.e., episodic memories) change qualitatively with time. Systems consolidation theories posit that organizational changes accompany qualitative shifts in memory resolution, but they differ as to the locus of this reorganization. Whereas some theories favor inter-regional changes in organization (e.g., hippocampus → cortex; multiple trace theory), others favor intra-regional reorganization (e.g., within-hippocampus; trace transformation theory). Using an engram-tagging and manipulation approach in mice, we here provide evidence that intra-regional changes in organization underlie shifts in memory resolution. We establish that contextual fear memories lose resolution as a function of time, with mice exhibiting conditioned freezing in both the training apparatus (context A) and a second apparatus (context B) at more remote delays (i.e., freezingA ≡ freezingB at remote delay). By tagging either hippocampal (dentate gyrus) or cortical (prelimbic cortex) neuronal ensembles in context A, and then pairing their optogenetic activation with shock (in context C), we tracked time-dependent changes in the resolution of either "context-only" or "context-shock" engrams by testing mice in contexts A and B. Hippocampal context-only or context-shock engrams were initially high resolution (i.e., recent delay: freezingA >> freezingB) but lost resolution with time (i.e., remote delay: freezingA ≡ freezingB). In contrast, cortical context-only or context-shock engrams were initially low resolution and remained low resolution over time (i.e., recent and remote delay: freezingA ≡ freezingB). Transformation of hippocampal engrams was dependent on adult hippocampal neurogenesis. Eliminating hippocampal neurogenesis arrested hippocampal engrams in a recent-like, high-resolution state in which mice continued to exhibit discriminative freezing at remote delays.

Episodic memories-high-fidelity memories for events that depend initially on the hippocampus-do not maintain their precision in perpetuity. One benefit of this time-dependent loss of precision is the emergence of event-linked gist memories that may be used to guide future behaviour in new but related situations (that is, generalization)1-3. Models of systems consolidation propose that memory reorganization accompanies this loss of memory precision1,4; however, the locus of this reorganization is unclear. Here we report that time-dependent reorganization of hippocampal engram circuitry is sufficient to explain shifts in memory precision associated with systems consolidation. Using engram labelling tools in mice, we demonstrate that the passage of time rewires hippocampal engram circuits, enabling hippocampal engram neurons to be promiscuously active and guide behaviour in related situations that do not match the original training conditions. Reorganization depends on hippocampal neurogenesis; eliminating hippocampal neurogenesis prevents reorganization and maintains precise, event memories. Conversely, promoting hippocampal neurogenesis accelerates memory reorganization and the emergence of event-linked gist memories in the hippocampus. Our results indicate that systems consolidation models require updating to account for within-hippocampus reorganization that leads to qualitative shifts in memory precision.

Stress induces aversive memory overgeneralization, a hallmark of many psychiatric disorders. Memories are encoded by a sparse ensemble of neurons active during an event (an engram ensemble). We examined the molecular and circuit processes mediating stress-induced threat memory overgeneralization in mice. Stress, acting via corticosterone, increased the density of engram ensembles supporting a threat memory in lateral amygdala, and this engram ensemble was reactivated by both specific and non-specific retrieval cues (generalized threat memory). Furthermore, we identified a critical role for endocannabinoids, acting retrogradely on parvalbumin-positive (PV+) lateral amygdala interneurons in the formation of a less-sparse engram and memory generalization induced by stress. Glucocorticoid receptor antagonists, endocannabinoid synthesis inhibitors, increasing PV+ neuronal activity, and knocking down cannabinoid receptors in lateral amygdala PV+ neurons restored threat memory specificity and a sparse engram in stressed mice. These findings offer insights into stress-induced memory alterations, providing potential therapeutic avenues for stress-related disorders.

Joseph LeDoux is a pioneering neuroscientist who has made profound discoveries that continue to impact our understanding of the neural basis of emotion and memory, particularly the role of the amygdala in threat conditioning. LeDoux's trailblazing and elegant studies were some of the first to examine the circuit basis of behavior. His work combined techniques to trace pathways into and out of the amygdala important for threat conditioning and related behaviors. Since that time, these types of circuit tracing studies have exploded in popularity across neuroscience, and I would argue, we all owe a debt to LeDoux for this. LeDoux has made numerous additional contributions to neuroscience and, by bringing emotion back to neuroscience, has helped unite neuroscience with psychology. A gifted writer with a knack for communicating complicated scientific ideas in an accessible manner, LeDoux has become an ambassador of science who uses his love of music to help educate and inspire. Perhaps more important than these laudable scientific achievements, LeDoux is also a true "gentleman" of science, showing that science need not be a contact sport. Here, I give a personal account on why Joseph LeDoux is one of my scientific role models.

The hippocampus is essential for episodic memory, yet its coding mechanism remains debated. In humans, two main theories have been proposed: one suggests that concept neurons represent specific elements of an episode, while another posits a conjunctive code, where index neurons code the entire episode. Here, we integrate new findings of index neurons in humans and other animals with the concept-specific memory framework, proposing that concept neurons evolve from index neurons through overlapping memories. This process is supported by engram literature, which posits that neurons are allocated to a memory trace based on excitability and that reactivation induces excitability. By integrating these insights, we connect two historically disparate fields of neuroscience: engram research and human single neuron episodic memory research.

Episodic-like memory is a later-developing cognitive function supported by the hippocampus. In mice, the formation of extracellular perineuronal nets in subfield cornu ammonis (CA) 1 of the dorsal hippocampus controls the emergence of episodic-like memory during the fourth post-natal week. Whether the timing of episodic-like memory onset is hard-wired, or flexibly set by early-life experiences during a critical or sensitive period for hippocampal maturation, is unknown. Here, we show that the trajectories for episodic-like memory development vary for mice given different sets of experiences spanning the second and third post-natal weeks. Specifically, episodic-like memory precision developed later in mice that experienced early-life adversity, while it developed earlier in mice that experienced early-life enrichment. Moreover, we demonstrate that early-life experiences set the timing of episodic-like memory development by modulating the pace of perineuronal net formation in dorsal CA1, which is dependent on the brain-derived neurotrophic factor (BDNF)-tropomysin receptor kinase B (TrkB) signaling pathway. These results indicate that the hippocampus undergoes a sensitive period during which early-life experiences determine the timing for episodic-like memory development.

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.

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’.

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.

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-beta 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. Coordinated neuronal activity may mediate memory in hippocampal CA1. Here, the authors use an array of machine-learning classifiers to reveal how higher-order population dynamics and learning-induced spine plasticity are disrupted in amnestic mice.

The mammalian inner ear houses the vestibular and cochlear sensory organs dedicated to sensing balance and sound, respectively. These distinct sensory organs arise from a common prosensory region, but the mechanisms underlying their divergence remain elusive. Here, we showed that two evolutionarily conserved homeobox genes, Irx3 and Irx5, are required for the patterning and segregation of the saccular and cochlear sensory domains, as well as for the formation of auditory sensory cells. Irx3/5 were highly expressed in the cochlea, their deletion resulted in a significantly shortened cochlea with a loss of the ductus reuniens that bridged the vestibule and cochlea. Remarkably, ectopic vestibular hair cells replaced the cochlear non-sensory structure, the Greater Epithelial Ridge. Moreover, most auditory sensory cells in the cochlea were transformed into hair cells of vestibular identity, with only a residual organ of Corti remaining in the mid-apical region of Irx3/5 double knockout mice. Conditional temporal knockouts further revealed that Irx3/5 are essential for controlling cochlear sensory domain formation before embryonic day 14. Our findings demonstrate that Irx3/5 regulate the patterning of vestibular and cochlear sensory cells, providing insights into the separation of vestibular and cochlear sensory organs during mammalian inner ear development.

Memories of events are linked to the contexts in which they were encoded. This contextual linking ensures enhanced access to those memories that are most relevant to the context at hand, including specific associations that were previously learned in that context. This principle, referred to as encoding specificity, predicts that context-specific neural states should bias retrieval of particular associations over others, potentially allowing for the disambiguation of retrieval cues that may have multiple associations or meanings. Using a context-odor paired associate learning paradigm in mice, here, we show that chemogenetic manipulation of dentate gyrus ensembles corresponding to specific contexts reinstates context-specific neural states in downstream CA1 and biases retrieval toward context-specific associations.