A One-Trial Mouse Model of Highly Stable Trauma-Induced Long-Term Memory.

The retention of information represents a defining trait of cognitive systems. The primary function of an organism’s memory is to retain engrams of unique experiences for extended periods, without interrupting memory trace during new learning processes. One of the cardinal unsolved issues in neuroscience lies in unveiling the mechanisms responsible for long-term memory. Despite this, a trustworthy experimental animal model for single-trial long-term memory remains undeveloped. To address this challenge, we aimed to create an animal model to study lifelong memory formation with a single trial. We used a mouse model of post-traumatic stress disorder (PTSD) for this purpose [1]. In this model, mice exposed to a powerful electrical foot shock develop highly persistent traumatic memories, which results in long-term behavioral changes [1]. Our hypothesis was that this model could uncover the mechanisms related to lifelong memory. The primary rationale for adopting the PTSD model as a model of lifelong memory pertains to the heightened durability of traumatic memories in comparison to conventional aversive memories [2]. Protein synthesis inhibitors, which are needed for long-term memory consolidation, can be used as amnesic agents to evaluate memory stability [3]. In a PTSD model using predator scent, the administration of a protein synthesis blocker prior to a traumatic experience disrupts the development of PTSD in mice [4]. However, it remains unclear how traumatic memory impairment affects PTSD development in the footshock model and whether the impairment persists long-term. Based on the evidence that the formation of PTSD necessitates the consolidation of associative memory, which is reliant on protein synthesis and coincides with changes in the stress response system, Siegmund and Watzhek [1] present a two-part proposal concerning the onset of PTSD. The two-part hypothesis regarding PTSD posits that PTSD formation involves sensory conditioning and sensitization processes that mutually reinforce one another. In line with this theorem, we considered the effects of studying context and exposure timing on the development of post-traumatic stress disorder, as it could potentially interfere with sensory conditioning formation [5]. The aim of this study is to create an experimental approach for developing enduring long-term memory through a single trial event on adult mice. We methodically analyzed behavioral expressions and the endurance of normal and traumatic fear memory, as well as their sensitivity to protein synthesis inhibition. Additionally, we investigated the effects of separating the timing of the associative and aversive elements of traumatic memory on PTSD development in a mouse model. The experiment involved male C57Bl/6 mice, aged between 3–4 months and 15–18 months (for an investigation into aged mice), which were placed in an electrified chamber. After 170 seconds, the mice experienced either one footshock (1.5 mA, 2 s) to elicit fear memory or three footshocks (1.5 mA, 10 s) for PTSD induction. After receiving the footshock, the mouse was held in the chamber for 60 seconds before being returned to its home cage. A memory test was conducted seven days later by placing the mice back in the same context. To assess the existence of standard PTSD symptoms, like sensitization and generalization, the creatures were exposed to an unfamiliar context and an unexpected auditory stimulus, respectively. In the experiment, animals were placed in unfamiliar or familiar safe contexts that differed from their previous ones. The duration of freezing was measured to assess fear and evaluate memory retention, sensitization, and generalization. Anxiety levels were evaluated using the elevated plus maze test. In the process of constructing a highly stable long-term memory model, we evaluated the behavioral performance of PTSD-induced (PTSD), fear-conditioned (FC), and active control (AC) groups of animals at 7 days, 1 month, and 3 months after exposure. As a result of PTSD induction, mice displayed increased fear levels for up to 3 months in the training context, along with heightened fear sensitization and generalization at 7 days following exposure, relative to the FC and AC groups. The PTSD group exhibited heightened freezing behavior within a month and a decreased number of entries to the closed arms during the initial three months following exposure when contrasted with the FC and AC groups. We additionally noted that the FC group displayed raised fear levels in contrast to the control animals up to 3 months post-exposure, albeit lower in magnitude than that of the PTSD group. The FC group, similar to the PTSD group, showed increased sensitization and generalization compared to control animals at 1 week post-exposure, but to a lesser extent than we observed in the PTSD group. The findings suggest that traumatic memory remains present for a minimum of three months, whereas fear memory in the fear conditioning paradigm diminishes in intensity during this time. Hence, PTSD induction effectively functions as an animal model of profoundly stable long-term memory, in opposition to the fear conditioning paradigm. As the high stable long-term memory model is designed for testing in senior animals, the impact of aging on traumatic memory formation and storage becomes an important inquiry at six months and one year following exposure. We analyzed the formation of both aversive and traumatic memory in mice aged 15–18 months, one week after footshock exposure. In the study involving older mice, the PTSD group exhibited increased levels of fear memory and sensitization when compared with both the FC and AC groups, in addition to displaying higher levels of generalization and anxiety when compared with the AC group. Furthermore, mice that underwent rear-conditioning showed heightened levels of fear during learning, fear sensitization, and generalization, although not anxiety. These findings demonstrate the formation of traumatic and aversive memory in aged mice, indicating that conducting memory tests six months and a year after exposure is an appropriate approach. To evaluate the durability of traumatic and aversive memory, we administered a protein synthesis inhibitor called cycloheximide (Chm) to interfere with memory formation. Mice were given a cycloheximide solution (95 mg/kg) via intraperitoneal injection 30 minutes before footshock (Chm-PTSD and Chm-FC groups) while control animals received a saline injection (Sal-PTSD, Sal-FC). After training, the Chm-FC group displayed a decrease in freezing rate when compared to the saline-injected group at the 7- and 30-day marks. The Chm-PTSD group exhibited fear levels comparable to those of the Sal-FC group in the training context, and did not display sensitization or generalization of fear. These findings suggest that inhibiting protein synthesis during memory formation resulted in complete amnesia for standard aversive memory and merely weakened traumatic memory. As a result, only the associative component of traumatic memory remained intact, while nonspecific symptoms of PTSD were absent. The strong resilience of memory in the PTSD model to severe disruptions, such as protein synthesis blockade, also confirms its stability, which facilitates exploration of lifelong memory mechanisms in the PTSD model. To examine the timing effect of the associative and aversive component of PTSD development, we evaluated traumatic memory formation when contextual memory formation was absent, and contextual exploration occurred three days before shock cessation. A week after exposure to immediate and intense footshock, mice in the experimental group exhibited lower levels of fear, reduced fear sensitization, and less anxiety compared to those in the PTSD-induced group. If an intense foot shock was preceded by a contextual study three days earlier, the animals exhibited lower levels of fear in an unfamiliar, safe context, and reduced anxiety compared to the mice induced with PTSD. In both cases, the fear level after the shock cessation was identical to that of the mice who experienced an immediate, moderate shock. Based on our findings, we concluded that aversive memory forms in the absence or prior formation of contextual memory in relation to traumatic exposure. However, PTSD induction does not occur under such circumstances. This indicates that contextual memory needs to be formed simultaneously with the traumatic event for the development of traumatic memory in the mouse model of PTSD. In summary, our findings indicate that aversive memory tends to fade over time, while traumatic memory remains stable for a minimum of 3 months following its induction. Therefore, PTSD proves to be a fitting candidate laboratory model for high, stable, and long-term memory. Aged mice aged between 15–18 months display the formation of traumatic memory and experience PTSD symptoms in a manner similar to adult animals aged between 3–4 months in the PTSD model. For the induction of PTSD in mice, contextual memory formation about the traumatic environment and the traumatic event occurrence may coincide, which is crucial.

The influence of circadian rhythms on memory has long been studied; however, the molecular prerequisites for their interaction remain elusive. The hippocampus, which is a region of the brain important for long-term memory formation and temporary maintenance, shows circadian rhythmicity in pathways central to the memory-consolidation process. As neuronal plasticity is the translation of numerous inputs, illuminating the direct molecular links between circadian rhythms and memory consolidation remains a daunting task. However, the elucidation of how clock genes contribute to synaptic plasticity could provide such a link. Furthermore, the idea that memory training could actually function as a zeitgeber for hippocampal neurons is worth consideration, based on our knowledge of the entrainment of the circadian clock system. The integration of many inputs in the hippocampus affects memory consolidation at both the cellular and the systems level, leaving the molecular connections between circadian rhythmicity and memory relatively obscure but ripe for investigation.

Mushroom-Body Memories: An Obituary Prematurely Written?

Encoding-Retrieval Similarity Reveals Distinct Neural Reinstatement of Safety Memories Following Counterconditioning in Posttraumatic Stress Disorder.

EphB2 receptor forward signaling is needed for normal long-term memory formation in aged mice

Memory is initially labile but can be consolidated into stable long-term memory (LTM) that is stored in the brain for extended periods. Despite recent progress, the molecular and cellular mechanisms underlying the intriguing neurobiological processes of LTM remain incompletely understood. Using the Drosophila courtship conditioning assay as a memory paradigm, here, we show that the LIM homeodomain (LIM-HD) transcription factor Apterous (Ap), which is known to regulate various developmental events, is required for both the consolidation and maintenance of LTM. Interestingly, Ap is involved in these 2 memory processes through distinct mechanisms in different neuronal subsets in the adult brain. Ap and its cofactor Chip (Chi) are indispensable for LTM maintenance in the Drosophila memory center, the mushroom bodies (MBs). On the other hand, Ap plays a crucial role in memory consolidation in a Chi-independent manner in pigment dispersing factor (Pdf)-containing large ventral–lateral clock neurons (l-LNvs) that modulate behavioral arousal and sleep. Since disrupted neurotransmission and electrical silencing in clock neurons impair memory consolidation, Ap is suggested to contribute to the stabilization of memory by ensuring the excitability of l-LNvs. Indeed, ex vivo imaging revealed that a reduced function of Ap, but not Chi, results in exaggerated Cl− responses to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in l-LNvs, indicating that wild-type (WT) Ap maintains high l-LNv excitability by suppressing the GABA response. Consistently, enhancing the excitability of l-LNvs by knocking down GABAA receptors compensates for the impaired memory consolidation in ap null mutants. Overall, our results revealed unique dual functions of the developmental regulator Ap for LTM consolidation in clock neurons and LTM maintenance in MBs.

Identifying the molecular mechanisms that underlie learning and memory is one of the major challenges in neuroscience. Synapses are highly specialized intercellular junctions specialized for transmission of signals between neuron and its target cells. One of the most profound characteristics of synapses is the extraordinary degree of morphological and functional plasticity under basal conditions and also in response to neuronal activity. Synaptic plasticity is a long studied mechanism that is thought to be in the center of memory formation and maintenance. The significance of synapse morphological dynamics for the synaptic plasticity and therefore memory still remains unclear. Taken the advantages of the nematode C. elegans, we investigated α-adducin (add-1) in aversive olfactory associative learning and memory. Loss of add-1 function selectively impaired short- and long-term memory without causing acquisition, sensory or motor deficits. We showed that α-adducin is required for consolidation of synaptic plasticity, for sustained synaptic increase of AMPA-type glutamate receptor (GLR-1) content and altered GLR-1 turnover dynamics. ADD-1 controlled the storage of memories presumably through actin capping activity in a splice form and tissue specific manner. In support of the C. elegans results, genetic variability of the human ADD1 gene was significantly associated with episodic memory performance in healthy young subjects. Finally, human ADD1 expression in nematodes restored loss of C. elegans add-1 gene function. Taken together, our findings support a role for α-adducin in memory from nematodes to humans. Studying the molecular and genetic underpinnings of memory over distinct species may be helpful in the development of novel strategies to treat memory-related diseases.
\n\tIn contrast to a relatively deep understanding of how memories are formed, is our limited understanding of how these same memories are maintained. Epigenetic modifications of DNA could be crucial for understanding the molecular basis of complex phenotypes. In the second project we tried to underpin the link between traumatic memories and posttraumatic stress disorder (PTSD) in genocide survivors and DNA methylation. Stress induces a complex set of mechanisms that affect the entire organism. The primary function of those changes is to prepare the organism for the direct consequences of stressful events and to ensure a quick return to homeostasis. Additionally, stress is triggering long-term adaptive responses, which result in enhanced memory of stressful events. Failing to recover from the initial response and to keep the adaptive biological alterations under control leads to impaired homeostasis, results in disorders like PTSD. One of the critical symptoms is loss of the auto-regulation of the stress-induced alterations in HPA (hypothalamic-pituitary- adrenal axis) signaling and increased inhibition of the HPA axis. These changes are maintained over a long period of time, although the underlying mechanisms remain unclear. We investigated epigenetic alterations of the glucocorticoid receptor (GR) gene promoter in saliva samples from survivors of the Rwandan genocide. We found a strong, negative correlation of PTSD symptoms like intrusions, avoidance, and PTSD diagnosis with DNA methylation of the GR gene promoter in genocide survivors. Furthermore, the epigenetic changes were specific to the NGFI transcriptional factor-binding site of the GR promoter and also correlate with GR gene expression. Additionally, we detected a significant negative correlation of LINE-1 element methylation with PTSD risk and avoidance symptoms. Together, our data suggests that epigenetic alterations of glucocorticoid receptor gene and genome-wide in LINE-1 elements could be important for pathophysiology of PTSD and may offer new targets for PTSD diagnosis and treatment. This study also suggests an intriguing possibility of using peripheral tissues for finding epigenetic signatures of some life experiences and complex memory processes. Finally, we took one-step ‘’back’’ to the context of the genomic DNA sequence. This revealed the association of genetic variation in the de-novo DNA methyltransferase 3B gene (DNMT3B) with PTSD symptom clusters and risk. Thus, our study suggests a possible mechanism that loops genetic variation and epigenetic mechanisms as driving forces of the phenotypic plasticity, with development, adaptation and disease. But, instead of revealing a simple predictive code that is shared by many genes, in-depth observation of epigenomic patterns highlights the unique complexity of each transcriptional unit and its associated transcriptional regulatory machinery.

Improved survival is likely linked to the ability to generate stable memories of significant experiences. Considerable evidence in humans and mammalian model animals shows that steroid hormones, which are released in response to emotionally arousing experiences, have an important role in the consolidation of memories of such events. In insects, ecdysone is the major steroid hormone, and it is well characterized with respect to its essential role in coordinating developmental transitions such as larval molting and metamorphosis. However, the functions of ecdysone in adult physiology remain largely elusive. Here, we show that 20-hydroxyecdysone (20E), the active metabolite of ecdysone that is induced by environmental stimuli in adult Drosophila, has an important role in the formation of long-term memory (LTM). In male flies, the levels of 20E were found to be significantly increased after courtship conditioning, and exogenous administration of 20E either enhanced or suppressed courtship LTM, depending on the timing of its administration. We also found that mutants in which ecdysone signaling is reduced were defective in LTM, and that an elevation of 20E levels was associated with activation of the cAMP response element binding protein (CREB), an essential regulator of LTM formation. Our results demonstrate that the molting steroid hormone ecdysone in adult Drosophila is critical to the evolutionarily conserved strategy that is used for the formation of stable memories. We propose that ecdysone is able to consolidate memories possibly by recapturing molecular and cellular processes that are used for normal neural development.

Many living organisms of the animal kingdom have the fundamental ability to form and retrieve memories. Most information is initially stored as short-term memory, which is then converted to a more stable long-term memory through a process called memory consolidation. At the neuronal level, synaptic plasticity is crucial for memory storage. It includes the formation of new spines, as well as the modification of existing spines, thereby tuning and shaping synaptic efficacy. Cofilin critically contributes to memory processes as upon activation, it regulates the shape of dendritic spines by targeting actin filaments. We previously found that prolonged activation of cofilin in hippocampal neurons attenuated the formation of long-term object-location memories. Because the modification of spine shape and structure is also essential for short-term memory formation, we determined whether overactivation of hippocampal cofilin also influences the formation of short-term memories. To this end, mice were either injected with an adeno-associated virus expressing catalytically active cofilin, or an eGFP control, in the hippocampus. We show for the first time that cofilin overactivation improves short-term memory formation in the object-location memory task, without affecting anxiety-like behavior. Surprisingly, we found no effect of cofilin overactivation on AMPA receptor expression levels. Altogether, while cofilin overactivation might negatively impact the formation of long-lasting memories, it may benefit short-term plasticity.

Fly Memory: A Mushroom Body Story in Parts

Memory is initially labile and gradually consolidated over time through new protein synthesis into a long-lasting stable form. Studies of odor-shock associative learning in Drosophila have established the mushroom body (MB) as a key brain structure involved in olfactory long-term memory (LTM) formation. Exactly how early neural activity encoded in thousands of MB neurons is consolidated into protein-synthesis-dependent LTM remains unclear. Here, several independent lines of evidence indicate that changes in two MB vertical lobe V3 (MB-V3) extrinsic neurons are required and contribute to an extended neural network involved in olfactory LTM: (i) inhibiting protein synthesis in MB-V3 neurons impairs LTM; (ii) MB-V3 neurons show enhanced neural activity after spaced but not massed training; (iii) MB-V3 dendrites, synapsing with hundreds of MB α/β neurons, exhibit dramatic structural plasticity after removal of olfactory inputs; (iv) neurotransmission from MB-V3 neurons is necessary for LTM retrieval; and (v) RNAi-mediated down-regulation of oo18 RNA-binding protein (involved in local regulation of protein translation) in MB-V3 neurons impairs LTM. Our results suggest a model of long-term memory formation that includes a systems-level consolidation process, wherein an early, labile olfactory memory represented by neural activity in a sparse subset of MB neurons is converted into a stable LTM through protein synthesis in dendrites of MB-V3 neurons synapsed onto MB α lobes.

Episodic memories acquired early in life are fragile and rapidly forgotten in both humans and nonhuman animals. However, early life experiences have been documented to profoundly affect brain function and physiology throughout life, suggesting that in certain circumstances, the developing brain is capable of producing long-term memory (LTM). In this study, we asked whether exposure to a novel environment, a process named "behavioral tagging," may promote the persistence of weak memories in male juvenile mice. Using a contextual fear conditioning (CFC) paradigm, we found that a weak training protocol, which typically induces a transient form of memory, results in LTM when paired with an exploration to a novel but not a familiar environment that occurs close in time to the training session. The promoting effect of the novel context exploration (NCE) on CFC-LTM formation is dependent on the activation of dopamine D1/D5 receptors and requires novel protein synthesis in the dorsal hippocampus. Moreover, NCE increases the size of overlapping CA1 neuronal ensembles engaged by CFC and NCE. These results provide direct support for the existence of a behavioral tagging process, in which exposure to novelty provides newly synthesized proteins to stabilize the contextual fear memory trace in juvenile mice.

The Long and the Short of It: Memory Signals in the Medial Temporal Lobe

Molecular Enhancement of Memory Formation

Similar to other invertebrate and vertebrate animals, cAMP-dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) and for the AC-activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this "strong" food-reward conditioning paradigm the activation of AC by PACAP was necessary for LTM to form. We found that in a "weak" multitrial food-reward conditioning paradigm, lip touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multitrial tactile conditioning accelerated the formation of transcription-dependent memory. Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning.