DNMT3a-mediated GluN2B desilencing in the anterior cingulate cortex underlies chemotherapy-induced neuropathic pain.

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders associated with genetic mutations, including some in GRIN genes encoding NMDA receptor (NMDA-R) subunits. Despite affecting the same receptor, each mutation may lead to distinct neurological disorders, emphasizing the necessity of understanding receptor dysfunction to tailor treatments effectively. In a genetic screen of DEEs patients, we identified a de novo pathogenic GRIN2B nonsense mutation, p.Glu839Ter (called GluN2B-E839*), which truncates the C-terminal domain (CTD) of the GluN2B subunit, a poorly characterized region critical for receptor function. This variant was prioritized for functional study due to the limited knowledge about the CTD's role. We fully characterized the clinical presentation of the patient, who displayed intellectual disability, epilepsy, and hyperkinetic behavioral disorders. Molecular and cellular analyses in heterologous systems and patient-derived neurons revealed that GluN2B-E839* subunit assembles correctly with other subunits to form NMDA-Rs but exhibits reduced surface expression, impaired interactions with PSD95, and altered biophysical properties, including reduced current amplitudes, increased magnesium sensitivity, and diminished calcium influx. These dysfunctions likely contribute to impaired synaptic plasticity and DEEs pathophysiology. Our findings highlight the critical role of the GluN2B CTD in NMDA-R function and neuronal signaling and underscore the need for systematic characterization of GRIN variants to improve diagnostic precision and therapeutic targeting for DEEs. This study establishes a robust framework combining complementary experimental approaches with patient-derived preclinical models to link molecular dysfunctions to clinical phenotypes.

GLP-1 receptor agonism reduces PTSD-like anxiety and alters amygdala-hippocampal activity patterns in a Chemogenetic mouse model.

The hippocampus exhibits marked structural, functional, and molecular heterogeneity. GluN3A, a non-classical N-methyl-D-aspartate receptor subunit enriched in hippocampal CA1, has a poorly understood role in stress-related behavior. Here, using chronic social defeat stress (CSDS), GluN3A knockout mice, and behavioral, molecular, genetic, pharmacological, and circuit analyses, we investigated the role of hippocampal GluN3A in stress coping. CSDS selectively reduced GluN3A expression in intermediate CA1 (CA1i), and both stress-induced and genetic GluN3A loss promoted passive coping, whereas CA1i-specific GluN3A overexpression reversed this phenotype. c-Fos staining, fiber photometry, and chemogenetic manipulations showed that GluN3A is required for CA1i pyramidal neuron activation during coping. In addition, local D-serine administration rapidly shifted coping from passive to active in a GluN3A-dependent manner. Circuit tracing showed that CA1i neurons project broadly, and GluN3A overexpression selectively enhanced downstream activity in CA3 and infralimbic cortex. Together, these findings identify GluN3A in CA1i as a key regulator of stress-coping behavior.

Our earlier human studies identified NRSN2 (Neurensin-2), a neuronal-specific vesicular protein, as a candidate gene contributing to 20p13 microdeletion syndrome, yet the functional consequences of NRSN2 deficiency in the nervous system remain poorly understood. To explore the role of Nrsn2 in neurodevelopment and cognitive function, we utilized previously generated homozygous Nrsn2 knockout mice (Nrsn2-/-) and performed a series of behavioral, morphological, and electrophysiological analyses. Behaviorally, Nrsn2-/- mice exhibited mild locomotor impairment, as assessed by gait analysis at 4 and 8 weeks of age, as well as significant deficits in spatial learning and memory (Morris water maze) and fear memory (passive avoidance test) at 8 weeks. Morphometric analysis suggested no overt alterations in dendritic complexity or spine density in hippocampal CA1 pyramidal neurons or cerebellar Purkinje cells without developmental malformation. Electrophysiological recordings and immunoblotting analyses may reflect region-specific synaptic alterations. In the hippocampus, expression levels of the NMDA receptor subunits GluN1 and GluN2A were reduced at 4 weeks of age. Consistently, CA1 pyramidal neurons displayed decreased sEPSC frequency with unchanged amplitude under the conditions examined. In addition, an imbalance in hippocampal excitatory/inhibitory transmission was observed, as reflected by altered sEPSC frequency in the absence of changes in sIPSC frequency. In cerebellar Purkinje cells, GluA1-containing AMPA receptors were selectively downregulated, accompanied by reduced frequency and amplitude of sEPSCs and a selective decrease in sIPSC frequency, indicating both excitatory and inhibitory synaptic dysfunction in this region. Collectively, these findings indicate that Nrsn2 deficiency is accompanied by altered excitatory synaptic transmission and reduced long-term potentiation (LTP) at 8 weeks of age, despite preserved dendritic architecture as assessed by Golgi staining. These synaptic and plasticity deficits occur alongside the observed cognitive and motor impairments in Nrsn2⁻/⁻ mice. This study provides a descriptive phenotypic characterization of Nrsn2 deficiency and offers initial insights into the neurobiological role of NRSN2 and its contribution to neurodevelopment, learning, and memory.

Sex differences in hippocampal and prefrontal gene expression of endocannabinoid, glutamatergic and GABAergic systems and amino acids profile underlie cocaine plus ethanol seeking incubation.

Schizophrenia is a chronic mental disorder characterized by abnormal synaptic connectivity. N-methyl-D-aspartate receptors (NMDARs) are essential for synaptic transmission and plasticity, and rare variants in the genes encoding NMDARs are linked to neurodevelopmental disorders. In this study, we examined rare pathogenic mutations in four GRIN2 genes, which encode the NMDAR subunit 2, in patients with schizophrenia using ion semiconductor technology and PCR-based fluorescence-based cycle sequencing. We identified 10 novel ultra-rare missense mutations in the GRIN2 genes, none of which were found in the GnomAD_genomes and the Taiwan BioBank. These findings suggest that ultra-rare pathogenic mutations may be present in some individuals with schizophrenia, supporting the existence of rare coding variants of synapse-associated genes that contribute to the genetic architecture of schizophrenia. Future research should explore the in-vitro and in-vivo effects of the identified mutations on the pathophysiology of schizophrenia for further insights.

Neuronal cilia have emerged as crucial signaling hubs, yet their molecular composition and integration with synaptic communication remain poorly understood. Using a newly developed Arl13b-TurboID mouse model, we achieved robust cilia-specific biotinylation and proteomic profiling across diverse tissues and cell types. Comparative proteomics revealed notable tissue-specific specialization, with neuronal cilia uniquely enriched in synaptic proteins, adhesion molecules, and neurotransmitter receptors. Unexpectedly, several signaling and adhesion molecules localize to neuronal cilia in discrete nanodomains maintained by active retrieval mechanisms. In the mouse cortex, expansion microscopy revealed that the NMDA receptor subunit GluN1 is organized in nanodomains on neuronal ciliary membranes, which are precisely positioned to sample neurotransmitter efflux from neighboring glutamatergic synapses. These findings establish neuronal cilia as specialized extrasynaptic signaling platforms, with nanoscale organization enabling them to integrate local synaptic cues and modulate neuronal connectivity.

Neural control of innate immunity must balance restraint of basal immune activity with rapid activation upon pathogen encounter. Glutamate, the primary excitatory neurotransmitter in the nervous system, has been implicated in several neurological disorders associated with inflammation, suggesting a potential link to immune regulation. However, how glutamatergic signaling contributes to immune balance remains unknown. Here, we demonstrated that the NMDA-type ionotropic glutamate receptor subunit NMR-2, a component of the NMDA receptor complex, acts in theC. elegansnervous system as a key regulator of pathogen-induced immune responses. Loss ofnmr-2enhanced resistance toStaphylococcus aureusandPseudomonas aeruginosa, without elevating basal immune gene expression. The pathogen-induced response controlled by NMR-2 required the conserved PMK-1/p38 MAPK, DAF-16/FOXO, and HLH-30/TFEB pathways. We identified the AVD interneuron as the site of action of NMR-2, where it integrates inputs from upstream sensory neurons ASE, ASK, AQR, and PQR. These findings uncover a neural circuit in which glutamatergic signaling distinguishes basal immune gene expression from pathogen-induced immune responses, revealing a mechanism that promotes effective defense without altering baseline immunity.

Chaihu Shugan San (CSS) is a classical traditional Chinese medicine formula widely used in improving depression. The purpose of this study was to investigate the molecular mechanisms underlying the antidepressant effects of CSS. Behavioral experiments and ELISA were employed to assess depressive-like behaviors and measure serum ACTH and CORT levels in chronic unpredictable mild stress (CUMS) rats. Western blotting, immunofluorescence, and Co-Immunoprecipitation were used to analyze α2δ-1 and NMDAR subunit NR1 expression and interactions in the hypothalamus. Cacna2d1 gene knockout and a disruptive peptide (α2δ-1 Tat) were applied in vitro to investigate the α2δ-1-NR1 complex. CSS ameliorated depressive-like behaviors in CUMS rats and decreased serum levels of ACTH and CORT. CSS reversed the upregulated expression of α2δ-1 and NR1 in the hypothalamus of CUMS rats, weakening the α2δ-1-NR1 complex binding. When the Cacna2d1 gene was knocked out or the α2δ-1-NR1 complex was disrupted with α2δ-1 Tat peptide, CSS-containing serum failed to significantly enhance NR1 expression in PC12 cells. CSS ameliorates depressive-like behaviors in CUMS rats by interfering with the α2δ-1-NR1 complex, reducing the expression levels of α2δ-1 and NR1 in the hypothalamus, and consequently inhibiting the excessive excitability of the hypothalamic-pituitary-adrenal axis.

Neuroexcitotoxicity mediated by NMDA receptor is a central contributor to ischemic stroke pathology. Neuroprotective effect and mechanism of the new compound GW201, which targets NMDA receptor, were studied. We evaluated the neuroprotective effects of GW201 using in vivo and in vitro models of ischemic stroke, including the middle cerebral artery occlusion/reperfusion (MCAOi/r) model, permanent MCAO (pMCAO) model and primary neuronal oxygen-glucose deprivation (OGD) model. Pharmacodynamic studies examined the dose-response relationship and therapeutic time window of GW201 neuroprotective effects. Electrophysiological membrane clamp experiments assessed the NMDA receptor subunit selectivity of GW201 and its influence on neuronal currents under ischemia-mimicking conditions. The mechanism of its action was further analysed using molecular docking technology. Live-cell fluorescence imaging was employed to evaluate the impact of GW201 on intracellular calcium levels in simulated ischemic stroke. Furthermore, bioinformatics analysis of the GEO database identified 16 significant calcium-related genes, with their regulation by GW201 validated in the MCAOi/r model. Across all models tested, GW201 exhibited marked neuroprotective effects. Membrane clamp studies identified GW201 as an allosteric modulator of the NMDA receptor GluN2A subunit reducing current amplitude under high glutamate/NMDA + glycine conditions and subsequently lowering intracellular calcium levels. Bioinformatics analysis revealed 16 calcium-related genes, with GW201 significantly modulating Ccl3, Stat3, Anxa1, Anxa2, Mgp, S100A8, S100A9 and Cacna1a. These results suggest that the neuroprotective effects of GW201 are mediated by its ability to allosterically modulate NMDA receptor activity, reducing calcium overload, influence downstream gene expression and suppress inflammatory responses.

The Arachidonic Acid (AA)-NMDA Receptor Axis describes a crucial neurochemical pathway where glutamate activation of NMDA receptors releases AA. AA binds directly to NMDA receptor subunits, increasing their open probability and also affecting other receptors and cellular processes, linking neuronal activity to lipid signalling.

Zika virus (ZIKV) infection is associated with severe neurodevelopmental disorders. However, the molecular mechanisms involved in the imbalance of excitatory and inhibitory neurotransmitter systems remain poorly understood. In this study, we evaluated the expression of components of the GABA–glutamate system in neonatal BALB/c mice inoculated with ZIKV at 10 days post-infection (dpi). Analysis of GABA-A and NMDA receptors revealed widespread downregulation of GABA-A and NMDA receptor subunits in the cerebral cortex and cerebellum, except for the alpha-5 and epsilon GABA-A subunits, which were upregulated in the cerebellum. Infected mice also showed increased GABA immunoreactivity and glutamate loss. The enzymes involved in neurotransmitter synthesis or transport confirmed these findings when assessed by qRT-PCR or Western blot, revealing increased expression of GAD-65/67, accompanied by a loss of glutamate dehydrogenase (GLUD) in the cerebral cortex and cerebellum, along with decreased expression of the glutamate transporter (VGLUT) in the cerebral cortex. These findings suggest that GABA synthesis increases during ZIKV infection, creating a neurochemical environment that favors viral replication and contributes to the migration and synaptic defects observed in congenital Zika syndrome.

Despite being essential mediators of pain processing, the molecular identity of N-methyl-D-aspartate receptor (NMDAR) subtypes in nociceptive dorsal horn circuits is poorly understood, especially between sexes and in humans. Given the importance of GluN2 subunits in shaping NMDAR function and plasticity, we investigated the expression and localization of specific GluN2 NMDAR variants in the dorsal horn of viable spinal cord tissue from male and female rodents and human organ donors. Analysis of single-cell/nuclei sequencing datasets and quantitative reverse transcriptase polymerase chain reactions (qRT-PCR) revealed that the GluN2A (GRIN2A) and GluN2B (GRIN2B) subunits are robustly expressed in dorsal horn neurons of mice, rats and humans, with moderate expression of GluN2D (GRIN2D). Immunohistochemistry (IHC) with antigen retrieval demonstrated that GluN2A, GluN2B, and GluN2D proteins are all preferentially localized to the superficial dorsal horn of both adult rats and humans, which is conserved between males and females. Surprisingly, we found that these GluN2 NMDAR subunits are enriched in the lateral superficial dorsal horn in rats but not in humans, while presynaptic and neuronal markers are symmetrically distributed across the rat mediolateral axis. A dramatic shift in localization of GluN2A to the lateral superficial dorsal horn was observed across later postnatal development (PD21-PD90) in both male and female rats, with a corresponding change in synaptic NMDAR currents. This discovery of changes in NMDAR subunit distribution during maturation and between species will shed light on the physiological roles of NMDARs and their potential as therapeutic targets for pain.Significance statement We used complementary single-cell/nuclei analysis, immunostaining, quantitative reverse transcriptase polymerase chain reactions, RNAscope in situ hybridization, and electrophysiological approaches to compare the relative expression of N-methyl-D-aspartate receptor (NMDAR) GluN2 subunits in dorsal horn spinal cord pain circuits of mouse, rat, and human spinal cord tissue. Through these comparisons, we find that the transcripts and proteins of the GluN2A, GluN2B, and GluN2D NMDAR subunits are robustly expressed in superficial dorsal horn neurons, with conserved expression across sex but important differences in expression and localization patterns across late development and between species. These discoveries shed light on the physiological roles of NMDARs and their utility as potential therapeutic targets for pain.

Aldehyde dehydrogenase 1A1-positive (ALDH1A1+) dopaminergic neurons (DANs) are preferentially vulnerable in Parkinson’s disease (PD), yet how their activity is modulated by presynaptic inputs remains poorly defined. Here we investigated the role of glutamatergic input by conditionally deleting Grin1, which encodes a critical NMDA receptor (NMDAR) subunit, in ALDH1A1+ DANs. Grin1 conditional knockout (cKO) mice displayed normal locomotion and motor learning; however, females exhibited enhanced operant reward acquisition and excessive feeding with transient weight gain following food restriction. To determine regional contributions, Grin1 was selectively knocked down in ALDH1A1+ DANs of either the ventral tegmental area (VTA) or substantia nigra pars compacta (SNc). VTA-specific knockdown in females was sufficient to reproduce the post-restriction feeding and weight gain phenotype. Bulk whole-brain mRNA sequencing revealed pronounced sex-dependent transcriptional changes, primarily in female Grin1 cKO mice after food restriction. Many differentially expressed genes were associated with mitochondrial function, energy metabolism, and synaptic signaling. Together, these findings reveal a sex-specific role for NMDAR–mediated glutamatergic input to ALDH1A1+ VTA DANs in regulating feeding behavior, providing mechanistic insight into how dysfunction of this vulnerable subpopulation may contribute to PD-associated compulsive eating disorders.

Pheromone signaling is pivotal in driving the social and reproductive behaviors of rodents. Learning and memorizing the pheromone locations involve olfactory subsystems. To study the neural basis of this behavior, we trained female heterozygous knockouts of GluA2 (AMPAR subunit) and NR1 (NMDAR subunit), targeting GAD65 interneuron population, in a pheromone location learning assay. We observed impaired memory of pheromone locations on early and late recall periods, pointing towards the possible role of ionotropic glutamate receptors (iGluRs) and thereby the synaptic inhibition in pheromone location learning. Correlated changes were observed in the expression levels of activity-regulated cytoskeletal (Arc) protein, which is critical for memory consolidation, in the associated brain areas. Further, to probe the involvement of the main and accessory olfactory bulbs (MOB and AOB) in pheromone location learning, we knocked out GluA2 and NR1 from MOB and/or AOB neuronal circuits by stereotaxic injection of Cre-dependent AAV5 viral particles. Perturbing the inhibitory circuits of MOB and AOB or AOB-alone resulted in pheromone location memory deficits. These results confirm the role of iGluRs and the synaptic inhibition exerted by the interneuron network of AOB in regulating learning and memory of pheromone locations.

Stress-induced neuroinflammation and synaptic dysregulation: Linking HPA axis to glutamate and NMDA receptors.

Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is an autoimmune disorder characterized by antibodies against the NR1 subunit of the NMDA receptor. It predominantly affects young women and is frequently associated with ovarian teratomas. Early recognition, tumor removal, and immunotherapy are critical for favorable outcomes. We report a 33-year-old woman with subacute headache, low-grade fever, generalized weakness, and mood disturbances, who developed generalized tonic-clonic seizures and cognitive impairment. Brain MRI and CSF were initially normal, and EEG showed diffuse slowing without epileptiform discharges. Pelvic imaging revealed a large right ovarian mass. She was treated with corticosteroids, plasmapheresis, and ultimately rituximab, alongside right salpingo-oophorectomy. Histopathology confirmed an immature teratoma. Anti-NMDAR antibodies were later detected in CSF, confirming the diagnosis. Following tumor removal and immunotherapy, she achieved complete neurological recovery and remained seizure-free at six months. This case highlights the diagnostic challenges of anti-NMDAR encephalitis, particularly when MRI and CSF are normal. Early tumor detection and removal, combined with prompt escalation to second-line immunotherapy when needed, are essential for favorable outcomes. Immature ovarian teratomas, although rare, can trigger severe disease and warrant careful evaluation in adult women presenting with acute neuropsychiatric symptoms.

Prenatal exposure to particulate matter impairs offspring behaviorvia hippocampal NMDA receptor reduction: An in vivo and ex vivostudy

Context-Dependent NMDA Receptor Dysfunction Predicts Seizure Treatment in Mice with Human GluN1 Variant Venkatesan S, Nazarkina D, Sullivan MT, Tan YF, Qu S, Ramsey AJ, Lambe EK.. iScience . 2025;29(1):114301. doi: 10.1016/j.isci.2025.114301. PMID: 41602908; PMCID: PMC12834114. Mutations in N-methyl-D-aspartate receptors (NMDARs) cause epilepsy and profound cognitive impairment, though the underlying subunit-specific vulnerabilities remain unclear. We investigate the impact of a severe human variant in the lurcher motif of obligate GluN1 NMDAR subunit using transgenic mice, revealing unexpected context-dependent phenotypes. We show that the GluN1 Y647S variant significantly reduces current flow through pharmacologically isolated synaptic NMDARs in prefrontal neurons. Yet in intact local circuits, this loss-of-function paradoxically extends NMDAR-dependent dendritic integration, causing prolonged circuit-wide excitation that promotes seizures. Mutant receptors appear deficient in engaging opposing dendritic ion channels that normally curtail NMDAR-dependent excitation. Boosting SK channel activity normalizes dendritic integration, whereas slight decreases in extracellular magnesium further extend abnormally prolonged integration in mutant mice. We find that magnesium supplementation successfully treats seizures in vivo in the transgenic mice, despite loss-of-function of NMDARs. Overall, we disentangle a GluN1 variant's receptor-level effects and its dendritic impact to treat seizures effectively.