GluN1 Subunit Research Articles

Cocaine preference and neuroadaptations are maintained by astrocytic NMDA receptors in the nucleus accumbens.

Cocaine-associated memories induce cravings and interfere with the ability of users to cease cocaine use. Reducing the strength of cue-drug memories by facilitating extinction may have therapeutic value for the treatment of cocaine addiction. Here, we demonstrate the expression of GluN1/2A/2C NMDA receptor currents in astrocytes in the nucleus accumbens core. Selective ablation of GluN1 subunit from astrocytes in the nucleus accumbens enhanced extinction of cocaine preference memory but did not affect cocaine conditioning or reinstatement. Repeated cocaine exposure up-regulated GluN2C subunit expression and increased astrocytic NMDA receptor currents. Furthermore, intra-accumbal inhibition of GluN2C/2D-containing receptors and GluN2C subunit deletion facilitated extinction of cocaine memory. Cocaine-induced neuroadaptations including dendritic spine maturation and AMPA receptor recruitment were absent in GluN2C knockout mice. Impaired retention of cocaine preference memory in GluN2C knockout mice was restored by exogenous administration of recombinant glypican 4. Together, these results identify a previously unknown astrocytic GluN2C-containing NMDA receptor mechanism underlying maintenance of cocaine preference memory.

Anti-N-Methyl-D-Aspartate Receptor Encephalitis in Pediatrics

Anti-N-methyl-D-aspartate receptor encephalitis (anti-NMDAR) is a severe autoimmune disease caused by the development of immunoglobulin G antibodies against the NMDAR's GluN1 subunit in the cerebrospinal fluid. It is characterized by a wide range of clinical features including psychological manifestations, dyskinesia, and epileptic seizures. Intravenous methylprednisolone, intravenous immunoglobulin, and plasma exchange are the first-line treatments. Early diagnosis, treatment, monitoring, and follow-up of the disease are crucial as it results in favorable prognosis. In the pediatric age group, relapse is possible.

Expression optimization, purification, and biophysical characterization of a GluN2D-containing NMDA receptor

N-methyl-d-aspartate (NMDA) receptors are hetero-tetrameric ion channels typically consisting of two GluN1 and two GluN2 subunits. A GluN2D subunit containing NMDA receptor dysfunction has been implicated in several neurological diseases, including schizophrenia; however, the lack of a purified GluN2D containing NMDA receptor has been a hurdle for structural and biophysical studies. Here, we present expression and purification strategies to generate human GluN2D containing NMDA receptor, confirm its hetero-tetrameric form using fluorescence size exclusion chromatography (FSEC) and evaluated its suitability for structural studies. The purification methodology outlined here will help in the development of GluN2D specific channel modulators and enable structure activity relationship (SAR) studies.

Clonidine ameliorates cerebral ischemia-reperfusion injury by up-regulating the GluN3 subunits of NMDA receptor.

This study aimed to investigate the protective effects of the alpha-2 adrenergic receptor (α2-AR) agonist, clonidine, on the cerebral ischemia-reperfusion (I/R) injury and elaborate the underlying mechanisms. Cerebral I/R model was established by middle cerebral artery occlusion (MCAO) for 2h followed by reperfusion for 4h in adult male SD rats. Saline, clonidine and yohimbine (an α2-AR antagonist) were intraperitoneally administered each day for one week before surgery. Neurological deficit was evaluated just before decapitation. TTC staining was applied for correlation of cerebral infarction volume.HE staining was performed to observe the neuron morphology. Immunohistochemical stainingwas performed to detect the localization and expression of GluN3 proteins. Western blot analysis also was used to detect the expression levels of GluN3 proteins. Our data showed that clonidine ameliorated neurological deficit and reduced the cerebral infarction volume of the rats with cerebral I/R. It is worth noting that treatment with clonidine up-regulated the protein expression of GluN3 in the rats with the cerebral I/R, especially in the cell membrane. Moreover, clonidine also up-regulated the transposition from cytoplasm to cell membrane of GluN3 after cerebral I/R. In addition, yohimbine abolished the neuroprotective effects of clonidine. The results indicated that clonidine played a protective role in cerebral I/R injury through regulation of the protein expression of GluN3 subunits of N-methyl-D-aspartate (NMDA) receptor.

Identification of a Subtype-Selective Allosteric Inhibitor of GluN1/GluN3 NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) are Ca2+-permeable ionotropic glutamate receptors (iGluRs) in the central nervous system and play important roles in neuronal development and synaptic plasticity. Conventional NMDARs, which typically comprise GluN1 and GluN2 subunits, have different biophysical properties than GluN3-containing NMDARs: GluN3-containing NMDARs have smaller unitary conductance, less Ca2+-permeability and lower Mg2+-sensitivity than those of conventional NMDARs. However, there are very few specific modulators for GluN3-containing NMDARs. Here, we developed a cell-based high-throughput calcium assay and identified 3-fluoro-1,2-phenylene bis (3-hydroxybenzoate) (WZB117) as a relatively selective inhibitor of GluN1/GluN3 receptors. The IC50 value of WZB117 on GluN1/GluN3A receptors expressed in HEK-293 cells was 1.15 ± 0.34 μM. Consistently, WZB117 exhibited strong inhibitory activity against glycine-induced currents in the presence of CGP-78608 but only slightly affected the NMDA-, KA- and AMPA-induced currents in the acutely isolated rat hippocampal neurons. Among the four types of endogenous currents, only the first one is primarily mediated by GluN1/GluN3 receptors. Mechanistic studies showed that WZB117 inhibited the GluN1/GluN3A receptors in a glycine-, voltage- and pH-independent manner, suggesting it is an allosteric modulator. Site-directed mutagenesis and chimera construction further revealed that WZB117 may act on the GluN3A pre-M1 region with key determinants different from those of previously identified modulators. Together, our study developed an efficient method to discover modulators of GluN3-containing NMDARs and characterized WZB117 as a novel allosteric inhibitor of GluN1/GluN3 receptors.

Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis

Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as "autoimmune encephalitis" (AE). There are currently 17 known AE syndromes, and all are associated with antibodies against the neuronal cell surface or synaptic proteins. The clinical syndromes are complex and vary according to the type of associated antibody. The best-known of these diseases is anti-N-methyl D-aspartate receptor (NMDAR) encephalitis, which is a prominent neuropsychiatric disorder associated with severe memory and behavioral impairments. The associated antibodies react with the GluN1 subunit of the NMDAR at the N-terminal domain. The approach most frequently used for the discovery and characterization of AE antibodies includes the culture of dissociated, fetal, rodent hippocampal neurons. During the process of antibody characterization, live neurons in culture are exposed to patients' serum or CSF, and the detection of reactivity indicates that the serum or CSF samples of the patient contain antibodies against neuronal surface antigens. Hippocampal cultures can also be used to determine whether the antibodies in patients are potentially pathogenic by examining if they cause structural or functional alterations of the neurons. The level of success of these studies depends on the quality of the cultures and on the protocols used to obtain and detect the reactivity of patient samples. This article provides an optimized protocol for primary cell culture of fetal rat hippocampal neurons combined with immunostaining to determine the presence of antibodies in the serum or CSF of patients. An example of how to examine the potential pathogenic effects of NMDAR antibodies using cultured neurons and calcium imaging is also presented.

Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis.

Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as "autoimmune encephalitis" (AE). There are currently 17 known AE syndromes, and all are associated with antibodies against the neuronal cell surface or synaptic proteins. The clinical syndromes are complex and vary according to the type of associated antibody. The best-known of these diseases is anti-N-methyl D-aspartate receptor (NMDAR) encephalitis, which is a prominent neuropsychiatric disorder associated with severe memory and behavioral impairments. The associated antibodies react with the GluN1 subunit of the NMDAR at the N-terminal domain. The approach most frequently used for the discovery and characterization of AE antibodies includes the culture of dissociated, fetal, rodent hippocampal neurons. During the process of antibody characterization, live neurons in culture are exposed to patients' serum or CSF, and the detection of reactivity indicates that the serum or CSF samples of the patient contain antibodies against neuronal surface antigens. Hippocampal cultures can also be used to determine whether the antibodies in patients are potentially pathogenic by examining if they cause structural or functional alterations of the neurons. The level of success of these studies depends on the quality of the cultures and on the protocols used to obtain and detect the reactivity of patient samples. This article provides an optimized protocol for primary cell culture of fetal rat hippocampal neurons combined with immunostaining to determine the presence of antibodies in the serum or CSF of patients. An example of how to examine the potential pathogenic effects of NMDAR antibodies using cultured neurons and calcium imaging is also presented.

NMDA Receptors in the Lateral Preoptic Hypothalamus Are Essential for Sustaining NREM and REM Sleep.

The lateral preoptic (LPO) hypothalamus is a center for NREM and REM sleep induction and NREM sleep homeostasis. Although LPO is needed for NREM sleep, we found that calcium signals were, surprisingly, highest in REM sleep. Furthermore, and equally surprising, NMDA receptors in LPO were the main drivers of excitation. Deleting the NMDA receptor GluN1 subunit from LPO abolished calcium signals in all cells and produced insomnia. Mice of both sexes had highly fragmented NREM sleep-wake patterns and could not generate conventionally classified REM sleep. The sleep phenotype produced by deleting NMDA receptors depended on where in the hypothalamus the receptors were deleted. Deleting receptors from the anterior hypothalamic area did not influence sleep-wake states. The sleep fragmentation originated from NMDA receptors on GABA neurons in LPO. Sleep fragmentation could be transiently overcome with sleeping medication (zolpidem) or sedatives (dexmedetomidine). By contrast, fragmentation persisted under high sleep pressure produced by sleep deprivation - mice had a high propensity to sleep but woke up. By analyzing changes in delta power, sleep homeostasis (also referred to as “sleep drive”) remained intact after NMDA receptor ablation. We suggest NMDA glutamate receptor activation stabilizes firing of sleep-on neurons, and that mechanisms of sleep maintenance differ from that of the sleep drive itself.

P.032 NMDA receptor encephalitis with severe orofacial dyskinesias treated tramadol and clonazepam

Background: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a neuroinflammatory disease mediated by antibodies targeting the GluN1 subunit of the NMDAR. It presents with well-defined neuropsychiatric symptoms, including psychosis, agitation, seizures, and memory disturbances. Movement disorders including orofacial dyskinesias are common, but often difficult to manage, with no specific published guidelines. Methods: A 23-year-old female was diagnosed with NMDAR encephalitis. She was treated with ovarian teratoma removal, corticosteroids, intravenous immunoglobulin therapy, rituximab, and tocilizumab. She continued to experience severe, self-mutilating orofacial dyskinesias. Tetrabenazine, haloperidol, and diazepam did not yield any sustained improvement. Tramadol was started based on a prior case report suggesting its efficacy. Results: Tramadol 50 mg po q6h led to immediate improvement in symptoms. Over the next 5 days, tramadol was increased to 150mg NG q6h and further reduced movements. When tramadol was held for one day, the movements significantly worsened and improved when it was restarted. Clonazepam 1mg NG QID also led to further improvement. Conclusions: Tramadol and clonazepam effectively treated severe orofacial dyskinesias in a patient with NMDAR encephalitis and refractory symptoms despite aggressive management. We propose early use of tramadol and clonazepam be considered for severe orofacial dyskinesias secondary to NMDAR encephalitis.

Activation of δ-opioid Receptors in Anterior Cingulate Cortex Alleviates Affective Pain in Rats

The negative emotions caused by persistent pain, called affective pain, are known to seriously affect human physical and mental health. The anterior cingulate cortex (ACC), especially the rostral ACC (rACC) plays a key role in the development of this affective pain. N-methyl-d-aspartate (NMDA) receptors, which are widely distributed in the ACC, are involved in the regulation of emotional behavior. It is well known that activation of opioid receptors can relieve pain, but whether it can alleviate affective pain is not clear. In the present study, conditioned place avoidance (CPA) responses induced by complete Freund's adjuvant (CFA) were used to represent the affective pain of place aversion. The behavioral measurements were synchronously combined with multichannel electrophysiological recordings of the discharge frequency of rACC pyramidal neurons to explore whether affective pain could be alleviated by the synthetic opioid [D-Ala2, D-Leu5]-Enkefalin (DADLE), an agonist of δ-opioid receptors. To further investigate this treatment as a mechanism for the relief of affective pain in CFA-treated animals, we used whole-cell patch recordings in slice preparations of the rACC region to determine the dose-dependent effects of DADLE on NMDA receptor-mediated currents. Then, western blot was used to determine levels of phosphorylated NMDA receptor subunits GluN1, GluN2 and GluN3 as affected by the δ-opioid receptor activation. The results showed that activation of δ-opioid receptors down-regulates the phosphorylation of NMDA receptor subunits, thereby inhibiting NMDA currents, decreasing the discharge frequency of rACC pyramidal neurons, and reversing the CPA response. Thus, δ-opioid receptor activation in the rACC region can alleviate affective pain.

Maternal Separation Alters Ethanol Drinking and Reversal Learning Processes in Adolescent Rats: The Impact of Sex and Glycine Transporter Type 1 (GlyT1) Inhibitor.

Adverse early life experiences are associated with an enhanced risk for mental and physical health problems, including substance abuse. Despite clinical evidence, the mechanisms underlying these relationships are not fully understood. Maternal separation (MS) is a commonly used animal model of early neglect. The aim of the current study is to determine whether the N-methyl-D-aspartate receptor (NMDAR)/glycine sites are involved in vulnerability to alcohol consumption (two-bottle choice paradigm) and reversal learning deficits (Barnes maze task) in adolescent rats subjected to the MS procedure and whether these effects are sex dependent. By using ELISA, we evaluated MS-induced changes in the NMDAR subunits (GluN1, GluN2A, GluN2B) expression, especially in the glycine-binding subunit, GluN1, in the prefrontal cortex (PFC) and ventral striatum (vSTR) of male/female rats. Next, we investigated whether Org 24598, a glycine transporter 1 (GlyT1) inhibitor, was able to modify ethanol drinking in adolescent and adult male/female rats with prior MS experience and reversal learning in the Barnes maze task. Our findings revealed that adolescent MS female rats consumed more alcohol which may be associated with a substantial increase in GluN1 subunit of NMDAR in the PFC and vSTR. Org 24598 decreased ethanol intake in both sexes with a more pronounced decrease in ethanol consumption in adolescent female rats. Furthermore, MS showed deficits in reversal learning in both sexes. Org 24598 ameliorated reversal learning deficits, and this effect was reversed by the NMDAR/glycine site inhibitor, L-701,324. Collectively, our results suggest that NMDAR/glycine sites might be targeted in the treatment of alcohol abuse in adolescents with early MS, especially females.

GluN3 subunit expression correlates with increased vulnerability of hippocampus and entorhinal cortex to neurodegeneration in a model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults that is often refractory to antiepileptic medication therapy. Neither the pathology nor the etiology of TLE is fully characterized, although recent studies have established that the two are causally related. TLE pathology entails a stereotypic pattern of neuron loss in hippocampal and parahippocampal regions, predominantly in CA1 subfield of the hippocampus and layer 3 of the medial entorhinal area (MEA), deemed hallmark pathological features of the disease. Through this work, we address the contribution of glutamatergic N-methyl-d-aspartate receptors (NMDARs) to the pathology (vulnerability and pattern of neuronal loss), and by extension to the pathophysiology (Ca2+-induced excitotoxicity), by assaying the spatial expression of their subunit proteins (GluN1, GluN2A, GluN2B, and GluN3A) in these regions using area-specific tissue analysis (ASTA), a novel methodology for harvesting brain chads from hard-to-reach regions within brain slices for Western blotting. Our data suggest gradient expression of the GluN3A subunit along the mid-lateral extent of layer 3 MEA and along the CA1-subicular axis in the hippocampus, unlike GluN1 or GluN2 subunits that are uniformly distributed. Incorporation of GluN3A in the subunit composition of conventional diheteromeric (d-) NMDARs yield triheteromeric (t-) NMDARs which by virtue of their increased selectivity for Ca2+ render neurons vulnerable to excitotoxic damage. Thus, the expression profile of this subunit sheds light on the spatial extent of the pathology observed in these regions and implicates the GluN3 subunit of NMDARs in hippocampal and entorhinal cortical pathology underlying TLE.NEW & NOTEWORTHY The role of the GluN3 subunit in NMDAR-mediated pathophysiology underlying TLE is not known. Here, we demonstrate using ASTA (area-specific tissue analysis) that its expression in specific regions of the entorhinal cortex and the hippocampus is correlated with significant cell loss and neurodegeneration, hallmark features of the disease.

Dietary zinc supplementation rescues fear-based learning and synaptic function in the Tbr1+/\u2212 mouse model of autism spectrum disorders

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by a dyad of behavioural symptoms—social and communication deficits and repetitive behaviours. Multiple aetiological genetic and environmental factors have been identified as causing or increasing the likelihood of ASD, including serum zinc deficiency. Our previous studies revealed that dietary zinc supplementation can normalise impaired social behaviours, excessive grooming, and heightened anxiety in a Shank3 mouse model of ASD, as well as the amelioration of synapse dysfunction. Here, we have examined the efficacy and breadth of dietary zinc supplementation as an effective therapeutic strategy utilising a non-Shank-related mouse model of ASD—mice with Tbr1 haploinsufficiency.MethodsWe performed behavioural assays, amygdalar slice whole-cell patch-clamp electrophysiology, and immunohistochemistry to characterise the synaptic mechanisms underlying the ASD-associated behavioural deficits observed in Tbr1+/− mice and the therapeutic potential of dietary zinc supplementation. Two-way analysis of variance (ANOVA) with Šídák's post hoc test and one-way ANOVA with Tukey’s post hoc multiple comparisons were performed for statistical analysis.ResultsOur data show that dietary zinc supplementation prevents impairments in auditory fear memory and social interaction, but not social novelty, in the Tbr1+/− mice. Tbr1 haploinsufficiency did not induce excessive grooming nor elevate anxiety in mice. At the synaptic level, dietary zinc supplementation reversed α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) hypofunction and normalised presynaptic function at thalamic-lateral amygdala (LA) synapses that are crucial for auditory fear memory. In addition, the zinc supplemented diet significantly restored the synaptic puncta density of the GluN1 subunit essential for functional NMDARs as well as SHANK3 expression in both the basal and lateral amygdala (BLA) of Tbr1+/− mice.LimitationsThe therapeutic effect of dietary zinc supplementation observed in rodent models may not reproduce the same effects in human patients. The effect of dietary zinc supplementation on synaptic function in other brain structures affected by Tbr1 haploinsufficiency including olfactory bulb and anterior commissure will also need to be examined.ConclusionsOur data further the understanding of the molecular mechanisms underlying the effect of dietary zinc supplementation and verify the efficacy and breadth of its application as a potential treatment strategy for ASD.

Amyloid-β oligomers interact with NMDA receptors containing GluN2B subunits and metabotropic glutamate receptor 1 in primary cortical neurons: Relevance to the synapse pathology of Alzheimer’s disease

Recent evidence suggests that soluble amyloid-β oligomers (AβOs) act as a key factor in the pathogenetic mechanism of Alzheimer's disease (AD). AβOs induce neurotoxic and synaptotoxic effects probably through binding to certain receptors, however it remains unclarified which receptors are most critically involved. In addition, dysregulation in glutamatergic signaling is implicated in AD. In this study, we used a rat primary cortical neuron model to investigate AβO-induced aberrations of synaptic proteins and binding of extracellular AβOs to candidate receptors in the glutamatergic system. Immunocytochemical analyses showed that both presynaptic (SNAP-25, synapsin I) and postsynaptic (spinophilin, homer 1b/c) proteins appeared to aberrantly dislocate from synapses upon AβO treatment. Double immunofluorescence staining of AβO-treated neurons without permeabilization pretreatment revealed that extracellular AβOs exist over neuronal soma and neurites and clearly colocalized with GluN1 and GluN2B subunits of NMDA receptors and metabotropic glutamate receptor 1 (mGluR1), but not with NMDA GluN2A subunits and mGluR5. AβO treatment altered neither total protein levels nor intracellular localizations of these receptors. These results suggest that extracellular AβOs specifically bind to both NMDA receptors containing GluN2B subunits and mGluR1. It is likely that binding of AβOs to these receptors induces various pathological responses, consequently leading to synaptic disruptions. Our study thus highlights the important roles of GluN2B-containing NMDA receptors and mGluR1 receptors in the synapse pathology in AD.

LRRK2-G2019S mice display alterations in glutamatergic synaptic transmission in midbrain dopamine neurons.

The progressive degeneration of dopamine (DA) neurons in the substantia nigra compacta (SNc) leads to the emergence of motor symptoms in patients with Parkinson's disease (PD). To propose neuroprotective therapies able to slow or halt the progression of the disease, it is necessary to identify cellular alterations that occur before DA neurons degenerate and before the onset of the motor symptoms that characterize PD. Using electrophysiological, histochemical, and biochemical approaches, we have examined if glutamatergic synaptic transmission in DA neurons in the SNc and in the adjacent ventral tegmental area (VTA) was altered in middle‐aged (10–12 months old) mice with the hG2019S point mutation (G2019S) in the leucine‐rich repeat kinase 2 (LRRK2) gene. G2019S mice showed increased locomotion and exploratory behavior compared with wildtype (WT) littermates, and intact DA neuron integrity. The intrinsic membrane properties and action potential characteristics of DA neurons recorded in brain slices were similar in WT and G2019S mice. Initial glutamate release probability onto SNc‐DA neurons, but not VTA‐DA neurons, was reduced in G2019S mice. We also found reduced protein amounts of the presynaptic marker of glutamatergic terminals, VGLUT1, and of the GluA1 and GluN1 subunits of AMPA and NMDA receptors, respectively, in the ventral midbrain of G2019S mice. These results identify alterations in glutamatergic synaptic transmission in DA neurons of the SNc and VTA before the onset of motor impairments in the LRRK2‐G2019S mouse model of PD.

De novo GRIN2A variants associated with epilepsy and autism and literature review

N-methyl-D-aspartate receptors (NMDAR) are di- or tri-heterotetrameric ligand-gated ion channels composed of two obligate glycine-binding GluN1 subunits and two glutamate-binding GluN2 or GluN3 subunits, encoded by GRIN1, GRIN2A–D, and GRIN3A–B receptor genes respectively. Each NMDA receptor subtype has different temporal and spatial expression patterns in the brain and varies in the cell types and subcellular localization resulting in different functions. They play a crucial role in mediating the excitatory neurotransmission, but are also involved in neuronal development and synaptic plasticity, essential for learning, memory, and high cognitive functions. Among genes coding NMDAR subunits, GRIN2B is predominantly associated with neurodevelopmental disorders such as intellectual disability, developmental delay, autism, attention-deficit/hyperactivity disorder and, further, schizophrenia, Alzheimer’s disease. The GRIN2A seems to be predominantly associated with a more definite phenotype including an epileptic spectrum ranging from Landau–Kleffner syndrome to benign childhood epilepsy with centrotemporal spikes, speech or language impairment, intellectual disability/developmental delay often in comorbidity. On the contrary, the occurrence of autism spectrum disorders, unlike GRIN2B-associated disorders, is questionable. To contribute to elucidate the latter issue and to better define the genotype/phenotype correlation, we report the clinical and neuropsychological profile of two patients featuring autism disorder, intellectual disability, language impairment, and focal epilepsy, associated with previously unreported heterozygous de novo GRIN2A pathogenic variants. We hypothesize that the unusual phenotype may be the result of interactions of tri-heterotetrameric 2GluN1/GluN2A-D/GluN3A-B subunits with mutated GluN2A subunit and/or the dysfunction may be influenced by other unknown modifier genes and/or environmental factors.

Nanoscale rules governing the organization of glutamate receptors in spine synapses are subunit specific

Heterotetrameric glutamate receptors are essential for the development, function, and plasticity of spine synapses but how they are organized to achieve this is not known. Here we show that the nanoscale organization of glutamate receptors containing specific subunits define distinct subsynaptic features. Glutamate receptors containing GluA2 or GluN1 subunits establish nanomodular elements precisely positioned relative to Synaptotagmin-1 positive presynaptic release sites that scale with spine size. Glutamate receptors containing GluA1 or GluN2B specify features that exhibit flexibility: GluA1-subunit containing AMPARs are found in larger spines, while GluN2B-subunit containing NMDARs are enriched in the smallest spines with neither following a strict modular organization. Given that the precise positioning of distinct classes of glutamate receptors is linked to diverse events including cell death and synaptic plasticity, this unexpectedly robust synaptic nanoarchitecture provides a resilient system, where nanopositioned glutamate receptor heterotetramers define specific subsynaptic regions of individual spine synapses.

Differential Expression of Presynaptic Munc13-1 and Munc13-2 in Mouse Hippocampus Following Ethanol Drinking

The Munc13 family of proteins is critically involved in synaptic vesicle priming and release in glutamatergic neurons in the brain. Munc13-1 binds to alcohol and, in Drosophila, modulates sedation sensitivity and self-administration. We examined the effect of alcohol consumption on the expression of Munc13-1 and Munc13-2, NMDA receptor subunits GluN1, GluN2A and GluN2B in the hippocampus-derived HT22 cells, hippocampal primary neuron culture, and wild-type and Munc13-1+/− male mouse hippocampus after ethanol consumption (Drinking in the Dark (DID) paradigm). In HT22 cells, Munc13-1 was upregulated following 25 mM ethanol treatment for 24 h. In the primary neuronal culture, however, the expression of both Munc13-1 and Munc13-2 increased after ethanol exposure. While Munc13-1 was upregulated in the hippocampus, Munc13-2 was downregulated following DID. This differential effect was found in the CA1 subfield of the hippocampus. Although Munc13-1+/− mice had approximately 50% Munc13-1 expression compared to wild-type, it was nonetheless significantly increased following DID. Munc13-1 and Munc13-2 were expressed in vesicular glutamate transporter1 (VGLUT1) immunoreactive neurons in the hippocampus, but ethanol did not alter the expression of VGLUT1. The NMDA receptor subunits, GluN1, GluN2A and GluN2B were upregulated in the hippocampal primary culture and in the CA1. Ethanol exerts a differential effect on the expression of Munc13-1 and Munc13-2 in the CA1 in male mice. Our study also found that ethanol’s effect on Munc13 expression is dependent on the experimental paradigm, and both Munc13-1 and Munc13-2 could contribute to the ethanol-induced augmentation of glutamatergic neurotransmission.

Opportunities for Precision Treatment of GRIN2A and GRIN2B Gain-of-Function Variants in Triheteromeric N-Methyl-D-Aspartate Receptors.

N-methyl-D-aspartate receptors (NMDARs) are tetrameric assemblies of two glutamate N-methyl-D-aspartate receptor subunits, GluN1 and two GluN2, that mediate excitatory synaptic transmission in the central nervous system. Four genes (GRIN2A-D) encode four distinct GluN2 subunits (GluN2A-D). Thus, NMDARs can be diheteromeric assemblies of two GluN1 plus two identical GluN2 subunits, or triheteromeric assemblies of two GluN1 subunits plus two different GluN2 subunits. An increasing number of de novo GRIN variants have been identified in patients with neurologic conditions and with GRIN2A and GRIN2B harboring the vast majority (> 80%) of variants in these cases. These variants produce a wide range of effects on NMDAR function depending upon its subunit subdomain location and additionally on the subunit composition of diheteromeric versus triheteromeric NMDARs. Increasing evidence implicates triheteromeric GluN1/GluN2A/GluN2B receptors as a major component of the NMDAR pool in the adult cortex and hippocampus. Here, we explore the ability of GluN2A- and GluN2B-selective inhibitors to reduce excess current flow through triheteromeric GluN1/GluN2A/GluN2B receptors that contain one copy of GRIN2A or GRIN2B gain-of-function variants. Our data reveal a broad range of sensitivities for variant-containing triheteromeric receptors to subunit-selective inhibitors, with some variants still showing strong sensitivity to inhibitors, whereas others are relatively insensitive. Most variants, however, retain sensitivity to non-selective channel blockers and the competitive antagonist D-(-)-2-amino-5-phosphonopentanoic acid. These results suggest that with comprehensive analysis, certain disease-related GRIN2A and GRIN2B variants can be identified as potential targets for subunit-selective modulation and potential therapeutic gain. SIGNIFICANCE STATEMENT: Triheteromeric NMDA receptors that contain one copy each of the GluN2A and GluN2B subunits show intermediate sensitivity to GluN2A- and GluN2B-selective inhibitors, making these compounds candidates for attenuating overactive, GRIN variant-containing NMDA receptors associated with neurological conditions. We show that functional evaluation of variant properties with inhibitor pharmacology can support selection of a subset of variants for which GluN2 subunit-selective agents remain effective inhibitors of variant-containing triheteromeric NMDA receptors.

Negative allosteric modulation of GluN3-containing NMDA receptors with a novel mechanism of action

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors involved in many physiological and neuropathological processes in the central nervous system. NMDARs are tertrameric assemblies of two GluN1 subunits and two GluN2 or two GluN3 subunits, with each receptor composition endowing distinct biophysical and pharmacological properties. GluN1 and GluN3A-B subunits bind glycine, while GluN2A-D subunits bind glutamate. The well understood GluN1/2 NMDARs require binding of both glutamate and glycine for activation, whereas the less studied GluN1/3 receptors only need glycine binding for activation.