N-methyl-D-aspartate Receptor Research Articles

Sevoflurane acts as an antidepressant by suppression of GluN2D-containing NMDA receptors on interneurons.

Sevoflurane, a commonly used inhaled anaesthetic known for its favourable safety profile and rapid onset and offset, has not been thoroughly investigated as a potential treatment for depression. In this study, we reveal the mechanism through which sevoflurane delivers enduring antidepressant effects. To assess the antidepressant effects of sevoflurane, behavioural tests were conducted, along with in vitro and ex vivo whole-cell patch-clamp recordings, to examine the effects on GluN1-GluN2 incorporated N-methyl-d-aspartate (NMDA) receptors (NMDARs) and neuronal circuitry in the medial prefrontal cortex (mPFC). Multiple-channel electrophysiology in freely moving mice was performed to evaluate sevoflurane's effects on neuronal activity, and GluN2D knockout (grin2d-/-) mice were used to confirm the requirement of GluN2D for the antidepressant effects. Repeated exposure to subanaesthetic doses of sevoflurane produced sustained antidepressant effects lasting up to 2 weeks. Sevoflurane preferentially inhibited GluN2C- and GluN2D-containing NMDARs, causing a reduction in interneuron activity. In contrast, sevoflurane increased action potentials (AP) firing and decreased spontaneous inhibitory postsynaptic current (sIPSC) in mPFC pyramidal neurons, demonstrating a disinhibitory effect. These effects were absent in grin2d-/- mice, and both pharmacological blockade and genetic knockout of GluN2D abolished sevoflurane's antidepressant actions, suggesting that GluN2D is essential for its antidepressant effect. Sevoflurane directly targets GluN2D, leading to a specific decrease in interneuron activity and subsequent disinhibition of pyramidal neurons, which may underpin its antidepressant effects. Targeting the GluN2D subunit could hold promise as a potential therapeutic strategy for treating depression.

Mechanisms of synapse-to-nucleus calcium signalling in striatal neurons and impairments in Huntington's disease.

Huntington's disease (HD) is a monogenic disorder with autosomal dominant inheritance. In HD patients, neurons in the striatum and cortex degenerate, leading to motor, psychiatric and cognitive disorders. Dysregulated synaptic function and calcium handling are common in many neurodegenerative diseases, including HD. N-methyl-D-aspartate (NMDA) receptor function is enhanced in HD at extrasynaptic sites, altering the balance of calcium-dependent neuronal survival versus death signalling pathways. Endoplasmic reticulum (ER) calcium handling is also abnormal in HD. The ER, which is continuous with the nuclear envelope, is purportedly involved in nuclear calcium signalling; based on this, we hypothesised that nuclear calcium signalling is altered in HD. We explored this hypothesis with calcium imaging techniques, including simultaneous epifluorescent imaging of cytosolic and nuclear calcium using jRCaMP1b and GCaMP3 sensors, respectively, in striatal spiny projection neurons in cortical-striatal co-cultures from the YAC128 mouse model of HD. Our data show contributions from a variety of calcium channels to nuclear calcium signalling. NMDA receptors (NMDARs) play an essential role in initiating action potential-dependent calcium signalling to the nucleus, and ryanodine receptors (RyR) contribute to both cytosolic and nuclear calcium signals. Unlike previous reports in glutamatergic hippocampal and cortical neurons, we found that in GABAergic striatal neurons, L-type voltage-gated calcium channels (CaV) contribute to cytosolic, but not nuclear calcium signalling. Calcium imaging also suggests impairments in nuclear calcium signalling in HD striatal neurons, where spontaneous action potential-dependent calcium transients in the nucleus were smaller in YAC128 striatal neurons compared to those of wild-type (WT). Our results elucidate mechanisms involved in action potential-dependent nuclear calcium signalling in GABAergic striatal neurons, and have revealed a clear deficit in this signalling in HD.

Ketamine can produce oscillatory dynamics by engaging mechanisms dependent on the kinetics of NMDA receptors

Ketamine is an N-methyl-D-aspartate (NMDA)-receptor antagonist that produces sedation, analgesia, and dissociation at low doses and profound unconsciousness with antinociception at high doses. At high and low doses, ketamine can generate gamma oscillations (>25 Hz) in the electroencephalogram (EEG). The gamma oscillations are interrupted by slow-delta oscillations (0.1 to 4 Hz) at high doses. Ketamine's primary molecular targets and its oscillatory dynamics have been characterized. However, how the actions of ketamine at the subcellular level give rise to the oscillatory dynamics observed at the network level remains unknown. By developing a biophysical model of cortical circuits, we demonstrate how NMDA-receptor antagonism by ketamine can produce the oscillatory dynamics observed in human EEG recordings and nonhuman primate local field potential recordings. We have identified how impaired NMDA-receptor kinetics can cause disinhibition in neuronal circuits and how a disinhibited interaction between NMDA-receptor-mediated excitation and GABA-receptor-mediated inhibition can produce gamma oscillations at high and low doses, and slow-delta oscillations at high doses. Our work uncovers general mechanisms for generating oscillatory brain dynamics that differs from ones previously reported and provides important insights into ketamine's mechanisms of action as an anesthetic and as a therapy for treatment-resistant depression.

GluN2A or GluN2B subunits of the NMDA receptor contribute to changes in neuronal excitability and impairments in LTP in the hippocampus of aging mice but do not mediate detrimental effects of oligomeric Aβ (1-42).

Studies in rodent models have revealed that oligomeric beta-amyloid protein [Aβ (1-42)] plays an important role in the pathogenesis of Alzheimer's disease. Early elevations in hippocampal neuronal excitability caused by Aβ (1-42) have been proposed to be mediated via enhanced activation of GluN2B-containing N-methyl-D-aspartate receptors (NMDAR). To what extent GluN2A or GluN2B-containing NMDAR contribute to Aβ (1-42)-mediated impairments of hippocampal function in advanced rodent age is unclear. Here, we assessed hippocampal long-term potentiation (LTP) and neuronal responses 4-5 weeks after bilateral intracerebral inoculation of 8-15 month old GluN2A+/- or GluN2B+/- transgenic mice with oligomeric Aβ (1-42), or control peptide. Whole-cell patch-clamp recordings in CA1 pyramidal neurons revealed a more positive resting membrane potential and increased total spike time in GluN2A+/-, but not GluN2B+/--hippocampi following treatment with Aβ (1-42) compared to controls. Action potential 20%-width was increased, and the descending slope was reduced, in Aβ-treated GluN2A+/-, but not GluN2B+/- hippocampi. Sag ratio was increased in Aβ-treated GluN2B+/--mice. Firing frequency was unchanged in wt, GluN2A+/-, and GluN2B+/-hippocampi after Aβ-treatment. Effects were not significantly different from responses detected under the same conditions in wt littermates, however. LTP that lasted for over 2 h in wt hippocampal slices was significantly reduced in GluN2A+/- and was impaired for 15 min in GluN2B+/--hippocampi compared to wt littermates. Furthermore, LTP (>2 h) was significantly impaired in Aβ-treated hippocampi of wt littermates compared to wt treated with control peptide. LTP induced in Aβ-treated GluN2A+/- and GluN2B+/--hippocampi was equivalent to LTP in control peptide-treated transgenic and Aβ-treated wt animals. Taken together, our data indicate that knockdown of GluN2A subunits subtly alters membrane properties of hippocampal neurons and reduces the magnitude of LTP. GluN2B knockdown reduces the early phase of LTP but leaves later phases intact. Aβ (1-42)-treatment slightly exacerbates changes in action potential properties in GluN2A+/--mice. However, the vulnerability of the aging hippocampus to Aβ-mediated impairments of LTP is not mediated by GluN2A or GluN2B-containing NMDAR.

Serine racemase expression profile in the prefrontal cortex and hippocampal subregions during aging in male and female rats.

Aging is associated with a decrease in N-methyl-D-aspartate (NMDA) receptor function, which is critical for maintaining synaptic plasticity, learning, and memory. Activation of the NMDA receptor requires binding of the neurotransmitter glutamate and also the presence of co-agonist D-serine at the glycine site. The enzymatic conversion of L-serine to D-serine is facilitated by the enzyme serine racemase (SR). Subsequently, SR plays a pivotal role in regulating NMDA receptor activity, thereby impacting synaptic plasticity and memory processes in the central nervous system. As such, age-related changes in the expression of SR could contribute to decreased NMDA receptor function. However, age-associated changes in SR expression levels in the medial and lateral prefrontal cortex (mPFC, lPFC), and in the dorsal hippocampal subfields, CA1, CA3, and dentate gyrus (DG), have not been thoroughly elucidated. Therefore, the current studies were designed to determine the SR expression profile, including protein levels and mRNA, for these regions in aged and young male and female Fischer-344 rats. Our results demonstrate a significant reduction in SR expression levels in the mPFC and all hippocampal subfields of aged rats compared to young rats. No sex differences were observed in the expression of SR. These findings suggest that the decrease in SR levels may play a role in the age-associated reduction of NMDA receptor function in brain regions crucial for cognitive function and synaptic plasticity.

Nobiletin regulates intracellular Ca2+ levels via IP3R and ameliorates neuroinflammation in Aβ42-induced astrocytes

Astrocytes are the major glial cells in the human brain and provide crucial metabolic and trophic support to neurons. The amyloid-β peptide (Aβ) alter the morphological and functional properties of astrocytes and induce inflammation and calcium dysregulation, contributing to Alzheimer's disease (AD) pathology. Recent studies highlight the role of Toll-like receptor (TLR) 4/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in inflammation. Reactive oxygen species (ROS) generated due to Aβ, induce apoptosis in the brain cells worsening AD progression. Astrocytic cell surface receptors, such as purinergic receptors (P2Y1 and P2Y2), metabotropic glutamate receptor (mGLUR)5, α7 nicotinic acetylcholine receptor (α7nAChR), and N-methyl-d-aspartate receptors (NMDARs), have been suggested to interact with inositol trisphosphate receptor (IP3R) on the endoplasmic reticulum (ER) to induce Ca2+ movement from ER to cytoplasm, causing Ca2+ dysregulation. We found that the citrus flavonoid nobiletin (NOB) protected primary astrocytes from Aβ42-induced cytotoxicity and inhibited TLR4/NF-κB signaling in Aβ42-induced primary rat astrocytes. NOB was found to regulate Aβ42-induced ROS levels through Keap1-Nrf2 pathway. The receptors P2Y1, P2Y2, mGLUR5, α7nAChR, and NMDARs induced intracellular Ca2+ levels by activating IP3R and NOB regulated them, thereby regulating intracellular Ca2+ levels. Molecular docking analysis revealed a possible interaction between NOB and IP3R in IP3R regulation. Furthermore, RNA sequencing revealed various NOB-mediated biological signaling pathways, such as the AD-presenilin, AD-amyloid secretase, and Wnt signaling pathway, suggesting possible neuroprotective roles of NOB. To conclude, NOB is a promising therapeutic agent for AD and works by modulating AD pathology at various levels in Aβ42-induced primary rat astrocytes.

NMDA Receptor Antagonists: Emerging Insights into Molecular Mechanisms and Clinical Applications in Neurological Disorders

Neurodegenerative disorders (NDs) include a range of chronic conditions characterized by progressive neuronal loss, leading to cognitive, motor, and behavioral impairments. Common examples include Alzheimer’s disease (AD) and Parkinson’s disease (PD). The global prevalence of NDs is on the rise, imposing significant economic and social burdens. Despite extensive research, the mechanisms underlying NDs remain incompletely understood, hampering the development of effective treatments. Excitotoxicity, particularly glutamate-mediated excitotoxicity, is a key pathological process implicated in NDs. Targeting the N-methyl-D-aspartate (NMDA) receptor, which plays a central role in excitotoxicity, holds therapeutic promise. However, challenges, such as blood–brain barrier penetration and adverse effects, such as extrapyramidal effects, have hindered the success of many NMDA receptor antagonists in clinical trials. This review explores the molecular mechanisms of NMDA receptor antagonists, emphasizing their structure, function, types, challenges, and future prospects in treating NDs. Despite extensive research on competitive and noncompetitive NMDA receptor antagonists, the quest for effective treatments still faces significant hurdles. This is partly because the same NMDA receptor that necessitates blockage under pathological conditions is also responsible for the normal physiological function of NMDA receptors. Allosteric modulation of NMDA receptors presents a potential alternative, with the GluN2B subunit emerging as a particularly attractive target due to its enrichment in presynaptic and extrasynaptic NMDA receptors, which are major contributors to excitotoxic-induced neuronal cell death. Despite their low side-effect profiles, selective GluN2B antagonists like ifenprodil and radiprodil have encountered obstacles such as poor bioavailability in clinical trials. Moreover, the selectivity of these antagonists is often relative, as they have been shown to bind to other GluN2 subunits, albeit minimally. Recent advancements in developing phenanthroic and naphthoic acid derivatives offer promise for enhanced GluN2B, GluN2A or GluN2C/GluN2D selectivity and improved pharmacodynamic properties. Additional challenges in NMDA receptor antagonist development include conflicting preclinical and clinical results, as well as the complexity of neurodegenerative disorders and poorly defined NMDA receptor subtypes. Although multifunctional agents targeting multiple degenerative processes are also being explored, clinical data are limited. Designing and developing selective GluN2B antagonists/modulators with polycyclic moieties and multitarget properties would be significant in addressing neurodegenerative disorders. However, advancements in understanding NMDA receptor structure and function, coupled with collaborative efforts in drug design, are imperative for realizing the therapeutic potential of these NMDA receptor antagonists/modulators.

Time-Dependent Long-Term Effect of Memantine following Repetitive Mild Traumatic Brain Injury.

Repetitive mild traumatic brain injury (rmTBI, e.g., sports concussions) may be associated with both acute and chronic symptoms and neurological changes. Despite the common occurrence of these injuries, therapeutic strategies are limited. One potentially promising approach is N-methyl-D-aspartate receptor (NMDAR) blockade to alleviate the effects of post-injury glutamatergic excitotoxicity. Initial pre-clinical work using the NMDAR antagonist, memantine, suggests that immediate treatment following rmTBI improves a variety of acute outcomes. It remains unclear (1) whether acute memantine treatment has long-term benefits and (2) whether delayed treatment following rmTBI is beneficial, which are both clinically relevant concerns. To test this, animals were subjected to rmTBI via a weight drop model with rotational acceleration (five hits in 5 days) and randomized to memantine treatment immediately, 3 months, or 6 months post-injury, with a treatment duration of one month. Behavioral outcomes were assessed at 1, 4, and 7 months post-injury. Neuropathological outcomes were characterized at 7 months post-injury. We observed chronic changes in behavior (anxiety-like behavior, motor coordination, spatial learning, and memory), as well as neuroinflammation (microglia, astrocytes) and tau phosphorylation (T231). Memantine treatment, either immediately or 6 months post-injury, appears to confer greater rescue of neuroinflammatory changes (microglia) than vehicle or treatment at the 3-month time point. Although memantine is already being prescribed chronically to address persistent symptoms associated with rmTBI, this study represents the first evidence of which we are aware to suggest a small but durable effect of memantine treatment in mild, concussive injuries. This effect suggests that memantine, although potentially beneficial, is insufficient to treat all aspects of rmTBI alone and should be combined with other therapeutic agents in a multi-therapy approach, with attention given to the timing of treatment.

GLP-1-directed NMDA receptor antagonism for obesity treatment

The N-methyl-d-aspartate (NMDA) receptor is a glutamate-activated cation channel that is critical to many processes in the brain. Genome-wide association studies suggest that glutamatergic neurotransmission and NMDA receptor-mediated synaptic plasticity are important for body weight homeostasis1. Here we report the engineering and preclinical development of a bimodal molecule that integrates NMDA receptor antagonism with glucagon-like peptide-1 (GLP-1) receptor agonism to effectively reverse obesity, hyperglycaemia and dyslipidaemia in rodent models of metabolic disease. GLP-1-directed delivery of the NMDA receptor antagonist MK-801 affects neuroplasticity in the hypothalamus and brainstem. Importantly, targeting of MK-801 to GLP-1 receptor-expressing brain regions circumvents adverse physiological and behavioural effects associated with MK-801 monotherapy. In summary, our approach demonstrates the feasibility of using peptide-mediated targeting to achieve cell-specific ionotropic receptor modulation and highlights the therapeutic potential of unimolecular mixed GLP-1 receptor agonism and NMDA receptor antagonism for safe and effective obesity treatment.

Clinical analysis of 9 children with refractory N-methyl-D-aspartate receptor antibody encephalitis children treated with tocilizumab

Objective: To analyze the clinical features of children with refractory N-methyl-D-aspartate (NMDA) receptor antibody encephalitis treated with tocilizumab. Methods: Demographic and clinical manifeatations, immunotherapy and prognosis data of 9 children with refractory NMDA receptor antibody encephalitis who received tocilizumab in the Department of Pediatrics Neurology, XiangYa Hospital of Central South University from August 2021 to September 2023 were collected retrospectively. Prognosis was evaluated using the modified Rankin scale at initial diagnosis, at the initiation of tocilizumab treatment, and at the last follow-up. Treatment related complications, neuroimaging, and electroencephalography data were analyzed. Results: Among the 9 children, 6 were male and 3 were female, with an onset age of 4.2 (2.8, 8.7) years. At the onset of the disease, 9 children had a modified Rankin scale score of 5. When tocilizumab treatment was initiated, 7 children had a score of 5, and 2 children had a score of 4. The interval between the onset and initiation of tocilizumab treatment was 12 (5, 27) months, and the treatment frequency was 8 (5, 13) times. The follow-up time was 2.8 (1.5, 3.7) years. At the last follow-up, the symptoms of 9 children, including movement disorder, sleep disorder, consciousness disorder, silence and autonomic dysfunction, were improved to varying degrees, and none of them had seizures. At the last follow-up, 4 cases with a modified Rankin scale score of 0, 1 case with a score of 1, 2 cases with a score of 3, 1 case with a score of 4 and 1 case with a score of 5. The modified Rankin scale at the last follow-up was significantly different from that at the start of tocilizumab (Z=-2.56, P=0.014). All children had no serious adverse reactions during the treatment. Conclusions: After treatment with tocilizumab, the symptoms in patievts with refractory NMDA receptor antibody encephalitis, including movement disorder, sleep disorder, consciousness disorder, silence and autonomic dysfunction were improved, and none of them had seizures. The modified Rankin scale were improved, and the safety was good.

New Insights on N-Methyl-D-Aspartate (NMDA) Receptor Under Combinatorial Molecular Docking and MD Simulation Studies Using Natural Bioactive Compounds Against Neurodegenerative Diseases

Neurodegenerative diseases pose a significant challenge, and novel therapeutic strategies are urgently needed. N-methyl-D-aspartate (NMDA) receptor is reported to play a critical role in the central nervous system and has emerged as a potential target for drug discovery. This study explored the potential scope of natural bioactive compounds as ligands for the NMDA receptor using current advances of docking studies with molecular dynamic (MD) simulations. An extensive virtual screening of 500 natural compounds were executed based on wide scientific literature and bibliography search. Docking simulations identified promising candidates with favorable binding affinities, with the top compounds - DL-Alanosine, and Zeinoxanthin (PubChem CIDs 153353 and 5281234) exhibiting exceptionally high docking scores of -6.6 and -6.4, against NMDA respectively. Further, MD simulations suggested the stability of the top-scoring compounds in complex with the NMDA receptor. These findings will provide a new insights to researchers and scientists on proceeding with new alternatives on the investigation of natural bioactive compounds as therapeutic lead candidates for targeting various receptors like NMDA in neurodegenerative diseases. However, in vitro and in vivo studies are warranted to validate these results and elucidate the underlying mechanisms of action.

Promising Effects of Casearins in Tumor-Bearing Mice and Antinociceptive Action against Oncologic Pain: Molecular Docking and In Vivo Findings.

Safer analgesic drugs remain a hard challenge because of cardiovascular and/or gastrointestinal toxicity, mainly. So, this study evaluated in vivo the antiproliferative actions of a fraction with casearins (FC) from Casearia sylvestris leaves against human colorectal carcinomas and antihyperalgesic effects on inflammatory- or opiate-based pain relief and oncologic pain in Sarcoma 180 (S180)-bearing mice. Moreover, docking investigations evaluated the binding among Casearin X and NMDA(N-methyl-D-aspartate)-type glutamate receptors. HCT-116 colorectal carcinoma-xenografted mice were treated with FC for 15 days. Antinociceptive assays included chemically induced algesia and investigated mechanisms by pharmacological blockade. Intraplantar region S180-bearing animals received a single dose of FC and were examined for mechanical allodynia and behavior alterations. AutoDock Vina determined molecular interactions among Cas X and NMDA receptor subunits. FC reduced tumor growth at i.p. (5 and 10 mg/kg) and oral (25 mg/kg/day) doses (31.12-39.27%). FC reduced abdominal pain, as confirmed by formalin and glutamate protocols, whose antinociception activity was blocked by naloxone and L-NAME (neurogenic phase) and naloxone, atropine, and flumazenil (inflammatory phase). Meanwhile, glibenclamide potentiated the FC analgesic effects. FC increased the paw withdrawal threshold without producing changes in exploratory parameters or motor coordination. Cas X generated a more stable complex with active sites of the NMDA receptor GluN2B subunits. FC is a promising antitumor agent against colorectal carcinomas, has peripheral analgesic effects by desensitizing secondary afferent neurons, and inhibits glutamate release from presynaptic neurons and/or their action on cognate receptors. These findings emphasize the use of clerodane diterpenes against cancer-related pain conditions.

Pathogenic MTOR somatic variant causing focal cortical dysplasia drives hyperexcitability via overactivation of neuronal GluN2C N-methyl-D-aspartate receptors.

Genetic variations in proteins of the mechanistic target of rapamycin (mTOR) pathway cause a spectrum of neurodevelopmental disorders often associated with brain malformations and with intractable epilepsy. The mTORopathies are characterized by hyperactive mTOR pathway and comprise tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) type II. How hyperactive mTOR translates into abnormal neuronal activity and hypersynchronous network remains to be better understood. Previously, the role of upregulated GluN2C-containing glutamate-gated N-methyl-D-aspartate receptors (NMDARs) has been demonstrated for germline defects in the TSC genes. Here, we questioned whether this mechanism would expand to other mTORopathies in the different context of a somatic genetic variation of the MTOR protein recurrently found in FCD type II. We used a rat model of FCD created by in utero electroporation of neural progenitors of dorsal telencephalon with expression vectors encoding either the wild-type or the pathogenic MTOR variant (p.S2215F). In this mosaic configuration, patch-clamp whole-cell recordings of the electroporated, spiny stellate neurons and extracellular recordings of the electroporated areas were performed in neocortical slices. Selective inhibitors were used to target mTOR activity and GluN2C-mediated currents. Neurons expressing the mutant protein displayed an excessive activation of GluN2C NMDAR-mediated spontaneous excitatory postsynaptic currents. GluN2C-dependent increase in spontaneous spiking activity was detected in the area of electroporated neurons in the mutant condition and was restricted to a critical time window between postnatal days P9 and P20. Somatic MTOR pathogenic variant recurrently found in FCD type II resulted in overactivation of GluN2C-mediated neuronal NMDARs in neocortices of rat pups. The related and time-restricted local hyperexcitability was sensitive to subunit GluN2C-specific blockade. Our study suggests that GluN2C-related pathomechanisms might be shared in common by mTOR-related brain disorders.

Sex-dependent effects of the uncompetitive N-methyl-D-aspartate receptor antagonist REL-1017 in G93A-SOD1 amyotrophic lateral sclerosis mice.

The pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the demise of motor neurons has been linked to excitotoxicity caused by excessive calcium influx via N-methyl-D-aspartate receptors (NMDARs), suggesting that uncompetitive NMDAR antagonism could be a strategy to attenuate motor neuron degeneration. REL-1017, the dextro-isomer of racemic methadone, is a low-affinity uncompetitive NMDAR antagonist. Importantly, in humans REL-1017 has shown excellent tolerability in clinical trials for major depression. Here, we tested if REL-1017 improves the disease phenotypes in the G93A SOD1 mouse, a well-established model of familial ALS, by examining survival and motor functions, as well as the expression of genes and proteins involved in neuroplasticity. We found a sex-dependent effect of REL-1017 in G93A SOD1 mice. A delay of ALS symptom onset, assessed as 10%-decrease of body weight (p < 0.01 vs. control untreated mice) and an extension of lifespan (p < 0.001 vs. control untreated mice) was observed in male G93A SOD1 mice. Female G93A SOD1 mice treated with REL-1017 showed an improvement of muscle strength (p < 0.01 vs. control untreated mice). Both males and females treated with REL-1017 showed a decrease in hind limb clasping. Sex-dependent effects of REL-1017 were also detected in molecular markers of neuronal plasticity (PSD95 and SYN1) in the spinal cord and in the GluN1 NMDAR subunit in quadricep muscles. In conclusion, this study provides preclinical in vivo evidence supporting the clinical evaluation of REL-1017 in ALS.

D-serine and D-amino acid oxidase levels in patients with schizophrenia spectrum disorders in the first episode and 6-month follow-up

BackgroundD-serine and the D-amino acid oxidase (DAO) enzyme, which breaks down d-amino acids, may be involved in the pathophysiology of schizophrenia by affecting the N-methyl-D-aspartate (NMDA) receptor. The exact role of D-serine and DAO, as well as the consequences of increased DAO activity in patients with schizophrenia, remain unclear. We aimed to investigate D-serine and DAO levels in patients with first-episode schizophrenia spectrum disorders before treatment and after six months of treatment. MethodComparisons for the serum levels of D-serine and DAO were made between 81 healthy controls and 89 patients with first-episode schizophrenia spectrum disorders without a history of treatment. Further comparisons were made after 6 months for changes in these levels in the 41 patients in follow-up. The Positive and Negative Syndrome Scale (PANNS), Calgary Scale for Depression in Schizophrenia (CDSS), Montreal Cognitive Assessment Scale (MoCA), Global Assessment Scale (GAS), and Clinical Global Impression Scale (CGI) were used to evaluate the symptom severity and functionality. Secondary results included comparisons related to antipsychotic equivalent doses. ResultsBefore treatment, patients had significantly lower levels of D-serine, DAO, and D-serine/DAO ratio compared to healthy individuals (p < 0.001; p < 0.001; p = 0.004). DAO and D-serine levels of the patients were higher after six months of treatment (p = 0.025; p = 0.001). There was correlation of DAO levels with antipsychotic dosage and with PANSS negative and total subscale scores (rho = 0.421, p = 0.01; rho = 0.280, p = 0.008; rho = 0.371, p = 0.000). No correlation was found between serum D-serine level, DAO level, and the D-serine/DAO ratio with cognitive function. ConclusionsThe results suggest that D-serine and DAO may play a role that is sensitive to treatment effects in schizophrenia spectrum disorders. To gain a more comprehensive understanding of the impact antipsychotic drugs have on NMDA receptor dysfunction, there is a requirement for studies that directly evaluates the activity of the DAO enzyme.

N-Methyl-D-aspartate receptor (NMDAR) activity in the nucleus tractus solitarii (nTS) is required for the full expression of phrenic long-term facilitation after acute intermittent hypoxia

Exposure to acute intermittent hypoxia (AIH) induces a persistent elevation of phrenic nerve activity during the normoxic period, or phrenic long-term facilitation (pLTF). We have previously shown that nTS activity is necessary for both the development and maintenance of pLTF. However, the underlying cellular mechanisms and pathways are not yet clear. Hypoxia induces the release of glutamate from chemoafferents in the nTS, where it binds to AMPA and NMDA receptors. Previous studies have shown that activation of NMDARs increases neuronal calcium influx and triggers long-term potentiation. The present study aimed to test the role of NMDARs in pLTF and, in particular, to determine whether activation of these receptors in the nTS is required for hypoxia-induced pLTF. Mean arterial pressure (MAP) and splanchnic sympathetic and phrenic nerve activity (sSNA and PhrNA) were recorded in anesthetized, vagotomized, neuromuscularly blocked, and artificially ventilated male Sprague-Dawley rats. Animals were exposed to 10 bouts of 10% O2 (45 sec, every 5 min; AIH). Time controls (TC) were exposed to a single hypoxia bout. Parameters were measured for an additional 1 hour after the end of AIH and compared to their initial baseline (Bsl) values or TC. The NMDAR antagonist MK-801 (3 mg kg (-1), i.v.) was injected systemically, or AP5 (10 mM, 60 nl) nanoinjected bilaterally into the nTS, after the development of AIH-induced pLTF. To test whether induction of AIH or blockade of NMDARs alters neuronal function of the nTS, nTS neuronal Ca2+ responses to AIH and MK-801 were measured by fiber photometry in 3 rats previously nanoinjected with a Ca2+ indicator GCaMP-expressing virus in the nTS. Minute PhrNA (MinPhrNA, frequency x amplitude) increased 1hr after AIH (% of Bsl, 438 ± 184%; mean ± SE; p &lt;0.01; t-test) and was significantly greater than TC (74 ± 15%Bsl p &lt;0.01; t-test), indicating pLTF. AIH also increased nTS neuronal Ca2+. Systemic blockade of NMDARs after the development of pLTF decreased MAP, sSNA, and nTS Ca2+ and eliminated pLTF, consistent with others. Bilateral nTS nanoinjection of AP5 reduced but did not abolish pLTF (before AP5: 449 ± 109% vs. after AP5: 371 ± 107%Bsl; p &lt;0.05; paired t-test), increased sSNA (before AP5: 192 ± 31% vs. after AP5: 219 ± 42%; p= 0.08; paired t-test) but did not alter MAP. nTS AP5 after TC had no effect on MAP, MinPhrNA (before AP5: 99 ± 46% vs. after AP5: 103 ± 43%; p = 0.44; t-test) or sSNA (before AP5: 244 ± 92% vs. after AP5: 245 ± 81%; =0.94; paired t-test). Altogether, these results show that systemic blockade of NMDARs eliminates pLTF, and our data suggest that nTS NMDARs are critical components for the maintenance of AIH-induced neuroplasticity. R01-HL-98602. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High throughput assay for detection of antibodies targeting multiple subunit membrane proteins of human neuron synapse in vitro

Abstract Synapses are essential to the transmission of nervous impulses from one neuron to another. There are more than 1000 multiple-subunit membrane proteins in synapse. Some of them are drug targets and/or autoantigens. Most autoantibodies against synapse membrane proteins recognize conformation of extracellular domains. Based on known structures and predicted structures by chatGPT, we used N-methyl-D-aspartate (NMDA)receptor as model for quantitative assay in vitro. In order to keep the native form of the extracellular domains of NMDA receptor, we modified some amino acids in transmembrane domains and intracellular domains, establishing a stable cell line expressing cluster NMDA receptor used to sensitively detect autoantibody in human serum and cerebrospinal fluid. We also prepared stable NMDA receptor tetramer complex from the stable cell line and prepared monoclonal antibodies (mAB) against subunit GluN1 and GluN2. Based on tetramer complex and mABs, we established a method to quantitatively detect antibodies from clinical samples.

Neuronal autoantibodies in the cerebrospinal fluid of 148 patients with schizophrenia and 151 healthy controls

Schizophrenia is a syndrome with multiple etiologies, one of which is the potential for an autoimmune disease of the brain such as N-methyl-d-aspartate receptor (NMDAR) encephalitis, which can induce psychosis resembling schizophrenia. Here, we examined anti-neuronal autoantibodies related to psychosis using both cell- (CBA) and tissue-based assays (TBA) in the cerebrospinal fluid (CSF) of patients with chronic schizophrenia and control participants. First, we screened for the antibodies against leucine-rich glioma-inactivated 1 (LGI1), γ-aminobutyric acid B receptor (GABABR), dipeptidyl aminopeptidase-like protein 6 (DPPX), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR1/R2), and contactin-associated protein-like 2 (CASPR2) in 148 patients with schizophrenia. No antibodies were detected. Next, we performed CBA for NMDAR antibodies in 148 patients with schizophrenia and 151 age- and sex-matched controls. Although we detected relatively weak immunoreactivity for NMDAR in the CSFs of two patients with schizophrenia and three controls, no samples were positive when strict criteria were applied. For TBA in the rat hippocampus and cerebellum, we detected positive signals in the CSFs of 13 patients with schizophrenia and eight controls. Positive samples were analyzed for paraneoplastic syndrome and antinuclear antibodies using immunoblotting. The CSFs of nine patients and six controls were positive for dense fine speckle 70 (DFS70) antibodies. Additionally, antibodies against centromere protein (CENP)-A and CENP-B were detected in patients with schizophrenia. Our results suggest that autoantibodies against NMDAR, LG1, GABABR, DPPX, AMPAR1/R2, and CASPR2 are not associated with the pathogenesis of chronic schizophrenia. Moreover, we emphasize the importance of considering the effect of anti-DFS70 antibodies when analyzing autoantibodies in CSF samples. Conclusively, we obtained no evidence suggesting that the most frequent neuronal autoantibodies in the CSF play a role in the pathogenesis of schizophrenia, even in our sample.

NMDA receptor within nucleus accumbens shell regulates propofol self-administration through D1R/ERK/CREB signalling pathway.

Addictive properties of propofol have been demonstrated in both humans and animals. The nucleus accumbens (NAc) shell (NAsh) in the brain, along with the interactions between N-methyl-D-aspartate receptor (NMDAR) and the dopamine D1 receptor (D1R), as well as their downstream ERK/CREB signalling pathway in the NAc, are integral in regulating reward-seeking behaviour. Nevertheless, it remains unclear whether NMDARs and the NMDAR-D1R/ERK/CREB signalling pathway in the NAsh are involved in mediating propofol addiction. To investigate it, we conducted experiments with adult male Sprague-Dawley rats to establish a model of propofol self-administration behaviour. Subsequently, we microinjected D-AP5 (a competitive antagonist of NMDARs, 1.0-4.0μg/0.3μL/site) or vehicle into bilateral NAsh in rats that had previously self-administered propofol to examine the impact of NMDARs within the NAsh on propofol self-administration behaviour. Additionally, we examined the protein expressions of NR2A and NR2B subunits, and the D1R/ERK/CREB signalling pathways within the NAc. The results revealed that propofol administration behaviour was enhanced by D-AP5 pretreatment in NAsh, accompanied by elevated expressions of phosphorylation of NR2A (Tyr1246) and NR2B (Tyr1472) subunits. There were statistically significant increases in the expressions of D1Rs, as well as in the phosphorylated ERK1/2 (p-ERK1/2) and CREB (p-CREB). This evidence substantiates a pivotal role of NMDARs in the NAsh, with a particular emphasis on the NR2A and NR2B subunits, in mediating propofol self-administration behaviour. Furthermore, it suggests that this central reward processing mechanism may operate through the NMDAR-D1R/ERK/CREB signal transduction pathway.

Abstract 145: Chronic Electronic Cigarette Exposure Accelerates SMC Phenotypic Modulation And Activates The NMDA-type Glutamate Receptor (NMDAR) Pathway In Mediating Vascular Calcification

Introduction: Electronic cigarettes pose a serious emerging threat to cardiovascular health. The use of electronic nicotine delivery systems (ENDS) such as electronic cigarettes (e-cigs) have increased exponentially, especially among adolescents. Emerging data suggest e-cig use has been implicated in promoting atherosclerosis. This work explores the mechanism of e-cig exposure accelerated the atherosclerotic lesion development and vascular remodeling. Methods: SMC-lineage tracing mice (n=16) on a ApoE null hyperlipidemic background were put on high fat diet for 12 weeks and exposed to pod-based e-cigs (Juul) three times a week for 12 weeks in a whole-body chamber (inExpose, Scireq) and compared to WT mice exposed to air (n=16). The aortic sinus was dissected and digested, FACS was performed to sort for live single cells, then single-cell RNA-Seq (sc-RNAseq) and ATAC-seq were performed on the 10X Genomics platform. Analyses were completed with Seurat and Signac tools. The osteogenic load was assessed by histology with alkaline phosphatase (AP) enzymatic assay using the Ferangi Blue Chromogen kit. Results: scRNA-Seq analysis revealed that e-cig accelerated the SMC transition to a chondrogenic phenotype (CMC). We found a significantly increased proportion of chondromyocytes (CMC) expressing specific markers such as Col2a1 and Acan in the e-cig exposed mice compared to control. The scATAC-seq profiles showed that e-cig exposed mice develop a cell cluster that enriched for markers related to nicotine, glutamatergic and dopaminergic pathways. The genes highly enriched for chromatin accessibility in this cluster included Grin2a , a gene that encodes a N-methyl-D-aspartate receptor (NMDAR) GluN2A, which was found to be expressed in the fibromyocyte and CMC population of the scRNA-seq. Consistent with our finding from the scRNA-Seq data showing a greater CMC proportion in the e-cig exposed mice, we found the relative AP-stained area to be larger in the exposed group than in the WT group. Conclusion: Overall, these data indicate that e-cig exposure can promote atherosclerosis through adverse SMC phenotypic modulation. Moreover, e-cig exposure induces NMDAR activation that may be implicated in SMC modulation to a chondrogenic phenotype.