GluN2A Subunits Research Articles

Impact of Mephedrone on Fear Memory in Adolescent Rats: Involvement of Matrix Metalloproteinase-9 (MMP-9) and N-Methyl-D-aspartate (NMDA) Receptor.

Treatment of Post-Traumatic Stress Disorder (PTSD) is complicated by the presence of drug use disorder comorbidity. Here, we examine whether conditioned fear (PTSD model) modifies the rewarding effect of mephedrone and if repeated mephedrone injections have impact on trauma-related behaviors (fear sensitization, extinction, and recall of the fear reaction). We also analyzed whether these trauma-induced changes were associated with exacerbation in metalloproteinase-9 (MMP-9) and the GluN2A and GluN2B subunits of N-methyl-D-aspartate (NMDA) glutamate receptor expression in such brain structures as the hippocampus and basolateral amygdala. Male adolescent rats underwent trauma exposure (1.5 mA footshock), followed 7 days later by a conditioned place preference training with mephedrone. Next, the post-conditioning test was performed. Fear sensitization, conditioned fear, anxiety-like behavior, extinction acquisition and relapse were then assessed to evaluate behavioral changes. MMP-9, GluN2A and GluN2B were subsequently measured. Trauma-exposed rats subjected to mephedrone treatment acquired a strong place preference and exhibited impairment in fear extinction and reinstatement. Mephedrone had no effect on trauma-induced MMP-9 level in the basolateral amygdala, but decreased it in the hippocampus. GluN2B expression was decreased in the hippocampus, but increased in the basolateral amygdala of mephedrone-treated stressed rats. These data suggest that the modification of the hippocampus and basolateral amygdala due to mephedrone use can induce fear memory impairment and drug seeking behavior in adolescent male rats.

Increasing Adiponectin Signaling by Sub-Chronic AdipoRon Treatment Elicits Antidepressant- and Anxiolytic-Like Effects Independent of Changes in Hippocampal Plasticity.

(1) Background: Adiponectin is an adipocyte-secreted hormone that has antidepressant- and anxiolytic-like effects in preclinical studies. Here, we investigated the antidepressant- and anxiolytic-like effects of sub-chronic treatment with AdipoRon, an adiponectin receptor agonist, and its potential linkage to changes in hippocampal adult neurogenesis and synaptic plasticity. (2) Methods: Different cohorts of wild-type C57BL/6J and CamKIIα-Cre male mice were treated with sub-chronic (7 days) AdipoRon, followed by behavioral, molecular, and electrophysiological experiments. (3) Results: 7-day AdipoRon treatment elicited antidepressant- and anxiolytic-like effects but did not affect hippocampal neurogenesis. AdipoRon treatment reduced hippocampal brain-derived neurotrophic factor (BDNF) levels, neuronal activation in the ventral dentate gyrus, and long-term potentiation of the perforant path. The knockdown of N-methyl-D-aspartate (NMDA) receptor subunits GluN2A and GluN2B in the ventral hippocampus did not affect the antidepressant- and anxiolytic-like effects of AdipoRon. (4) Conclusions: Increasing adiponectin signaling through sub-chronic AdipoRon treatment results in antidepressant- and anxiolytic-like effects independent of changes in hippocampal structural and synaptic function.

NMDA Receptor Activation-Dependent Antidepressant-Relevant Behavioral and Synaptic Actions of Ketamine.

Ketamine is a well-characterized NMDA receptor (NMDAR) antagonist, although the relevance of this pharmacology to its rapid (within hours of administration) antidepressant actions, which depend on mechanisms convergent with strengthening of excitatory synapses, is unclear. Activation of synaptic NMDARs is necessary for the induction of canonical long-term potentiation (LTP) leading to a sustained expression of increased synaptic strength. We tested the hypothesis that induction of rapid antidepressant effects requires NMDAR activation, by using behavioral pharmacology, western blot quantification of hippocampal synaptoneurosomal protein levels, and ex vivo hippocampal slice electrophysiology in male mice. We found that ketamine exerts an inverted U-shaped dose-response in antidepressant-sensitive behavioral tests, suggesting that an excessive NMDAR inhibition can prevent ketamine's antidepressant effects. Ketamine's actions to induce antidepressant-like behavioral effects, up-regulation of hippocampal AMPAR subunits GluA1 and GluA2, as well as metaplasticity measured ex vivo using electrically-stimulated LTP, were abolished by pretreatment with other non-antidepressant NMDAR antagonists, including MK-801 and CPP. Similarly, the antidepressant-like actions of other putative rapid-acting antidepressant drugs (2R,6R)-hydroxynorketamine (ketamine metabolite), MRK-016 (GABAAα5 negative allosteric modulator), and LY341495 (mGlu2/3 receptor antagonist) were blocked by NMDAR inhibition. Ketamine acted synergistically with an NMDAR positive allosteric modulator to exert antidepressant-like behavioral effects and activation of the NMDAR subunit GluN2A was necessary and sufficient for such relevant effects. We conclude rapid-acting antidepressant compounds share a common downstream NMDAR-activation dependent effector mechanism, despite variation in initial pharmacological targets. Promoting NMDAR signaling or other approaches that enhance NMDAR-dependent LTP-like synaptic potentiation may be an effective antidepressant strategy.SIGNIFICANCE STATEMENT The anesthetic and antidepressant drug ketamine is well-characterized as an NMDA receptor (NMDAR) antagonist; though, the relevance and full impact of this pharmacology to its antidepressant actions is unclear. We found that NMDAR activation, which occurs downstream of their initial actions, is necessary for the beneficial effects of ketamine and several other putative antidepressant compounds. As such, promoting NMDAR signaling, or other approaches that enhance NMDAR-dependent long-term potentiation (LTP)-like synaptic potentiation in vivo may be an effective antidepressant strategy directly, or acting synergistically with other drug or interventional treatments.

Design and synthesis of 6-methylpyridin-2-one derivatives as novel and potent GluN2A positive allosteric modulators for the treatment of cognitive impairment

N-Methyl-D-aspartate receptors (NMDARs) are key regulators of synaptic plasticity in the central nervous system. Potentiation of NMDARs containing GluN2A subunit has been recently recognized as a promising therapeutic approach for neurological disorders. We identified a novel series of GluN2A positive allosteric modulator (PAM) with a pyridin-2-one scaffold. Initial lead compound 1 was discovered through in silico-based screening of virtual ligands with various monocyclic scaffolds. GluN2A PAM activity was increased by introduction of a methyl group at the 6-position of the pyridin-2-one ring and a cyano group in the side chain. Modification of the aromatic ring led to the identification of potent and brain-penetrant 6-methylpyridin-2-one 17 with a negligible binding activity for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Oral administration of 17 significantly enhanced rat hippocampal long-term potentiation (LTP). Thus, 17 would be a useful in vivo pharmacological tool to investigate complex NMDAR functions for the discovery of therapeutics toward diseases associated with NMDAR dysfunction

Identification of homologous GluN subunits variants accelerates GRIN variants stratification.

The clinical spectrum of GRIN-related neurodevelopmental disorders (GRD) results from gene- and variant-dependent primary alterations of the NMDA receptor, disturbing glutamatergic neurotransmission. Despite GRIN gene variants' functional annotations being dually critical for stratification and precision medicine design, genetically diagnosed pathogenic GRIN variants currently outnumber their relative functional annotations. Based on high-resolution crystal 3D models and topological domains conservation between GluN1, GluN2A, and GluN2B subunits of the NMDAR, we have generated GluN1-GluN2A-GluN2B subunits structural superimposition model to find equivalent positions between GluN subunits. We have developed a GRIN structural algorithm that predicts functional changes in the equivalent structural positions in other GluN subunits. GRIN structural algorithm was computationally evaluated to the full GRIN missense variants repertoire, consisting of 4,525 variants. The analysis of this structure-based model revealed an absolute predictive power for GluN1, GluN2A, and GluN2B subunits, both in terms of pathogenicity-association (benign vs. pathogenic variants) and functional impact (loss-of-function, benign, gain-of-function). Further, we validated this computational algorithm experimentally, using an in silico library of GluN2B-equivalent GluN2A artificial variants, designed from pathogenic GluN2B variants. Thus, the implementation of the GRIN structural algorithm allows to computationally predict the pathogenicity and functional annotations of GRIN variants, resulting in the duplication of pathogenic GRIN variants assignment, reduction by 30% of GRIN variants with uncertain significance, and increase by 70% of functionally annotated GRIN variants. Finally, GRIN structural algorithm has been implemented into GRIN variants Database (http://lmc.uab.es/grindb), providing a computational tool that accelerates GRIN missense variants stratification, contributing to clinical therapeutic decisions for this neurodevelopmental disorder.

Individual behavioral profiling as a translational approach to assess treatment efficacy in an animal model of post-traumatic stress disorder.

A major challenge in treating post-traumatic stress disorder (PTSD) continues to be the large variability in responsiveness to pharmacotherapy. Only 20-30% of patients experience total remission to a specific treatment, while others demonstrate either partial remission or no response. However, this heterogeneity in response to pharmacotherapy has not been adequately addressed in animal models, since these analyze the averaged group effects, ignoring the individual variability to treatment response, which seriously compromises the translation power of such models. Here we examined the possibility of employing an "individual behavioral profiling" approach, originally developed to differentiate between "affected" and "exposed-unaffected" individuals in an animal model of PTSD, to also enable dissociating "responders" or "non-responders" after SSRI (fluoxetine) treatment. Importantly, this approach does not rely on a group averaged response to a single behavioral parameter, but considers a cluster of behavioral parameters, to individually characterize an animal as either "responder" or "non-responder" to the treatment. The main variable to assess drug efficacy thus being the proportion of "responders" following treatment. Alteration in excitatory/inhibitory (E/I) balance has been proposed as being associated with stress-related psychopathology. Toward a functional proof of concept for our behaviorally-based characterization approach, we examined the expression patterns of α1 and α2 subunits of GABAA receptor, and GluN1 and GluN2A subunits of the NMDAR receptor in the ventral hippocampus, as well as electrophysiologically local circuit activity in the dorsal dentate gyrus (DG). We demonstrate that with both parameters, treatment "responders" differed from treatment "non-responders," confirming the functional validity of the behavior-based categorization. The results suggest that the ability to respond to fluoxetine treatment may be linked to the ability to modulate excitation-inhibition balance in the hippocampus. We propose that employing the "individual behavioral profiling" approach, and the resultant novel variable of the proportion of "recovered" individuals following treatment, offers an effective translational tool to assess pharmacotherapy treatment efficacy in animal models of stress and trauma-related psychopathology.

Sex-specific role of hippocampal NMDA-Erk-mTOR signaling in fear extinction of adolescent mice

Adolescence is a key phase of development for perturbations in fear extinction, with inability to adequately manage fear a potent factor for developing psychiatric disorders in adulthood. However, while behavioral correlates of adolescent fear regulation are established to a degree, molecular mediators of extinction learning in adolescence remain largely unknown. In this study, we observed fear acquisition and fear extinction (across 4 and 7 days) of adolescent and adult mice of both sexes and investigated how hippocampal levels of different plasticity markers relate to extinction learning. While fear was acquired evenly in males and females of both ages, fear extinction was found to be impaired in adolescent males. We also observed lower levels of GluA1, GLUN2A and GLUN2B subunits in male adolescents following fear acquisition, with an increase in their expression, as well as the activity of Erk-mTOR pathway over subsequent extinction sessions, which was paralleled with improved extinction learning. On the other hand, we detected no changes in plasticity-related proteins after fear acquisition in females, with alterations in GluA1, GluA4 and GLUN2B levels across fear extinction sessions. Additionally, we did not discern any pattern regarding the Erk-mTOR activity in female mice associated with their extinction performance. Overall, our research identifies sex-specific synaptic properties in the hippocampus that underlie developmentally regulated differences in fear extinction learning. We also point out hippocampal NMDA-Erk-mTOR signaling as the driving force behind successful fear extinction in male adolescents, highlighting this pathway as a potential therapeutic target for fear-related disorders in the adolescent population.

Effects of ethanol on GluN1/GluN2A and GluN1/GluN2B NMDA receptor-ion channel gating kinetics.

The N-methyl-D-aspartate receptor (NMDAR) is a major molecular target of alcohol action in the central nervous system, yet many aspects of alcohol's modulation of the activity of this ion channel remain unclear. We and others have shown that ethanol inhibition of NMDAR involves alterations in gating, especially a reduction in mean open time. However, a full description of ethanol's effects on NMDAR kinetics, including fitting them to a kinetic model, has not been reported. To determine ethanol's effects on NMDAR kinetics, we used steady-state single-channel recording in outside-out patches from HEK-293 cells transfected with recombinant GluN1/GluN2A or GluN1/GluN2B NMDAR subunits. Very low glutamate concentrations were used to isolate individual activations of the receptor. In both subunit types, ethanol, at approximate whole-cell IC50 values (156 mM, GluN2A; 150 mM, GluN2B), reduced open probability (po ) by approximately 50% and decreased mean open time without changing the frequency of opening. Open and shut time distributions exhibited two and five components, respectively; ethanol selectively decreased the time constant and relative proportion of the longer open time component. In the GluN2A subunit, ethanol increased the time constants of all but the longest shut time components, whereas in the GluN2B subunit, shut times were unchanged by ethanol. Fitting of bursts of openings (representing individual activations of the receptor) to the gating portion of a kinetic model revealed that ethanol altered two rates: the rate associated with activation of the GluN2A or GluN2B subunit, and the rate associated with the closing of the longer of the two open states. These results demonstrate that ethanol selectively alters individual kinetic rates and thus appears to selectively affect distinct conformational transitions involved in NMDAR gating.

GluN2A-Selective NMDA Receptor Antagonists: Mimicking the U-Shaped Bioactive Conformation of TCN-201 by a [2.2]Paracyclophane System.

Under physiological conditions, N-Methyl-D-Aspartate (NMDA) receptors play a crucial role for synaptic plasticity, long-term potentiation and long-term depression. However, overactivation of NMDA receptors can result in excitotoxicity, which is associated with various neurological and neurodegenerative diseases. The physiological properties of NMDA receptors are strongly dependent on the GluN2 subunit incorporated into the heterotetrameric NMDA receptor. Therefore, subtype selective NMDA receptor modulators are of high interest. Since prototypical GluN2A-NMDA receptor antagonists TCN-201 and its MPX-analogs adopt a U-shaped conformation within the binding pocket, paracyclophanes were designed containing the phenyl rings in an already parallel orientation. Docking studies of the designed paracyclophanes show a similar binding pose as TCN-201. [2.2]Paracyclophanes with a benzoate or benzamide side chain were prepared in four-step synthesis, respectively, starting with a radical bromination in benzylic 1-position of [2.2]paracyclophane. In two-electrode voltage clamp experiments using Xenopus laevis oocytes transfected with cRNAs for the GluN1-4a and GluN2A subunits, the esters and amides (conc. 10 μM) did not show considerable inhibition of ion flux. It can be concluded that the GluN2A-NMDA receptor does not accept ligands with a paracyclophane scaffold functionalized in benzylic 1-position, although docking studies had revealed promising binding poses for benzoic acid esters and benzamides.

Zn2+ inhibits spatial memory and hippocampal place cell representation through high-affinity binding to the NMDA receptor GluN2A subunit

A subset of glutamatergic neurons in the forebrain uses labile Zn2+ as a co-transmitter alongside glutamate. Synaptic Zn2+ plays a key role in learning and memory processes, but its mechanisms of action remain poorly understood. Here, we used a knock-in (KI) mouse line carrying a point mutation at the GluN2A Zn2+ binding site that selectively eliminates zinc inhibition of NMDA receptors. Ablation of Zn2+-GluN2A binding improves spatial memory retention and contextual fear memory formation. Electrophysiological recording of hippocampal neurons in the CA1 area revealed a greater proportion of place cells and substantial place field remapping in KI mice compared to wildtype littermates. Persistent place cell remapping was also seen in KI mice upon repeated testing suggesting an enhanced ability to maintain a distinct representation across multiple overlapping experiences. Together, these findings reveal an original molecular mechanism through which synaptic Zn2+ negatively modulates spatial cognition by dampening GluN2A-containing NMDA receptor signaling.

The Promising Epigenetic Regulators for Refractory Epilepsy: An Adventurous Road Ahead.

The attribution of seizure freedom is yet to be achieved for patients suffering from refractory epilepsy, e.g. DravetSyndrome (DS). The confined ability of mono-chemical entity-based antiseizure drugs (ASDs) to act directly at genomic level is one of the factors, combined with undetermined seizure triggers lead to recurrent seizure (RS) in DS, abominably affecting the sub-genomic architecture of neural cells. Thus, the RS and ASD appear to be responsible for the spectrum of exorbitant clinical pathology. The RS distresses the 5-HT-serotonin pathway, hypomethylates genes of CNS, and modulates the microRNA (miRNA)/long non-coding RNA (lncRNA), eventually leading to frozen molecular alterations. These changes shall be reverted by compatible epigenetic regulators (EGR) like, miRNA and lncRNA from Breast milk (BML) andBacopa monnieri(BMI). The absence of studious seizure inSCN1Amutation-positivebabies for the first 6 monthsraises the possibility that the consequences of mutation in SCN1A are subsidized by EGRs from BML. EGR-dependent-modifier gene effect is likely imposed by the other members of the SCN family. Therefore, we advocate that miRNA/lncRNA from BML and bacosides/miRNA from BMI buffer the effect of SCN1Amutation by sustainably maintaining modifier gene effect in the aberrant neurons. The presence of miRNA-155-5p, -30b-5p, and -30c-5p family in BML and miR857, miR168, miR156, and miR158 in BMI target at regulatingSCNfamily andCLCN5as visualized by Cystoscope. Thus, we envisage that the possible effects of EGR might include (a) upregulating the haploinsufficient SCN1A strand, (b) down-regulating seizure-elevated miRNA, (c) suppressing the seizure-induced methyltransferases, and (d) enhancing the GluN2A subunit of NMDA receptor to improve cognition. The potential of these EGRs from BML and BML is to further experimentally strengthen, long-haul step forward in molecular therapeutics.

Disrupted Maturation of Prefrontal Layer 5 Neuronal Circuits in an Alzheimer's Mouse Model of Amyloid Deposition.

Mutations in genes encoding amyloid precursor protein (APP) and presenilins (PSs) cause familial forms of Alzheimer's disease (AD), a neurodegenerative disorder strongly associated with aging. It is currently unknown whether and how AD risks affect early brain development, and to what extent subtle synaptic pathology may occur prior to overt hallmark AD pathology. Transgenic mutant APP/PS1 over-expression mouse lines are key tools for studying the molecular mechanisms of AD pathogenesis. Among these lines, the 5XFAD mice rapidly develop key features of AD pathology and have proven utility in studying amyloid plaque formation and amyloid β (Aβ)-induced neurodegeneration. We reasoned that transgenic mutant APP/PS1 over-expression in 5XFAD mice may lead to neurodevelopmental defects in early cortical neurons, and performed detailed synaptic physiological characterization of layer 5 (L5) neurons from the prefrontal cortex (PFC) of 5XFAD and wild-type littermate controls. L5 PFC neurons from 5XFAD mice show early APP/Aβ immunolabeling. Whole-cell patch-clamp recording at an early post-weaning age (P22-30) revealed functional impairments; although 5XFAD PFC-L5 neurons exhibited similar membrane properties, they were intrinsically less excitable. In addition, these neurons received smaller amplitude and frequency of miniature excitatory synaptic inputs. These functional disturbances were further corroborated by decreased dendritic spine density and spine head volumes that indicated impaired synapse maturation. Slice biotinylation followed by Western blot analysis of PFC-L5 tissue revealed that 5XFAD mice showed reduced synaptic AMPA receptor subunit GluA1 and decreased synaptic NMDA receptor subunit GluN2A. Consistent with this, patch-clamp recording of the evoked L23>L5 synaptic responses revealed a reduced AMPA/NMDA receptor current ratio, and an increased level of AMPAR-lacking silent synapses. These results suggest that transgenic mutant forms of APP/PS1 overexpression in 5XFAD mice leads to early developmental defects of cortical circuits, which could contribute to the age-dependent synaptic pathology and neurodegeneration later in life.

Knockdown of astrocytic Grin2a exacerbated sleep deprivation-induced cognitive impairments and elevation of amyloid-beta

Sleep disorders are associated with cognitive impairments, greater amyloid-β (Aβ) burden and increased risk of developing Alzheimer’s disease, while the underlying mechanism is unclear. N-methyl-d-aspartate receptors (NMDARs), as vital modulators of cognition, are sensitive to sleep disturbance. Sleep deprivation (SD) could induce the alterations of neuronal NMDAR subunits expression, however the alterations of astrocytic NMDARs in SD have not been reported. Our previous study has demonstrated knockdown of astrocytic Grin2a (gene encoding NMDAR subunit GluN2A) could aggravate Aβ-induced cognitive impairments, but what role astrocytic GluN2A may play in SD is unknown. Here we focused on the changes and roles of hippocampal astrocytic GluN2A in SD. Our results showed SD increased the expression of astrocytic GluN2A. Specific knockdown of hippocampal astrocytic Grin2a aggravated SD-induced cognitive decline, elevated Aβ, and attenuated the SD-induced increase in autophagy flux. Our finding, for the first time, revealed a novel neuroprotective role for astrocytic GluN2A in SD, which may be helpful for developing new preventive and therapeutic targets to sleep disorders.

Changes in NMDA Receptor Function in Rapid Ischemic Tolerance: A Potential Role for Tri-Heteromeric NMDA Receptors.

In this study, we characterize biophysical changes in NMDA receptor function in response to brief non-injurious ischemic stress (ischemic preconditioning). Electrophysiological studies show NMDA receptor function is reduced following preconditioning in cultured rat cortical neurons. This functional change is not due to changes in the reversal potential of the receptor, but an increase in desensitization. We performed concentration–response analysis of NMDA-evoked currents, and demonstrate that preconditioned neurons show a reduced potency of NMDA to evoke currents, an increase in Mg2+ sensitivity, but no change in glycine sensitivity. Antagonists studies show a reduced inhibition of GluN2B antagonists that have an allosteric mode of action (ifenprodil and R-25-6981), but competitive antagonists at the GluR2A and 2B receptor (NVP-AMM077 and conantokin-G) appear to have similar potency to block currents. Biochemical studies show a reduction in membrane surface GluN2B subunits, and an increased co-immunoprecipitation of GluN2A with GluN2B subunits, suggestive of tri-heteromeric receptor formation. Finally, we show that blocking actin remodeling with jasplakinolide, a mechanism of rapid ischemic tolerance, prevents NMDA receptor functional changes and co-immunoprecipitation of GluN2A and 2B subunits. Together, this study shows that alterations in NMDA receptor function following preconditioning ischemia are associated with neuroprotection in rapid ischemic tolerance.

Activation of non-classical NMDA receptors by glycine impairs barrier function of brain endothelial cells

Blood–brain barrier (BBB) integrity is necessary to maintain homeostasis of the central nervous system (CNS). NMDA receptor (NMDAR) function and expression have been implicated in BBB integrity. However, as evidenced in neuroinflammatory conditions, BBB disruption contributes to immune cell infiltration and propagation of inflammatory pathways. Currently, our understanding of the pathophysiological role of NMDAR signaling on endothelial cells remains incomplete. Thus, we investigated NMDAR function on primary mouse brain microvascular endothelial cells (MBMECs). We detected glycine-responsive NMDAR channels, composed of functional GluN1, GluN2A and GluN3A subunits. Importantly, application of glycine alone, but not glutamate, was sufficient to induce NMDAR-mediated currents and an increase in intracellular Ca2+ concentrations. Functionally, glycine-mediated NMDAR activation leads to loss of BBB integrity and changes in actin distribution. Treatment of oocytes that express NMDARs composed of different subunits, with GluN1 and GluN3A binding site inhibitors, resulted in abrogation of NMDAR signaling as measured by two-electrode voltage clamp (TEVC). This effect was only detected in the presence of the GluN2A subunits, suggesting the latter as prerequisite for pharmacological modulation of NMDARs on brain endothelial cells. Taken together, our findings argue for a novel role of glycine as NMDAR ligand on endothelial cells shaping BBB integrity.

P140Cap Regulates the Composition and Localization of the NMDAR Complex in Synaptic Lipid Rafts.

The NMDARs are key players in both physiological and pathologic synaptic plasticity because of their involvement in many aspects of neuronal transmission as well as learning and memory. The contribution in these events of different types of GluN2A-interacting proteins is still unclear. The p140Cap scaffold protein acts as a hub for postsynaptic complexes relevant to psychiatric and neurologic disorders and regulates synaptic functions, such as the stabilization of mature dendritic spine, memory consolidation, LTP, and LTD. Here we demonstrate that p140Cap directly binds the GluN2A subunit of NMDAR and modulates GluN2A-associated molecular network. Indeed, in p140Cap KO male mice, GluN2A is less associated with PSD95 both in ex vivo synaptosomes and in cultured hippocampal neurons, and p140Cap expression in KO neurons can rescue GluN2A and PSD95 colocalization. p140Cap is crucial in the recruitment of GluN2A-containing NMDARs and, consequently, in regulating NMDARs' intrinsic properties. p140Cap is associated to synaptic lipid-raft (LR) and to soluble postsynaptic membranes, and GluN2A and PSD95 are less recruited into synaptic LR of p140Cap KO male mice. Gated-stimulated emission depletion microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in LR in an activity-dependent fashion. In the synaptic compartment, p140Cap influences the association between GluN2A and PSD95 and modulates GluN2A enrichment into LR. Overall, such increase in these membrane domains rich in signaling molecules results in improved signal transduction efficiency.SIGNIFICANCE STATEMENT Here we originally show that the adaptor protein p140Cap directly binds the GluN2A subunit of NMDAR and modulates the GluN2A-associated molecular network. Moreover, we show, for the first time, that p140Cap also associates to synaptic lipid rafts and controls the selective recruitment of GluN2A and PSD95 to this specific compartment. Finally, gated-stimulated emission depletion microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in lipid rafts in an activity-dependent fashion. Overall, our findings provide the molecular and functional dissection of p140Cap as a new active member of a highly dynamic synaptic network involved in memory consolidation, LTP, and LTD, which are known to be altered in neurologic and psychiatric disorders.

Memantine ameliorates the impairment of social behaviors induced by a single social defeat stress as juveniles

Clinically, juveniles are more sensitive to stress than adults, and exposure to stress as juveniles prolongs psychiatric symptoms and causes treatment resistance. However, the efficacy of antidepressants for juveniles with psychiatric disorders is unknown. In the present study, we investigated whether the expression or development of impaired social behavior was attenuated by memantine, a non-competitive NMDA receptor antagonist. In addition, we clarified the molecular mechanisms related to intracellular signal transduction through NMDA receptors and the ameliorating effect of memantine in mice with impaired social behavior. Acute administration of memantine before the social interaction test, but not before exposure to social defeat stress, attenuated social behavioral impairment. A single social defeat stress increased the phosphorylation of NMDA receptor subunit GluN2A and extracellular-signal-related kinase 1/2 (ERK1/2). Memantine inhibited the increase of phosphorylated GluN2A and ERK1/2 resulting from social interaction behavior. In both GluN2A deficient and pharmacological blockaded mice, social behavioral impairment was not observed in the social interaction test through regulation of ERK1/2 phosphorylation. These findings suggest that memantine ameliorates social behavioral impairment in mice exposed to a single social defeat stress as juveniles by regulating the NMDA receptor and subsequent ERK1/2 signaling activation. Memantine may constitute a novel therapeutic drug for stress-related psychiatric disorders in juveniles with adverse juvenile experiences.

Sevoflurane Exposure in the Developing Brain Induces Hyperactivity, Anxiety-Free, and Enhancement of Memory Consolidation in Mice.

BackgroundSevoflurane exposure at brain developmental stages has been reported to induce neurotoxicity and, subsequently, results in learning deficits at the juvenile age. In this study, we aimed to investigate the effects of prior early-age sevoflurane exposure on locomotor activity, anxiety, CA1-dependent learning, and spatial memory, as well as synapse changes in mice.MethodsTotally, 3% sevoflurane was given to neonatal mice at postnatal day 7 for 4 h. These sevoflurane-treated mice were later subjected to open field and Morris water maze tests at their adult age (postnatal days 60–90) to assess their motor activity and spatial learning ability, respectively. The brain slices of sevoflurane-treated and control mice were examined for dendritic spine density and long-term potentiation (LTP) features following behavior tests (postnatal day 60). Protein levels of N-methyl-D-aspartate (NMDA) receptor subtypes and PSD95 in brain lysate were measured by using immunoblotting at the same age (postnatal day 60).ResultsPrior early-age sevoflurane exposure increased the overall moving distance, prolonged the central-area lingering time, and increased the central-area entries of adult mice. Sevoflurane-treated mice spent more time in the target quadrant during the probe test. An increase of the spine density of pyramidal neurons in the CA1 region was observed in sevoflurane-treated mice. NMDA receptor GluN2A subunit, but not the GluN2B or PSD95, was increased in the brain lysate of sevoflurane-treated mice compared with that of control mice. LTP in the hippocampus did not significantly differ between sevoflurane-treated and control mice.ConclusionExposure to sevoflurane for mice during an early brain developmental stage (P7) induces later-on hyperactivity, anxiety-free, and enhancement of memory retention. These observations shed light on future investigations on the underlying mechanisms of sevoflurane’s effect on neuronal development.

Synaptic Dysfunction by Mutations in GRIN2B: Influence of Triheteromeric NMDA Receptors on Gain-of-Function and Loss-of-Function Mutant Classification.

GRIN2B mutations are rare but often associated with patients having severe neurodevelopmental disorders with varying range of symptoms such as intellectual disability, developmental delay and epilepsy. Patient symptoms likely arise from mutations disturbing the role that the encoded NMDA receptor subunit, GluN2B, plays at neuronal connections in the developing nervous system. In this study, we investigated the cell-autonomous effects of putative gain- (GoF) and loss-of-function (LoF) missense GRIN2B mutations on excitatory synapses onto CA1 pyramidal neurons in organotypic hippocampal slices. In the absence of both native GluN2A and GluN2B subunits, functional incorporation into synaptic NMDA receptors was attenuated for GoF mutants, or almost eliminated for LoF GluN2B mutants. NMDA-receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) from synaptic GoF GluN1/2B receptors had prolonged decays consistent with their functional classification. Nonetheless, in the presence of native GluN2A, molecular replacement of native GluN2B with GoF and LoF GluN2B mutants all led to similar functional incorporation into synaptic receptors, more rapidly decaying NMDA-EPSCs and greater inhibition by TCN-201, a selective antagonist for GluN2A-containing NMDA receptors. Mechanistic insight was gained from experiments in HEK293T cells, which revealed that GluN2B GoF mutants slowed deactivation in diheteromeric GluN1/2B, but not triheteromeric GluN1/2A/2B receptors. We also show that a disease-associated missense mutation, which severely affects surface expression, causes opposing effects on NMDA-EPSC decay and charge transfer when introduced into GluN2A or GluN2B. Finally, we show that having a single null Grin2b allele has only a modest effect on NMDA-EPSC decay kinetics. Our results demonstrate that functional incorporation of GoF and LoF GluN2B mutants into synaptic receptors and the effects on EPSC decay times are highly dependent on the presence of triheteromeric GluN1/2A/2B NMDA receptors, thereby influencing the functional classification of NMDA receptor variants as GoF or LoF mutations. These findings highlight the complexity of interpreting effects of disease-causing NMDA receptor missense mutations in the context of neuronal function.

GluN3A excitatory glycine receptors control adult cortical and amygdalar circuits

SummaryGluN3A is an atypical glycine-binding subunit of NMDA receptors (NMDARs) whose actions in the brain are mostly unknown. Here, we show that the expression of GluN3A subunits controls the excitability of mouse adult cortical and amygdalar circuits via an unusual signaling mechanism involving the formation of excitatory glycine GluN1/GluN3A receptors (eGlyRs) and their tonic activation by extracellular glycine. eGlyRs are mostly extrasynaptic and reside in specific neuronal populations, including the principal cells of the basolateral amygdala (BLA) and SST-positive interneurons (SST-INs) of the neocortex. In the BLA, tonic eGlyR currents are sensitive to fear-conditioning protocols, are subject to neuromodulation by the dopaminergic system, and control the stability of fear memories. In the neocortex, eGlyRs control the in vivo spiking of SST-INs and the behavior-dependent modulation of cortical activity. GluN3A-containing eGlyRs thus represent a novel and widespread signaling modality in the adult brain, with attributes that strikingly depart from those of conventional NMDARs.