NCKX4 regulates hippocampal Ca2+ homeostasis and contributes to contextual memory and anxiety-related behaviours.

Evidence from human studies suggests that high expression of brain mineralocorticoid receptors (MR) may promote resilience against negative consequences of stress exposure, including childhood trauma. We examined, in mice, whether brain MR overexpression can alleviate the effects of chronic early life stress (ELS) on contextual memory formation under low and high stress conditions, and neurogenesis and synaptic function of dentate gyrus granular cells. Male mice were exposed to ELS by housing the dam with limited nesting and bedding material from postnatal day (PND) 2 to 9. We investigated the moderating role of MRs by using forebrain-specific transgenic MR overexpression (MR-tg) mice. Low-stress contextual (i.e., object relocation) memory formation was hampered by ELS in wildtype but not MR-tg mice. Anxiety like behavior and high-stress contextual (i.e., fear) memory formation were unaffected by ELS and/or MR expression level. At the cellular level, an interaction effect was observed between ELS and MR overexpression on the number of doublecortin-positive cells, with a significant difference between the wildtype ELS and MR-tg ELS groups. No interaction was found regarding Ki-67 and BrdU staining. A significant interaction between ELS and MR expression was further observed with regard to mEPSCs and mIPSC frequency. The ratio of evoked EPSC/IPSC or NMDA/AMPA responses was unaffected. Overall, these results suggest that ELS affects contextual memory formation under low stress conditions as well as neurogenesis and synaptic transmission in dentate granule cells, an effect that can be alleviated by MR-overexpression.

The roles of Eph receptors in contextual fear conditioning memory formation

The development of dendritic arborizations and spines is essential for neuronal information processing, and abnormal dendritic structures and/or alterations in spine morphology are consistent features of neurons in patients with mental retardation. We identify the neural EGF family member CALEB/NGC as a critical mediator of dendritic tree complexity and spine formation. Overexpression of CALEB/NGC enhances dendritic branching and increases the complexity of dendritic spines and filopodia. Genetic and functional inactivation of CALEB/NGC impairs dendritic arborization and spine formation. Genetic manipulations of individual neurons in an otherwise unaffected microenvironment in the intact mouse cortex by in utero electroporation confirm these results. The EGF-like domain of CALEB/NGC drives both dendritic branching and spine morphogenesis. The phosphatidylinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway and protein kinase C (PKC) are important for CALEB/NGC-induced stimulation of dendritic branching. In contrast, CALEB/NGC-induced spine morphogenesis is independent of PI3K but depends on PKC. Thus, our findings reveal a novel switch of specificity in signaling leading to neuronal process differentiation in consecutive developmental events.

Polysialic acid (PSA) regulates functions of the neural cell adhesion molecule (NCAM) during development and in neuroplasticity in the adult; the underlying mechanisms at different phases of learning and memory consolidation are, however, unknown. To investigate the contributions of PSA versus the extracellular domain of the NCAM glycoprotein backbone to synaptic plasticity, we applied NCAM, PSA-NCAM, and PSA to acute slices of the hippocampal CA1 region of NCAM-deficient mice and measured their effects on long-term potentiation (LTP). Remarkably, only PSA and PSA-NCAM, but not NCAM restored normal LTP. Application of these molecules to the dorsal hippocampus of wild-type mice showed that PSA-NCAM and PSA, but not NCAM, injected before fear conditioning, impaired formation of hippocampus-dependent contextual memory. Consolidation of contextual memory was affected by PSA-NCAM only when injected during its late, but not early phases. None of the tested compounds disturbed extrahippocampal-cued memory. Mice lacking the polysialyltransferase (ST8SialV/PST) responsible for attachment of PSA to NCAM in adulthood showed a mild deficit only in hippocampal contextual learning, when compared with NCAM-deficient mice that were disturbed in both contextual and cued memories. Contextual and tone memory in NCAM-deficient mice could be partially restored by injection of PSA-NCAM, but not of NCAM, into the hippocampus, suggesting that the impact of PSA-NCAM in synaptic plasticity and learning is not mediated by modulation of NCAM-NCAM homophilic interactions. In conclusion, our data support the view that polysialylated NCAM is involved in both formation and late consolidation of contextual memory.

Background: With the global popularity of communication devices such as mobile phones, there are increasing concerns regarding the effect of radiofrequency electromagnetic radiation (RF-EMR) on the brain, one of the most important organs sensitive to RF-EMR exposure at 1,800 MHz. However, the effects of RF-EMR exposure on neuronal cells are unclear. Neurite outgrowth plays a critical role in brain development, therefore, determining the effects of 1,800 MHz RF-EMR exposure on neurite outgrowth is important for exploring its effects on brain development.Objectives: We aimed to investigate the effects of 1,800 MHz RF-EMR exposure for 48 h on neurite outgrowth in neuronal cells and to explore the associated role of the Rap1 signaling pathway.Material and Methods: Primary hippocampal neurons from C57BL/6 mice and Neuro2a cells were exposed to 1,800 MHz RF-EMR at a specific absorption rate (SAR) value of 4 W/kg for 48 h. CCK-8 assays were used to determine the cell viability after 24, 48, and 72 h of irradiation. Neurite outgrowth of primary hippocampal neurons (DIV 2) and Neuro2a cells was observed with a 20 × optical microscope and recognized by ImageJ software. Rap1a and Rap1b gene expressions were detected by real-time quantitative PCR. Rap1, Rap1a, Rap1b, Rap1GAP, and p-MEK1/2 protein expressions were detected by western blot. Rap1-GTP expression was detected by immunoprecipitation. The role of Rap1-GTP was assessed by transfecting a constitutively active mutant plasmid (Rap1-Gly_Val-GFP) into Neuro2a cells.Results: Exposure to 1,800 MHz RF-EMR for 24, 48, and 72 h at 4 W/kg did not influence cell viability. The neurite length, primary and secondary neurite numbers, and branch points of primary mouse hippocampal neurons were significantly impaired by 48-h RF-EMR exposure. The neurite-bearing cell percentage and neurite length of Neuro2a cells were also inhibited by 48-h RF-EMR exposure. Rap1 activity was inhibited by 48-h RF-EMR with no detectable alteration in either gene or protein expression of Rap1. The protein expression of Rap1GAP increased after 48-h RF-EMR exposure, while the expression of p-MEK1/2 protein decreased. Overexpression of constitutively active Rap1 reversed the decrease in Rap1-GTP and the neurite outgrowth impairment in Neuro2a cells induced by 1,800 MHz RF-EMR exposure for 48 h.Conclusion: Rap1 activity and related signaling pathways are involved in the disturbance of neurite outgrowth induced by 48-h 1,800 MHz RF-EMR exposure. The effects of RF-EMR exposure on neuronal development in infants and children deserve greater focus.

Npas4 Is a Critical Regulator of Learning-Induced Plasticity at Mossy Fiber-CA3 Synapses during Contextual Memory Formation

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 dentate gyrus is critical for spatial memory formation and shows task related activation of cellular ensembles considered as memory engrams. Semilunar granule cells (SGCs), a sparse dentate projection neuron subtype distinct from granule cells (GCs), were recently reported to be enriched among behaviorally activated neurons. However, the mechanisms governing SGC recruitment during memory formation and their role in engram refinement remains unresolved. By examining neurons labeled during contextual memory formation in TRAP2 mice, we empirically tested competing hypotheses for GC and SGC recruitment into memory ensembles. In support of the proposal that more excitable neurons are preferentially recruited into memory ensembles, SGCs showed greater sustained firing than GCs. Additionally, SGCs labeled during memory formation showed less adapting firing than unlabeled SGCs. Our recordings did not reveal glutamatergic connections between behaviorally labeled SGCs and GCs, providing evidence against SGC driven local circuit feedforward excitation in ensemble recruitment. Contrary to a leading hypothesis, there was little evidence for individual SGCs or labeled neuronal ensembles supporting lateral inhibition of unlabeled neurons. Instead, labeled GCs and SGCs received more spontaneous excitatory synaptic inputs than their unlabeled counterparts. Moreover, pairs of GCs and SGCs within labeled neuronal cohorts received more temporally correlated spontaneous excitatory synaptic inputs than labeled-unlabeled neuronal pairs, validating a role for correlated afferent inputs in neuronal ensemble selection. These findings challenge the proposal that SGCs drive dentate GC ensemble refinement, while supporting a role for intrinsic active properties and correlated inputs in preferential SGC recruitment to contextual memory engrams.Evaluation of semilunar granule cell involvement in dentate gyrus contextual memory processing supports recruitment based on intrinsic and input characteristics while revealing limited contribution to ensemble refinement.

The dentate gyrus is critical for spatial memory formation and shows task related activation of cellular ensembles considered as memory engrams. Semilunar granule cells (SGCs), a sparse dentate projection neuron subtype distinct from granule cells (GCs), were recently reported to be enriched among behaviorally activated neurons. However, the mechanisms governing SGC recruitment during memory formation and their role in engram refinement remains unresolved. By examining neurons labeled during contextual memory formation in TRAP2 mice, we empirically tested competing hypotheses for GC and SGC recruitment into memory ensembles. In support of the proposal that more excitable neurons are preferentially recruited into memory ensembles, SGCs showed greater sustained firing than GCs. Additionally, SGCs labeled during memory formation showed less adapting firing than unlabeled SGCs. Our recordings did not reveal glutamatergic connections between behaviorally labeled SGCs and GCs, providing evidence against SGCs driving local circuit feedforward excitation in ensemble recruitment. Contrary to a leading hypothesis, there was little evidence for individual SGCs or labeled neuronal ensembles supporting lateral inhibition of unlabeled neurons. Instead, pairs of GCs and SGCs within labeled neuronal cohorts received more temporally correlated spontaneous excitatory synaptic inputs than labeled-unlabeled neuronal pairs, validating a role for correlated afferent inputs in neuronal ensemble selection. These findings challenge the proposal that SGCs drive dentate GC ensemble refinement, while supporting a role for intrinsic active properties and correlated inputs in preferential SGC recruitment to contextual memory engrams.Evaluation of semilunar granule cell involvement in dentate gyrus contextual memory processing supports recruitment based on intrinsic and input characteristics while revealing limited contribution to ensemble refinement.

BackgroundEpigenetic modulation may play a role in anesthesia related phenotypes, such as cognitive impairment or memory loss, especially with exposure to anesthetics in the vulnerable phase of brain development. While isoflurane anesthesia can evoke neuroinflammation and neuroapoptosis in young animals, we investigated in a permanent hippocampal cell line (HT22) and in primary hippocampal neurons in an a priori in vitro analysis, whether isoflurane exposure 1) evokes DNA methylation changes in genes involved in apoptosis and inflammation, and 2) results observed in a permanent hippocampal cell line are comparable to primary hippocampal neurons. In case of methylation changes in specific genes, (3) mRNA analysis was performed to assess possible effects on gene expression.MethodsHT22 cells and primary mouse hippocampal neurons were exposed to 3% isoflurane for 4 h and DNA (each 6 single experiments) and RNA (3 single independent experiments) were extracted. Methylation analysis (EpiTect Methyl II PCR Array Systems, Qiagen) included the methylation status of 66 genes involved in apoptosis, cytokine production, inflammatory response, and autoimmunity. Quantitative Real-Time PCR was performed using the Quantitect SYBR Green Kit on a Step One Plus.ResultsMethylation status was markedly different between immortalized HT22 cells and cultured primary hippocampal neurons without isoflurane exposure. Of 66 genes investigated, 29 were methylated to a significantly greater degree in HT22 cells compared to primary hippocampal neurons. In cultured primary hippocampal neurons, in contrast, there was a greater methylation in several genes involved in inflammation, accompanied with significant downregulation of C-X-C motif chemokine 12 with isoflurane exposure (p = 0.023).ConclusionsWe demonstrate marked differences in gene methylation between HT22 cells and cultured primary hippocampal neurons without isoflurane exposure, with a greater methylation of several genes involved in inflammation upon isoflurane exposure and significant downregulation of Cxcl12 mRNA expression in primary hippocampal neurons. Accordingly, further investigations of anesthesia related DNA methylation should be performed with special consideration being given to the choice of cells targeted for such investigations.

Contextual learning is a critical component of episodic memory and important for living in any environment. Context can be described as the attributes of a location that are not the location itself. This includes a variety of non-spatial information that can be derived from sensory systems (sounds, smells, lighting, etc.) and internal state. In this review, we first address the behavioral underpinnings of contextual memory and the development of context memory theory, with a particular focus on the contextual fear conditioning paradigm as a means of assessing contextual learning and the underlying processes contributing to it. We then present the various neural centers that play roles in contextual learning. We continue with a discussion of the current knowledge of the neural circuitry and physiological processes that underlie contextual representations in the Entorhinal cortex-Hippocampal (EC-HPC) circuit, as the most well studied contributor to contextual memory, focusing on the role of ensemble activity as a representation of context with a description of remapping, and pattern separation and completion in the processing of contextual information. We then discuss other critical regions involved in contextual memory formation and retrieval. We finally consider the engram assembly as an indicator of stored contextual memories and discuss its potential contribution to contextual memory.

Hippocampal pyramidal cells encode memory engrams, which guide adaptive behavior. Selection of engram-forming cells is regulated by somatostatin-positive dendrite-targeting interneurons, which inhibit pyramidal cells that are not required for memory formation. Here, we found that γ-aminobutyric acid (GABA)-releasing neurons of the mouse nucleus incertus (NI) selectively inhibit somatostatin-positive interneurons in the hippocampus, both monosynaptically and indirectly through the inhibition of their subcortical excitatory inputs. We demonstrated that NI GABAergic neurons receive monosynaptic inputs from brain areas processing important environmental information, and their hippocampal projections are strongly activated by salient environmental inputs in vivo. Optogenetic manipulations of NI GABAergic neurons can shift hippocampal network state and bidirectionally modify the strength of contextual fear memory formation. Our results indicate that brainstem NI GABAergic cells are essential for controlling contextual memories.

Activation of NMDA receptors (NMDAR) in the hippocampus is essential for the formation of contextual and trace memory. However, the role of individual NMDAR subunits in the molecular mechanisms contributing to these memory processes is not known. Here we demonstrate, using intrahippocampal injection of subunit-selective compounds, that the NR2A-preferring antagonist impaired contextual and trace fear conditioning as well as learning-induced increase of the nuclear protein c-Fos. The NR2B-specific antagonist, on the other hand, selectively blocked trace fear conditioning without affecting c-Fos levels. Studies with cultured primary hippocampal neurons, further showed that synaptic and extrasynaptic NR2A and NR2B differentially regulate the extracellular signal-regulated kinase 1 and 2/mitogen- and stress-activated protein kinase 1 (ERK1/2/MSK1)/c-Fos pathway. Activation of the synaptic population of NMDAR induced cytosolic, cytoskeletal, and perinuclear phosphorylation of ERK1/2 (pERK1/2). The nuclear propagation of pERK1/2 signals, revealed by upregulation of the downstream nuclear targets pMSK1 and c-Fos, was blocked by a preferential NR2A but not by a specific NR2B antagonist. Conversely, activation of total (synaptic and extrasynaptic) NMDAR engaged receptors with NR2B subunits, and resulted in membrane retention of pERK1/2 without inducing pMSK1 and c-Fos. Stimulation of extrasynaptic NMDAR alone was consistently ineffective at activating ERK signaling. The discrete contribution of synaptic and total NR2A- and NR2B-containing NMDAR to nuclear transmission vs. membrane retention of ERK signaling may underlie their specific roles in the formation of contextual and trace fear memory.

Effects of membrane androgen receptor binding on synaptic plasticity in primary hippocampal neurons

Exposure Effects of Terahertz Waves on Primary Neurons and Neuron-like Cells Under Nonthermal Conditions