Molecular insights from the crab Neohelice memory model.

Memory acquisition, formation and maintenance depend on synaptic post-translational machinery and regulation of gene expression triggered by several transduction pathways. In turns, these processes lead to stabilization of synaptic modifications in neurons in the activated circuits. In order to study the molecular mechanisms involved in acquisition and memory, we have taken advantage of the context-signal associative learning and, more recently, the place preference task, of the crab Neohelice granulata. In this model organism, we studied several molecular processes, including activation of extracellular signal-regulated kinase (ERK) and the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) transcription factor, involvement of synaptic proteins such as NMDA receptors and neuroepigenetic regulation of gene expression. All these studies allowed description of key plasticity mechanisms involved in memory, including consolidation, reconsolidation and extinction. This article is aimed at review the most salient findings obtained over decades of research in this memory model.

Characteristics of the Reminder that Triggers Object Recognition Memory Reconsolidation in Mice.

Memories are initially labile and become stable through consolidation. Once consolidated, a memory can be destabilized by a reminder, requiring reconsolidation to become stable again. Memory reconsolidation has been evidenced in several learning tasks, including novel object recognition (NOR). But the features of the reminder that trigger memory destabilization and reconsolidation in this task are poorly characterized. Memory reconsolidation can be evidenced by delivering either an amnesic agent or a memory enhancer after reactivation and testing the resulting long-term memory alteration. Here we trained male mice for 15 min to induce a strong memory formation. Sulfasalazine, a specific inhibitor of the NF-κB pathway, was administered as an amnesic agent in the dorsal hippocampus. NF-κB is a key transcription factor required for consolidation and reconsolidation. We found that reconsolidation was induced when animals were re-exposed for 5 min to a combination of novel and familiar objects, but not to either two familiar or two novel objects. No destabilization was induced by re-exposure to the context without objects. Re-exposure to a combination of novel and familiar objects induced destabilization with a reactivation session as brief as 1 min. One minute of training induced a weak memory that could be enhanced by sodium butyrate, an inhibitor of histone deacetylases (HDACs), after 1 min of re-exposure. Histone acetylation is an epigenetic mechanism involved in gene expression regulation which positively correlates with memory. Thus, in this study we have performed an accurate characterization of the features of the reminder effective in triggering hippocampal NF-κB-dependent reconsolidation.

Two spaced training trials induce associative ERK-dependent long term memory in Neohelice granulata.

Memory formation depends upon several parametric training conditions. Among them, trial number and inter-trial interval (ITI) are key factors to induce long-term retention. However, it is still unclear how individual training trials contribute to mechanisms underlying memory formation and stabilization. Contextual conditioning in Neohelice granulata has traditionally elicited associative long-term memory (LTM) after 15 spaced (ITI = 3 min) trials. Here, we show that LTM in crabs can be induced after only two training trials by increasing the ITI to 45 min (2t-LTM) and maintaining the same training duration as in traditional protocols. This newly observed LTM was preserved for at least 96 h, exhibiting protein synthesis dependence during consolidation and reconsolidation as well as context-specificity. Moreover, we demonstrate that 2t-LTM depends on inter-trial and post-training ERK activation showing a faster phosphorylation after the second trial compared to the first one. In summary, we present a new training protocol in crabs through a reduced number of trials showing associative features similar to traditional spaced training. This novel protocol allows for intra-training manipulation and the assessment of individual trial contribution to LTM formation.

LIMK, Cofilin 1 and actin dynamics involvement in fear memory processing.

Long-term memory has been associated with morphological changes in the brain, which in turn tightly correlate with changes in synaptic efficacy. Such plasticity is proposed to rely on dendritic spines as a neuronal canvas on which these changes can occur. Given the key role of actin cytoskeleton dynamics in spine morphology, major regulating factors of this process such as Cofilin 1 (Cfl1) and LIM kinase (LIMK), an inhibitor of Cfl1 activity, are prime molecular targets that may regulate dendritic plasticity. Using a contextual fear conditioning paradigm in mice, we found that pharmacological induction of depolymerization of actin filaments through the inhibition of LIMK causes an impairment in memory reconsolidation, as well as in memory consolidation. On top of that, Cfl1 activity is inhibited and its mRNA is downregulated in CA1 neuropil after re-exposure to the training context. Moreover, by pharmacological disruption of actin cytoskeleton dynamics, the process of memory extinction can either be facilitated or impaired. Our results lead to a better understanding of the role of LIMK, Cfl1 and actin cytoskeleton dynamics in the morphological and functional changes underlying the synaptic plasticity of the memory trace.

The lateral neocortex is critical for contextual fear memory reconsolidation.

Memories are a product of the concerted activity of many brain areas. Deregulation of consolidation and reprocessing of mnemonic traces that encode fearful experiences might result in fear-related psychopathologies. Here, we assessed how pre-established memories change with experience, particularly the labilization/reconsolidation of memory, using the whole-brain analysis technique of positron emission tomography in male mice. We found differences in glucose consumption in the lateral neocortex, hippocampus and amygdala in mice that underwent labilization/reconsolidation processes compared to animals that did not reactivate a fear memory. We used chemogenetics to obtain insight into the role of cortical areas in these phases of memory and found that the lateral neocortex is necessary for fear memory reconsolidation. Inhibition of lateral neocortex during reconsolidation altered glucose consumption levels in the amygdala. Using an optogenetic/neuronal recording-based strategy we observed that the lateral neocortex is functionally connected with the amygdala, which, along with retrograde labeling using fluorophore-conjugated cholera toxin subunit B, support a monosynaptic connection between these areas and poses this connection as a hot-spot in the circuits involved in reactivation of fear memories.

Sustained CaMKII Delta Gene Expression Is Specifically Required for Long-Lasting Memories in Mice.

Although important information is available on the molecular mechanisms of long-term memory formation, little is known about the processes underlying memory persistence in the brain. Here, we report that persistent gene expression of CaMKIIδ isoform participates in object recognition long-lasting memory storage in mice hippocampus. We found that CaMKIIδ mRNA expression was sustained up to one week after training and paralleled memory retention. Antisense DNA infusion in the hippocampus during consolidation or even after consolidation impairs 7-day- but not 1-day-long memory, supporting a role of CaMKIIδ in memory persistence. CaMKIIδ gene expression was accompanied by long-lasting nucleosome occupancy changes at its promoter. This epigenetic mechanism is described for the first time in a memory process and offers a novel mechanism for persistent gene expression in neurons. CaMKIIδ protein is mainly present in nucleus and presynaptic terminals, suggesting a role in these subcellular compartments for memory persistence. All these results point to a key function of the sustained gene expression of this overlooked CaMKII isoform in long-lasting memories.

CaMKII Isoforms in Learning and Memory: Localization and Function.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key protein kinase in neural plasticity and memory, as have been shown in several studies since the first evidence in long-term potentiation (LTP) 30 years ago. However, most of the studies were focused mainly in one of the four isoforms of this protein kinase, the CaMKIIα. Here we review the characteristics and the role of each of the four isoforms in learning, memory and neural plasticity, considering the well known local role of α and ß isoforms in dendritic terminals as well as recent findings about the γ isoform as calcium signals transducers from synapse to nucleus and δ isoform as a kinase required for a more persistent memory trace.

Effects of Hippocampal LIMK Inhibition on Memory Acquisition, Consolidation, Retrieval, Reconsolidation, and Extinction.

Long-lasting changes in dendritic spines provide a physical correlate for memory formation and persistence. LIM kinase (LIMK) plays a critical role in orchestrating dendritic actin dynamics during memory processing, since it is the convergent downstream target of both the Rac1/PAK and RhoA/ROCK pathways that in turn induce cofilin phosphorylation and prevent depolymerization of actin filaments. Here, using a potent LIMK inhibitor (BMS-5), we investigated the role of LIMK activity in the dorsal hippocampus during contextual fear memory in rats. We first found that post-training administration of BMS-5 impaired memory consolidation in a dose-dependent manner. Inhibiting LIMK before training also disrupted memory acquisition. We then demonstrated that hippocampal LIMK activity seems to be critical for memory retrieval and reconsolidation, since both processes were impaired by BMS-5 treatment. Contextual fear memory extinction, however, was not sensitive to the same treatment. In conclusion, our findings demonstrate that hippocampal LIMK activity plays an important role in memory acquisition, consolidation, retrieval, and reconsolidation during contextual fear conditioning.

Requirement of NF-kappa B Activation in Different Mice Brain Areas during Long-Term Memory Consolidation in Two Contextual One-Trial Tasks with Opposing Valences.

NF-kappa B is a transcription factor whose activation has been shown to be necessary for long-term memory consolidation in several species. NF-kappa B is activated and translocates to the nucleus of cells in a specific temporal window during consolidation. Our work focuses on a one trial learning tasks associated to the inhibitory avoidance (IA) setting. Mice were trained either receiving or not a footshock when entering a dark compartment (aversive vs. appetitive learning). Regardless of training condition (appetitive or aversive), latencies to step-through during testing were significantly different to those measured during training. Additionally, these testing latencies were also different from those of a control group that only received a shock unrelated to context. Moreover, nuclear NF-kappa B DNA-binding activity was augmented in the aversive and the appetitive tasks when compared with control and naïve animals. NF-kappa B inhibition by Sulfasalazine injected either in the Hippocampus, Amygdala or Nucleus accumbens immediately after training was able to impair retention in both training versions. Our results suggest that NF-kappa B is a critical molecular step, in different brain areas on memory consolidation. This was the case for both the IA task and also the modified version of the same task where the footshock was omitted during training. This work aims to further investigate how appetitive and aversive memories are consolidated.

Heterozygous Che-1 KO mice show deficiencies in object recognition memory persistence.

Transcriptional regulation is a key process in the formation of long-term memories. Che-1 is a protein involved in the regulation of gene transcription that has recently been proved to bind the transcription factor NF-κB, which is known to be involved in many memory-related molecular events. This evidence prompted us to investigate the putative role of Che-1 in memory processes. For this study we newly generated a line of Che-1(+/-) heterozygous mice. Che-1 homozygous KO mouse is lethal during development, but Che-1(+/-) heterozygous mouse is normal in its general anatomical and physiological characteristics. We analyzed the behavioral characteristic and memory performance of Che-1(+/-) mice in two NF-κB dependent types of memory. We found that Che-1(+/-) mice show similar locomotor activity and thigmotactic behavior than wild type (WT) mice in an open field. In a similar way, no differences were found in anxiety-like behavior between Che-1(+/-) and WT mice in an elevated plus maze as well as in fear response in a contextual fear conditioning (CFC) and object exploration in a novel object recognition (NOR) task. No differences were found between WT and Che-1(+/-) mice performance in CFC training and when tested at 24h or 7days after training. Similar performance was found between groups in NOR task, both in training and 24h testing performance. However, we found that object recognition memory persistence at 7days was impaired in Che-1(+/-) heterozygous mice. This is the first evidence showing that Che-1 is involved in memory processes.

NF-κB transcription factor role in consolidation and reconsolidation of persistent memories.

Transcriptional regulation is an important molecular process required for long-term neural plasticity and long-term memory (LTM) formation. Thus, one main interest in molecular neuroscience in the last decades has been the identification of transcription factors that are involved in memory processes. Among them, the nuclear factor κB (NF-κB) family of transcription factors has gained interest due to a significant body of evidence that supports a key role of these proteins in synaptic plasticity and memory. In recent years, the interest was particularly reinforced because NF-κB was characterized as an important regulator of synaptogenesis. This function may be explained by its participation in synapse to nucleus communication, as well as a possible local role at the synapse. This review provides an overview of experimental work obtained in the last years, showing the essential role of this transcription factor in memory processes in different learning tasks in mammals. We focus the review on the consolidation and reconsolidation memory phases as well as on the regulation of immediate-early and late genes by epigenetic mechanisms that determine enduring forms of memories.

Nuclear factor kappa B-dependent Zif268 expression in hippocampus is required for recognition memory in mice.

Long-term memory formation requires gene expression after acquisition of new information. The first step in the regulation of gene expression is the participation of transcription factors (TFs) such as nuclear factor kappa B (NF-кB), which are present before the neuronal activity induced by training. It was proposed that the activation of these types of TFs allows a second step in gene regulation by induction of immediate-early genes (IEGs) whose protein products are, in turn, TFs. Between these IEGs, zif268 has been found to play a critical role in long-term memory formation and reprocessing after retrieval. Here we found in mice hippocampus that, on one hand, NF-кB was activated 45 min after training in a novel object recognition (NOR) task and that inhibiting NF-кB immediately after training by intrahippocampal administration of NF-кB Decoy DNA impaired NOR memory consolidation. On the other hand, Zif268 protein expression was induced 45 min after NOR training and the administration of DNA antisense to its mRNA post-training impaired recognition memory. Finally, we found that the inhibition of NF-кB by NF-кB Decoy DNA reduced significantly the training-induced Zif268 increment, indicating that NF-кB is involved in the regulation of Zif268 expression. Thus, the present results support the involvement of NF-кB activity-dependent Zif268 expression in the hippocampus during recognition memory consolidation.

Protein degradation by ubiquitin-proteasome system in formation and labilization of contextual conditioning memory.

The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates.

Calcineurin phosphatase as a negative regulator of fear memory in hippocampus: control on nuclear factor-κB signaling in consolidation and reconsolidation.

Protein phosphatases are important regulators of neural plasticity and memory. Some studies support that the Ca(2+) /calmodulin-dependent phosphatase calcineurin (CaN) is, on the one hand, a negative regulator of memory formation and, on the other hand, a positive regulator of memory extinction and reversal learning. However, the signaling mechanisms by which CaN exerts its action in such processes are not well understood. Previous findings support that CaN negatively regulate the nuclear factor kappaB (NF-κB) signaling pathway during extinction. Here, we have studied the role of CaN in contextual fear memory consolidation and reconsolidation in the hippocampus. We investigated the CaN control on the NF-κB signaling pathway, a key mechanism that regulates gene expression in memory processes. We found that post-training intrahippocampal administration of the CaN inhibitor FK506 enhanced memory retention one day but not two weeks after training. Accordingly, the inhibition of CaN by FK506 increased NF-κB activity in dorsal hippocampus. The administration of the NF-κB signaling pathway inhibitor sulfasalazine (SSZ) impeded the enhancing effect of FK506. In line with our findings in consolidation, FK506 administration before memory reactivation enhanced memory reconsolidation when tested one day after re-exposure to the training context. Strikingly, memory was also enhanced two weeks after training, suggesting that reinforcement during reconsolidation is more persistent than during consolidation. The coadministration of SSZ and FK506 blocked the enhancement effect in reconsolidation, suggesting that this facilitation is also dependent on the NF-κB signaling pathway. In summary, our results support a novel mechanism by which memory formation and reprocessing can be controlled by CaN regulation on NF-κB activity.

Epigenetic mechanisms and memory strength: a comparative study.

Memory consolidation requires de novo mRNA and protein synthesis. Transcriptional activation is controlled by transcription factors, their cofactors and repressors. Cofactors and repressors regulate gene expression by interacting with basal transcription machinery, remodeling chromatin structure and/or chemically modifying histones. Acetylation is the most studied epigenetic mechanism of histones modifications related to gene expression. This process is regulated by histone acetylases (HATs) and histone deacetylases (HDACs). More than 5 years ago, we began a line of research about the role of histone acetylation during memory consolidation. Here we review our work, presenting evidence about the critical role of this epigenetic mechanism during consolidation of context-signal memory in the crab Neohelice granulata, as well as during consolidation of novel object recognition memory in the mouse Mus musculus. Our evidence demonstrates that histone acetylation is a key mechanism in memory consolidation, functioning as a distinctive molecular feature of strong memories. Furthermore, we found that the strength of a memory can be characterized by its persistence or its resistance to extinction. Besides, we found that the role of this epigenetic mechanism regulating gene expression only in the formation of strongest memories is evolutionarily conserved.

Synaptic NF-kappa B pathway in neuronal plasticity and memory.

Several transcription factors are present at the synapse, and among these are the Rel-NF-kappa B pathway components. NF-kappa B is a constitutive transcription factor, with a strong presence in the brain of which a considerable part is located in the neuropiles. This localization of the transcription factor, plus evidence pointing to different functions, is what gave place to two general hypotheses for synaptic NF-kappa B: (a) The transcription factor plays a role in the synapse to nucleus communication, and it is retrogradely transported from polarized localizations to regulate gene expression; (b) The transcription factor modulates the synaptic function locally. Evidence indicates that both mechanisms can operate simultaneously; here we will present different possibilities of these hypotheses that are supported by an increasing amount of data. We pay special attention to the local role of the transcription factor at the synapse, and based in the described evidence from different animal models, we propose several processes in which the transcription factor may change the synaptic strength.

Decrease of ERK/MAPK overactivation in prefrontal cortex reverses early memory deficit in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) can be considered as a disease of memory in its initial clinical stages. Amyloid-ß (Aß) peptide accumulation is central to the disease initiation leading later to intracellular neurofibrillary tangles (NFTs) of cytoskeletal tau protein formation. It is under discussion whether different Aß levels of aggregation, concentration, brain area, and/or time of exposure might be critical to the disease progression, as well as which intracellular pathways it activates. The aim of the present work was to study memory-related early molecular and behavioral alterations in a mouse model of AD, in which a subtle deregulation of the physiologic function of Aß can be inferred. For this purpose we used triple-transgenic (3xTg) mice, which develop Aß and tau pathology resembling the disease progression in humans. Memory impairment in novel object recognition task was evident by 5 months of age in 3xTg mice. Hippocampus and prefrontal cortex extra-nuclear protein extracts developed differential patterns of Aß aggregation. ERK1/MAPK showed higher levels of cytosolic activity at 3 months and higher levels of nuclear activity at 6 months in the prefrontal cortex. No significant differences were found in JNK and NF-κB activity and in calcineurin protein levels. Finally, intra-PFC administration of a MEK inhibitor in 6-month-old 3xTg mice was able to reverse memory impairment, suggesting that ERK pathway alterations might at least partially explain memory deficits observed in this model, likely as a consequence of memory trace disruption.

Nuclear factor κB-dependent histone acetylation is specifically involved in persistent forms of memory.

Memory consolidation requires gene expression regulation by transcription factors, which eventually may induce chromatin modifications as histone acetylation. This mechanism is regulated by histone acetylases and deacetylases. It is not yet clear whether memory consolidation always recruits histone acetylation or it is only engaged in more persistent memories. To address this question, we used different strength of training for novel object recognition task in mice. Only strong training induced a long-lasting memory and an increase in hippocampal histone H3 acetylation. Histone acetylase inhibition in the hippocampus during consolidation impaired memory persistence, whereas histone deacetylase inhibition caused weak memory to persist. Nuclear factor κB (NF-κB) transcription factor inhibition impaired memory persistence and, concomitantly, reduced the general level of H3 acetylation. Accordingly, we found an important increase in H3 acetylation at a specific NF-κB-regulated promoter region of the Camk2d gene, which was reversed by NF-kB inhibition. These results show for the first time that histone acetylation is a specific molecular signature of enduring memories.