Learning & Memory

Recent studies have shown that a shared neuronal ensemble in the hippocampus links distinct contextual memories encoded within a certain time window (specifically, 5 hours, 2 and 7 days). Here we explored the temporal dynamics of two contextual (neutral and aversive) memories linking and analysed neuronal ensembles in the hippocampus, amygdala and different cortical regions reactivated during retrieval. Firstly, we have found that memories integrated across different time-points including several hours, days and weeks but not if learning phases was separated by short-term and very long-term time intervals. Secondly, we have demonstrated a higher neuronal co-reactivation in the hippocampus and amygdala during retrieval in case of memories integration that supports the hypothesis that shared neuronal ensembles link distinct memories. Finally, we have elicited that proportions of reactivated neuronal ensembles in these brain regions are greater in case of contextual memories integration.

Aversive associative learning paradigms such as inhibitory avoidance (IA) are frequently used to examine episodic-like memories in rodents. In IA, rodents learn to associate a context with a footshock, followed by testing for memory strength in the original training context and for memory precision in a similar yet distinct neutral context. The present work assessed the effects of different contextual exposure procedures on memory strength and precision in IA at both recent and remote time points using male and female Long-Evans rats. An initial experiment found that rats kept in the lit (non-shock) compartment of the IA apparatus for 60 s during training, as opposed to 10 s, displayed enhanced memory strength, with discrimination between both chambers at the recent retention test and generalization at the remote retention test. Subsequent experiments investigated the effects of contextual pre-exposure the day before training. The results indicate that pre-exposure to the neutral context promoted generalization without altering memory strength compared to the first experiment. In contrast, pre-exposure to the aversive chamber promoted discrimination and enhanced memory strength. Notably, the different procedures yielded similar effects in both sexes. However, the results also indicate an overall pattern of greater contextual discrimination in females compared to males. These findings provide evidence for how different contextual exposures influence the degree of encoding at the time of training and a behavioral foundation for future studies examining the neurobiological mechanisms underlying memory strength and precision in IA, while highlighting the importance of using both sexes in initial behavioral work. Significance StatementStrength and precision are two fundamental properties of memory that can be simultaneously measured using inhibitory avoidance (IA), a type of context-footshock association task. However, little is known about how different context exposures alter rats encoding of these memories, thereby influencing subsequent memory strength and precision. Here, we found that pre-exposure to the neutral IA chamber decreased memory precision, whereas pre-exposure to the aversive IA chamber promoted memory strength and precision. Additionally, females demonstrated overall enhanced memory precision compared to males. These results indicate that different types of contextual exposures influence initial IA encoding and add to a limited body of research examining memory strength and precision in IA in both sexes.

Forming a long-term memory requires changes in neuronal transcription. What happens, though, as the memory is forgotten? And how does the transcriptional state relate to the maintenance and recall of the long-term memory? To answer these questions we have been systematically tracing the time-course of transcriptional changes evoked by long-term sensitization in the marine mollusk Aplysia californica. Our approach captures transcriptional changes in neurons of known behavioral relevance using a within-subjects design, delineating patterns of transcriptional change that are comprehensive and reproducible. We have previously reported that within 1 day of long-term sensitization training there is a widespread transcriptional response involving robust changes in over 5% of tested transcripts (1,252 of [~]22k; Conte, 2017). Within 1 week, however, memory strength fades and nearly all transcriptional changes relapse to baseline (Perez, 2018). Here we report microarray analysis (N = 16) of transcriptional changes 5 days post-learning, a time-point when memory strength has weakened but is still robust. Remarkably, we find that at this intermediate behavioral stage nearly all transcriptional changes have fully decayed, even in subsets of animals that have shown very little forgetting. Thus, most transcriptional changes seem to decay more rapidly than memory expression. We discuss several possible ways that memory expression could become decoupled from detectable transcriptional regulation. HighlightsO_LILong-term sensitization training produces a memory that then fades over the course of a week, with behavioral expression at 5 days at an intermediate stage of partly forgotten with continued clear sensitization and considerable variety across animals. C_LIO_LIThe transcriptional response to sensitization training fades more quickly than behavioral expression, with nearly all transcripts regulated 1 day after training showing a statistically significant decline in regulation, even amongst animals that had shown little forgetting. C_LIO_LITranscription does not seem to have a straightforward relationship with the expression of sensitization memory, with a small set of transcripts consistently regulated even as behavioral expression changes and strong behavioral expression possible without most of the transcriptional changes observed during early maintenance. C_LI

The hippocampus plays a critical role in storing and retrieving spatial information. By targeting the dorsal hippocampus and manipulating specific "candidate" molecules using pharmacological and genetic manipulations, we have previously discovered that long-term active place avoidance memory requires transient activation of particular molecules in dorsal hippocampus. These molecules include amongst others, the persistent kinases Ca-calmodulin kinase II (CaMKII) and the atypical protein kinase C isoform PKC{iota} /{lambda} for acquisition of the conditioned behavior, whereas persistent activation of the other atypical PKC, protein kinase M zeta (PKM{zeta}) is necessary for maintaining the memory for at least a month. It nonetheless remains unclear what other molecules and their interactions maintain active place avoidance long-term memory, and the candidate molecule approach is both impractical and inadequate to identify new candidates since there are so many to survey. Here we use a complementary approach to identify candidates by transcriptional profiling of hippocampus subregions after formation of the long-term active place avoidance memory. Interestingly, 24-h after conditioning and soon after expressing memory retention, immediate early genes were upregulated in the dentate gyrus but not Ammons horn of the memory expressing group. In addition to determining what genes are differentially regulated during memory maintenance, we performed an integrative, unbiased survey of the genes with expression levels that covary with behavioral measures of active place avoidance memory persistence. Gene Ontology analysis of the most differentially expressed genes shows that active place avoidance memory is associated with activation of transcription and synaptic differentiation in dentate gyrus but not CA3 or CA1, whereas hypothesis-driven candidate molecule analyses identified insignificant changes in the expression of many LTP-associated molecules in the various hippocampal subfields, nor did they covary with active place avoidance memory expression, ruling out strong transcriptional regulation but not translational regulation, which was not investigated. These findings and the data set establish an unbiased resource to screen for molecules and evaluate hypotheses for the molecular components of a hippocampus-dependent, long-term active place avoidance memory.

Memories are often categorized into types, reflecting their behavioral, anatomical and molecular diversity: these classifications both aid understanding of the differences among varieties of memory and help delineate the unifying cross-species principles underlying them. In the nematode worm Caenorhabditis elegans, we find that an associative memory of the pairing of the normally attractive odorant benzaldehyde and starvation depends on de novo translation, is independent of CREB, and is produced by massed training: a pattern which does not correspond to any of the well-characterized molecular categories of invertebrate memory. Further, as has been shown for many memories in vertebrates, but not previously in nematodes, we find that formation of this memory continues after removal of the stimuli initially causing it, and that it is labile to disruption through protein synthesis inhibition following training, but that inhibition of proteasomal activity does not extend the duration of the memory. Previous findings have implicated insulin pathway signaling, which canonically regulates the transcription factor DAF- 16, as a key component of this benzaldehyde/starvation memory, however our results suggest that transcriptional inhibition has, at most, only moderate effects on memory formation. We find that insulin signaling instead acts to regulate phospholipase C, which in turn regulates memory through diacylglycerol signaling. These findings better characterize this model associative memory in relation to other invertebrate memory types and identify ways in which it both shares their traits and differs from them, as well as revealing a more complete picture of the molecular pathway underlying it.

Although many studies have addressed the role of the amygdala in modulating long-term memory, it is not known whether weak training plus amygdala stimulation can transform a short-term memory into a remote memory. Object place recognition (OPR) memory after strong training remains hippocampus-dependent through the persistent action of PKM{zeta} for at least 6 days, but it is unknown whether weak training plus amygdala stimulation can transform short-term memory into an even longer memory, and whether such memory is stored through more persistent action of PKM{zeta} in hippocampus. We trained rats to acquire OPR and 15 min or 5 h later induced a brief pattern of electrical stimulation in basolateral amygdala (BLA). Our results reveal that a short-term memory lasting < 4 h can be converted into remote memory lasting at least 3 weeks if the BLA is activated 15 min, but not 5 h after learning. To examine how this remote memory is maintained, we injected ZIP, an inhibitor of atypical PKCs (aPKCs), PKM{zeta} and PKC{iota}/{lambda}, into either hippocampal CA1, dentate gyrus (DG), or anterior cingulate cortex (ACC). Our data reveal amygdala stimulation produces consolidation into remote memory, not by persistent aPKC activation and capture by synaptic tagging processes in the hippocampal formation, but in ACC. Our data establish a powerful modulating role of the BLA in forming remote memory and open a path in the search for neurological restoration of memory, based on enhancing synaptic plasticity in aging or neurodegenerative disorders such as Alzheimers disease.

The acquisition of temporally proximate information can impair the brains ability to consolidate earlier experiences, resulting in retroactive interference (RI). Recognition-based behavioral paradigms are well-suited for investigating RI in rodents, particularly those involving sequential learning episodes. The medial prefrontal cortex (mPFC) integrates multimodal information relevant to the regulation of memory interference and is strongly modulated by the serotonergic system. Serotonin 2A receptors (5-HT2AR), which are densely expressed in the mPFC, have been shown to influence the retrieval of competing object-recognition memories. However, their role in other phases of memory processing, particularly in modulating RI, remains unclear. Using a modified version of the object recognition task designed to induce RI, combined with pharmacological manipulation of 5-HT2AR, we demonstrate that RI specifically impairs the object-related component of memory. Moreover, serotonin signaling through 5-HT2AR is necessary to prevent RI. Strikingly, activating 5-HT2AR before retrieval can rescue the expression of memories affected by RI, suggesting that RI may not erase memory traces but rather hinder their access.

We acquire perceptual skills through experience to adapt ourself to the changing environment. Accomplishing an effective skill acquisition is a main purpose of perceptual learning research. Given the often observed learning effect specificity, multiple perceptual learnings with shared parameters could serve to improve the generalization of the learning effect. However, the interference between the overlapping memory traces of different learnings may impede this effort. Here, we trained human participants on an orientation discrimination task. We observed a proactive interference effect that the first training blocked the second training at its untrained location. This was a more pronounced effect than the well-known location specificity in perceptual learning. We introduced a short reactivation of the first training before the second training and successfully eliminated the proactive interference when the second training was inside the reconsolidation time window of the reactivated first training. Interestingly, we found that practicing an irrelevant task at the location of the second training immediately after the reactivation of the first training could also restore the effect of the second training but in a smaller magnitude, even if the second training was conducted outside of the reconsolidation window. We proposed a two-level mechanism of reactivation-induced memory integration to account for these results. The reactivation-based procedure could integrate either the previously trained and untrained locations or the two trainings at these locations, depending on the activated representations during the reconsolidation process. The findings provide us with new insight into the roles of long-term memory mechanisms in perceptual learning.

Contextual fear conditioning is a classical laboratory task that tests associative memory formation and recall. Techniques such as multi-photon microscopy and holographic stimulation offer tremendous opportunities to understand the neural underpinnings of these memories. However, these techniques generally require animals to be head-fixed. There are few paradigms that test contextual fear conditioning in head-fixed mice, and none where the behavioral outcome following fear conditioning is freezing, the most common measure of fear in freely moving animals. To address this gap, we developed a contextual fear conditioning paradigm in head-fixed mice using virtual reality (VR) environments. We designed an apparatus to deliver tail shocks (unconditioned stimulus, US) while mice navigated a VR environment (conditioned stimulus, CS). The acquisition of contextual fear was tested when the mice were reintroduced to the shock-paired VR environment the following day. We tested three different versions of this paradigm and, in all of them, observed an increased conditioned fear response characterized by increased freezing behavior. This was especially prominent during the first trial in the shock-paired VR environment, compared to a neutral environment where the mice received no shocks. Our results demonstrate that head-fixed mice can be fear conditioned in VR, discriminate between a feared and neutral VR context, and display freezing as a conditioned response, similar to freely behaving animals. Furthermore, using a two-photon microscope, we imaged from large populations of hippocampal CA1 neurons before, during, and following contextual fear conditioning. Our findings reconfirmed those from the literature on freely moving animals, showing that CA1 place cells undergo remapping and show narrower place fields following fear conditioning. Our approach offers new opportunities to study the neural mechanisms underlying the formation, recall, and extinction of contextual fear memories. As the head-fixed preparation is compatible with multi-photon microscopy and holographic stimulation, it enables long-term tracking and manipulation of cells throughout distinct memory stages and provides subcellular resolution for investigating axonal, dendritic, and synaptic dynamics in real-time.

The anticipation of extrinsic reward facilitates memory formation. However, it is unclear how reward following memory retrieval influences the information that is retrieved and later remembered. Here, we conducted four behavioral experiments (N=42 male/female young adults per experiment) in which we manipulated retrieval practice reward delivery. Across all experiments, participants studied word-image pairs and then completed two rounds of retrieval practice, followed by a final recognition test. Participants made vividness judgments during retrieval practice and in three of four experiments each response had a 50% chance of yielding positive feedback. We find that repeated rewards following retrieval practice facilitate later memory whereas low vivid retrieval practice impairs later memory. Together, these results suggest that the benefit of both retrieval practice and reward may be dependent on the strength of the memory that is retrieved.

New information is rarely learned in isolation, instead most of what we experience can be incorporated into or uses previous knowledge networks in some form. However, most rodent laboratory tasks assume the animal to be naive with no previous experience influencing the results. Previous knowledge in form of a schema can facilitate knowledge acquisition and accelerate systems consolidation: memories become more rapidly hippocampal independent and instead rely more on the prefrontal cortex. Here, we developed a new spatial navigation task where food locations are learned in a large, gangway maze - the HexMaze. Analysing performance across sessions as well as on specific trials, we can show simple memory effects as well as multiple effects of previous knowledge accelerating both online learning and performance increases over offline periods. Importantly, we are the first to show that schema build-up is dependent on how much time passes, not how often the animal is trained.

Stress and emotional arousal interfere with encoding of temporal context memory for episodic events. However, it remains unclear how stress affects more fine-grained temporal memory, such as episodic events sequences and event times. Here, 86 healthy participants (M age = 22.5; 46% women, 54% men) were subjected to either a stress condition (socially evaluated cold pressor test) or a control condition, directly after or at a delay of 30 minutes they were presented the temporal structure of four virtual days. In these virtual days, time was scaled and participants could use clock cues to construe the passage of time within a day. We examined whether acute stress would interfere with encoding of episodic event sequences and temporal memory. Our results show that when learning took place directly after a stressor, virtual time estimates were more strongly biased towards a generalized timeline but temporal memory overall was not differentially affected between the stress and control groups. Exploratory analyses suggest that memory accuracy improved in men and deteriorated in women as a function of subjective stress levels following acute stress. In conclusion, acute stress amplified memory generalization but we found no stress related differences in memory accuracy across levels of temporal granularity.

Head-fixed linear treadmill tasks have been used to study hippocampal physiology in mice. Although some hippocampal neurons establish place fields along linear treadmills, it is not clear if the hippocampus is required for spatial memory on this task. Using a Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) approach, we found that silencing hippocampal output on rewarded treadmill tasks impaired search for rewards signaled by spatial cues but did not impair search for rewards signaled by local cues, recapitulating findings from other behavior tasks. These findings serve to contextualize data on hippocampal physiology from mice performing this task.

In extinction learning, contextual renewal occurs when an extinguished behavior reemerges after a context change. A key question is how stimuli become integrated as contextual cues. While contingency, the predictive relationship between stimuli and outcomes; is known to be important, its precise contribution remains unclear. Using a ABA renewal design with pigeons in operant chambers, we systematically violated contingency by probabilistically reinforcing responses during extinction. Our results show that partial violations of contingency modulated extinction learning but did not abolish contextual renewal. Instead, pigeons developed meta-learning strategies, adapting their behavior across sessions to optimize reward despite extinction conditions. These findings highlight that context formation is sensitive to contingency levels, but also that animals can flexibly reorganize their learning strategies when contingency is unstable.

While forgetting has been studied extensively in various organisms, its precise nature has often been unclear. Here, we used behavioral experiments in Drosophila to determine that a significant aspect of forgetting consists of a decrease in the ability of a memory to induce an appropriate behavior. We tested flies for memory retention at various times after training and then separately retested both flies that chose correctly and those that chose incorrectly. Although the ability to choose correctly decreased over time, we could not measure any differences in memory between flies that initially chose correctly and those that chose incorrectly upon retest. This suggests that forgetting is unlikely to consist of a spontaneous loss of a memory but instead consists of a decrease in the probability of flies that remember choosing the correct behavioral response. Thus, although flies maintain memory over time, there is an increase in uncertainty associated with this memory. We find that forgetting of long-term memories and accelerated forgetting in old flies occur in a similar manner.

Mammalian brain has evolved to infer from past experiences and elicit context relevant novel behavioural responses hitherto unexpressed by the animal. However, little is known about how prior knowledge influences the emergence of such responses. Remarkably, the brain not only arrives at these responses through logical inferences based on previous leanings, but also acquire new related information, without causing catastrophic interference. Mental schemas have often been proposed as the framework for this phenomenon. In this study, using mice as a model animal, we show that schematic networks not only enhance the cognitive load handling capacity (CLHC) and prevent catastrophic interference, but also facilitate the generation of novel, contextually relevant responses. Interestingly, when the animals were trained in a paradigm that did not invoke the pre-formed mental schema, we observed neither an enhancement to CLHC nor a generation of novel context relevant responses. Based on the principles of mental schemas discovered in our animal experiments, we developed a biologically plausible artificial neural network (ANN) that avoids catastrophic interference and captures the learning properties observed in our experiments. The custom architecture of this ANN enables it to generate responses similar to those of animals in novel scenarios. Significance StatementLittle is known about the role of mental schemas in preventing memory interference--a process in which overlapping or similar memories hinder the acquisition and retention of related information. In this study, we demonstrate that mental schemas enhance cognitive load handling capacity and improve the ability to solve novel but related problems. Using mice as a model, we show that the mere existence of a mental schema is not enough for improved cognitive load handling; instead, the relationship between existing and new information must be explicitly established during the learning process. Based on these findings, we developed a minimalistic artificial neural network (ANN) that effectively mimics this behaviour. These insights pave the way for developing more efficient learning and teaching strategies.

Experience can change how individuals learn. Learning to solve a new problem can be accelerated by generalizing known rules in the new context, but the impact of experience on solving problems where generalization cannot be applied remains unclear. To study the impact of experience on solving new problems that are distinct from previously learned tasks, we examined how rats learned a new spatial navigation task after having previously learned different sets of spatial navigation tasks. The new task differed from the previous tasks in spatial layout and navigation rule, and could not be solved by applying previously learned rules. We found that different experience histories did not impact task performance in the new task. However, by examining navigation choices made by rats, we found exploration patterns during the early stage of learning in the new task was dependent on experience history. We identified these behavioral differences by analyzing each rats navigation choices and by modeling their choice sequences with a modified distance dependent Chinese restaurant process. We further pinpointed the behavioral difference to sequential turn/no turn decisions made at choice points. Our results indicate that experience can influence problem-solving strategies when learning to solve new problems. Individuals with distinct experience histories can approach new problems from different starting points but converge on the same solution.

Associative recognition memory allows us to form representations of items and their environment and to judge the novelty of such representations. This memory is dependent on a brain circuit that includes interactions between medial prefrontal cortex (mPFC) and lateral entorhinal cortex (LEC); however it is unknown whether the interaction of these brain areas is required for memory encoding, retrieval or both processes. Furthermore, little is known as to whether indirect or direct mPFC-LEC connections are critical for associative recognition memory and, if the latter, in which direction information travels. To address these questions, we first performed pharmacological disconnection of mPFC and LEC, finding that mPFC-LEC interaction is required for both memory encoding and retrieval. Next, we optogenetically inhibited projections from mPFC to LEC, showing that this projection was crucial for both encoding and retrieval of both object-in-place and object-in-context recognition memory when a 1 h, but not a 5 min, memory retention delay was used. These data show that a direct connection from mPFC to LEC is critical for associative recognition memory, in a delay-dependent manner.

Many newly encoded memories are labile when acquired but then consolidate to more stable states. Reconsolidation theory posits that reactivating a consolidated memory again destabilizes it, increasing its vulnerability to interference from competing memories. In a series of 3-day experiments, we investigated the fate of a motor memory when it is reactivated and challenged with a competing one. We pursued a modular design in which humans adapted to a visuomotor rotation A (day 1), then an opposite rotation B (day 2), followed by a retest on A (day 3). We first found that reactivating A before learning B (A-AB-A) caused no greater impairment in A retention than non-reactivation (A-B-A). That is, while interference occurred, it appeared to be uninfluenced by reactivation, contradicting reconsolidation predictions. We then tested an alternate idea, that reactivation might serve to protect the original memory from interference. In subsequent experiments, we introduced no rotation (N) trials either prior to A relearning (A-AB-NA and A-B-NA groups), or immediately after B learning (A-ABN-A and A-BN-A groups). Here, we observed that reactivation served a protective function, but only when B was washed out immediately, preventing its consolidation (A-ABN-A group). Collectively, our results show that reactivation does not necessarily increase the susceptibility of a motor memory to interference but may rather shield it from degradation by competing learning. Our findings align with theories positing memory transitions between active and inactive states, and hold implications for strategies focused on improving memory retention in rehabilitation, sports and skills training. SIGNIFICANCE STATEMENTConsolidated memories are widely thought to undergo destabilization when reactivated, which then increases their susceptibility to interference from competing inputs. Diverging from this established model, we demonstrate that reactivation, or active retrieval, of a motor memory does not induce vulnerability, but rather protects the original memory from degradation. This protection occurs specifically when the competing learning remains unconsolidated, indicating some temporal constraints on the mechanism. Our findings question reconsolidation theory in motor systems and support an alternative framework in which reactivation promotes memory stability. They also suggest new principles for optimizing skill retention in rehabilitation, athletic training, and skill acquisition, where protecting existing memories is perhaps as critical as forming new ones.

Learning to detect and respond to threats is fundamental for survival and is often modeled through threat conditioning (TC) paradigms. While these paradigms reliably produce implicit memories that elicit physiological and behavioral responses to conditioned stimuli (CS), less is explored about how TC influences cognitive and emotional biases, particularly those implicated in anxiety disorders, such as threat overestimation and negative stimulus representation. In this study, we investigated the dynamic interaction between the reactivation of the implicit threat memory and these cognitive biases using a validated TC paradigm in humans. In Experiment 1, participants underwent TC on Day 1, followed by a memory reactivation session (incomplete reminder: one unreinforced CS+) and a highly demanding working memory (HWM) task, used as an amnesic manipulation, or a control condition on Day 2. On Day 3, memory retention was tested using a simplified, single-trial protocol (one CS+, one CS-, and one neutral CS), followed by tasks assessing threat valuation and representation. Results indicated that the HWM task administered post-reactivation significantly reduced skin conductance responses (SCRs) and attenuated cognitive biases, without altering expectancy of the unconditioned stimulus (US). In Experiment 2, we evaluated the effect of varying reactivation frequency (none, one, or two reminders) on implicit memory and cognitive biases. While repeated reactivations generalized the conditioned response to other stimuli, cognitive and emotional biases remained stable, suggesting a dissociation between memory generalization and evaluative processing. These findings demonstrate that implicit threat memories can be selectively modified through post-reactivation interventions, affecting both physiological and cognitive-emotional domains. Importantly, the distinct effects of memory reactivation and reconsolidation on physiological versus cognitive outcomes support the existence of temporally and functionally dissociable mechanisms. This research highlights the need to consider cognitive biases alongside physiological responses when evaluating memory-based interventions and offers novel insight into mechanisms underlying anxiety maintenance and treatment.