NMDA Receptor Signaling Research Articles

S62. Computational diagnosis of ion channel dysfunction in NMDA receptor antibody encephalitis

Introduction NMDA receptor (NMDAR) antibody encephalitis is an autoimmune disorder causing acute psychosis, confusion and seizures. Electroencephalography (EEG) findings are usually non-specific, with diagnosis confirmed by demonstration of serum/CSF autoantibodies. Improved clinical outcomes depend on early institution of immunotherapies. A non-invasive measure of NMDAR function could facilitate early treatment, and enable tracking of disease activity. Recent biophysical modeling advances allow inference of ion channel dynamics from macroscopic measures of brain activity. Here, we show that clinically relevant diagnoses of NMDAR ion channel dysfunction can be obtained using a microcircuit model fitted to individual human EEG data. Methods We compared resting state EEG cross-spectra from patients with NMDAR encephalitis, encephalopathy unrelated to NMDAR dysfunction, and non-encephalopathic controls. We fit biophysical models (dynamic causal models – DCMs) of connected neuronal ensembles, simulating post-synaptic membrane potentials and EEG spectra through lead field projection. We used canonical variate analysis (CVA) and an empirical Bayesian approach searching candidate models across the groups to reveal specific synaptic deficits. Results Clinical severity was matched between encephalopathic groups. However, there were clear differences in EEG cross-spectra at low (2–4 Hz) and high (28–40 Hz) frequencies, with increased beta-gamma power present in NMDAR antibody encephalitis patients. Subject-specific DCMs mapped model parameters representing dynamics at NMDA, AMPA and GABA receptors to spectral data features. Four inter-connected sources described delta-theta and beta-gamma cross-spectra observed at scalp sensors. Each source comprised a neural mass model with four neuronal subpopulations, with dynamics determined by intrinsic connections mediated via ionotropic receptors. Dynamics along intrinsic and extrinsic connections could reproduce scalp-based EEG recordings. Parameter estimate CVA showed significant differences between patients with and without NMDAR encephalitis in NMDAR parameters. A parametric empirical Bayes (PEB) analysis demonstrated that the group effects of NMDAR encephalitis were specific to NMDA model parameters, in contrast to other encephalopathies where differences were seen across receptor subtypes. Conclusion Neurological and psychiatric practice lack diagnostic probes to assess disease mechanisms at the neuronal level non-invasively in humans. We show that biophysical models of EEG applied to clinical EEG data identify abnormal NMDAR signaling in patients with NMDAR encephalitis but not in patients with other encephalopathies or neurological controls. By comparing model parameter estimates from these patient groups, we selectively identify NMDAR dysfunction in a multi ion-channel model. Given that EEG is ubiquitously available, our findings suggest the feasibility of EEG neuromodeling as a unique assay of brain dysfunction at the molecular-level.

NMDA Receptor Signaling Is Important for Neural Tube Formation and for Preventing Antiepileptic Drug-Induced Neural Tube Defects.

Failure of neural tube closure leads to neural tube defects (NTDs), which can have serious neurological consequences or be lethal. Use of antiepileptic drugs (AEDs) during pregnancy increases the incidence of NTDs in offspring by unknown mechanisms. Here we show that during Xenopus laevis neural tube formation, neural plate cells exhibit spontaneous calcium dynamics that are partially mediated by glutamate signaling. We demonstrate that NMDA receptors are important for the formation of the neural tube and that the loss of their function induces an increase in neural plate cell proliferation and impairs neural cell migration, which result in NTDs. We present evidence that the AED valproic acid perturbs glutamate signaling, leading to NTDs that are rescued with varied efficacy by preventing DNA synthesis, activating NMDA receptors, or recruiting the NMDA receptor target ERK1/2. These findings may prompt mechanistic identification of AEDs that do not interfere with neural tube formation.SIGNIFICANCE STATEMENT Neural tube defects are one of the most common birth defects. Clinical investigations have determined that the use of antiepileptic drugs during pregnancy increases the incidence of these defects in the offspring by unknown mechanisms. This study discovers that glutamate signaling regulates neural plate cell proliferation and oriented migration and is necessary for neural tube formation. We demonstrate that the widely used antiepileptic drug valproic acid interferes with glutamate signaling and consequently induces neural tube defects, challenging the current hypotheses arguing that they are side effects of this antiepileptic drug that cause the increased incidence of these defects. Understanding the mechanisms of neurotransmitter signaling during neural tube formation may contribute to the identification and development of antiepileptic drugs that are safer during pregnancy.

Protective influences of N-acetylcysteine against alcohol abstinence-induced depression by regulating biochemical and GRIN2A, GRIN2B gene expression of NMDA receptor signaling pathway in rats

Evidences have indicated a high degree of comorbidity of alcoholism and depression. N-acetylcysteine (NAC) has shown its clinical efficiency in the treatment of several psychiatric disorders and is identified as a multi-target acting drug. The ability of NAC to prevent alcohol abstinence-induced depression-like effects and underlying mechanism(s) have not been adequately addressed. This study was aimed to investigate the beneficial effects of NAC in the alcohol abstinence-induced depression developed following long-term voluntary alcohol intake. For evaluation of the effects of NAC, Sprague–Dawley rats were enabled to voluntary drinking of 4.5%, 7.5% and 9% v/v alcohol for fifteen days. NAC (25, 50, and 100 mg/kg) and fluoxetine (5 mg/kg) were injected intraperitoneally for three consecutive days during the alcohol abstinence period on the days 16, 17, 18. The behavioral studies were conducted employing forced swim test (FST), and tail suspension test (TST) on day 18 to determine the effects of N-acetylcysteine and fluoxetine in the ethanol withdrawal induced-depression. Blood alcohol concentration, alcohol biomarkers like SGPT, SGOT, ALP, GGT, and MCV were estimated by using commercially available kits. Serotonin concentrations were measured in the plasma, hippocampus and pre-frontal cortex using the rat ELISA kit. The expression of GRIN1, GRIN2A, GRIN2B genes for the N-methyl d-aspartate receptors (NMDAR) subunits in the hippocampus and the prefrontal cortex were also examined by reverse-transcription quantitative polymerase chain reaction. The results revealed that alcohol abstinence group depicted increased immobility time in FST and TST. Further, NAC exerted significant protective effect at the doses 50 mg/kg and 100 mg/kg, but 25 mg/kg showed insignificant protection against alcohol abstinence-induced depression. The increased level of biochemical parameters following ethanol abstinence were also reversed by NAC at the dose of 100 mg/kg. The significant reversal effect of NAC on the serotonin level following alcohol abstinence was greater in the hippocampus as compared to the third-day alcohol withdrawal group. The increased expression levels of GRIN2A and GRIN2B following ethanol abstinence were reversed with a higher dose of NAC (100 mg/kg) treatment. In conclusion, the results of the study reveal that NAC has remarkable protective effects in the alcohol abstinence-induced depression by modulating alcohol markers, serotonin levels and GRIN2A, GRIN2B gene expression of NMDAR signaling pathway in rats.

VOLTAGE‐GATED SODIUM CHANNEL ACTIVATION TRIGGERS CALCIUM INFLUX AND HIPPOCAMPAL NEURON COMPLEXITY

Background and significanceAfter stroke, the molecular mechanisms underlying recovery are similar to the development of the nervous system. During development, neuronal activity regulates both the morphology and connectivity of neurons. Neuronal activity primarily involves voltage‐gated sodium channel (VGNaC) activation. Calcium signaling through the N‐methyl‐D‐aspartate receptor (NMDAR) drives morphological changes such as, dendritic arborization, synaptogenesis, and spinogenesis. VGNaC activation influences NMDAR function making this pathway an attractive therapeutic target for stroke recovery.HypothesisActivation of VGNaCs by PbTx‐2 augments NMDAR signaling resulting in and increased Ca2+ influx leading to dendritic arborization and spinogenesis in hippocampal neurons.Experimental design and MethodsWe used the VGNaC gating modifier, brevetoxin (PbTx‐2), to assess the effects of VGNaC activation on neuronal morphology. We defined the calcium‐signaling pathway triggered by PbTx‐2 in a population of hippocampal neurons (HNs), and evaluated PbTx‐2‐induced effects on dendritic arborization and spinogenesis in organotypic hippocampal slice culture (OHSC).HNscells were prepared from E18 Swiss Webster mouse embryos and plated in 96‐well plates. All experiments were run on (10 Days in vitro). A FlexStation2 was used to monitor PbTx‐2‐induced Ca2+ influx.OHSCSlices were prepared from P5, Thy‐1 YFP transgenic, mouse pups. Slices were treated with Pbtx‐2 18H after plating. A Leica SP8 confocal microscope was used to generate Z‐stack images of neurons or dendrites. Imaris‐XT software was used to reconstruct 3D‐images of the dendritic arbor, run Sholl analysis, and measure spine density.Results and ConclusionIn HNs, the EC50 for PbTx‐2‐induced Ca2+ influx was 376 nM (95% CI, 281–504 nM). The PbTx‐2 triggered Ca2+ influx in HNs was through the reverse mode of operation of Na+/Ca2+ exchanger, the NMDAR and the L‐type Ca2+ channel pathways. In OHSC, four‐day treatment with PbTx‐2 (100 nM) produced significant increases in dendritic arbor complexity as determined by Sholl analysis (p<0.005, ANOVA). PbTx‐2 treatment (300 nM) produced significant increases in spine density (p<0.001, ANOVA). Modifying the gating properties of VGNaC using PbTx‐2 enhanced the morphology of hippocampal neurons. Overall, these studies are consistent with the hypothesis that VGNaC activation may represent a novel pharmacological strategy to regulate NMDAR function and aid in recovery post‐stroke.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GKAP Acts as a Genetic Modulator of NMDAR Signaling to Govern Invasive Tumor Growth

SummaryGenetic linkage analysis previously suggested that GKAP, a scaffold protein of the N-methyl-D-aspartate receptor (NMDAR), was a potential modifier of invasion in a mouse model of pancreatic neuroendocrine tumor (PanNET). Here, we establish that GKAP governs invasive growth and treatment response to NMDAR inhibitors of PanNET via its pivotal role in regulating NMDAR pathway activity. Combining genetic knockdown of GKAP and pharmacological inhibition of NMDAR, we implicate as downstream effectors FMRP and HSF1, which along with GKAP demonstrably support invasiveness of PanNET and pancreatic ductal adenocarcinoma cancer cells. Furthermore, we distilled genome-wide expression profiles orchestrated by the NMDAR-GKAP signaling axis, identifying transcriptome signatures in tumors with low/inhibited NMDAR activity that significantly associate with favorable patient prognosis in several cancer types.

D-Serine administration affects nitric oxide synthase 1 adaptor protein and DISC1 expression in sex-specific manner

Antipsychotic medications are inefficient at treating symptoms of schizophrenia (SCZ), and N-methyl d-aspartate receptor (NMDAR) agonists are potential therapeutic alternatives. As such, these agonists may act on different pathways and proteins altered in the brains of patients with SCZ than do antipsychotic medications. Here, we investigate the effects of administration of the antipsychotic haloperidol and NMDAR agonist d-serine on function and expression of three proteins that play significant roles in SCZ: nitric oxide synthase 1 adaptor protein (NOS1AP), dopamine D2 (D2) receptor, and disrupted in schizophrenia 1 (DISC1). We administered haloperidol or d-serine to male and female Sprague Dawley rats via intraperitoneal injection for 12 days and subsequently examined cortical expression of NOS1AP, D2 receptor, and DISC1. We found sex-specific effects of haloperidol and d-serine treatment on the expression of these proteins. Haloperidol significantly reduced expression of D2 receptor in male, but not female, rats. Conversely, d-serine reduced expression of NOS1AP in male rats and did not affect D2 receptor expression. d-serine treatment also reduced expression of DISC1 in male rats and increased DISC1 expression in female rats. As NOS1AP is overexpressed in the cortex of patients with SCZ and negatively regulates NMDAR signaling, we subsequently examined whether treatment with antipsychotics or NMDAR agonists can reverse the detrimental effects of NOS1AP overexpression in vitro as previously reported by our group. NOS1AP overexpression promotes reduced dendrite branching in vitro, and as such, we treated cortical neurons overexpressing NOS1AP with different antipsychotics (haloperidol, clozapine, fluphenazine) or d-serine for 24 h and determined the effects of these drugs on NOS1AP expression and dendrite branching. While antipsychotics did not affect NOS1AP protein expression or dendrite branching in vitro, d-serine reduced NOS1AP expression and rescued NOS1AP-mediated reductions in dendrite branching. Taken together, our data suggest that d-serine influences the function and expression of NOS1AP, D2 receptor, and DISC1 in a sex-specific manner and reverses the effects of NOS1AP overexpression on dendrite morphology.

Functional characterization of α7 nicotinic acetylcholine and NMDA receptor signaling in SH-SY5Y neuroblastoma cells in an ERK phosphorylation assay

In the present study, the functional properties of α7 nicotinic acetylcholine receptors (α7 nAChRs) and N-methyl-D-aspartate receptors (NMDARs) endogenously expressed in SH-SY5Y human neuroblastoma cells were characterized in an extracellular-signal regulated kinase (ERK) phosphorylation assay. Both choline and N-methyl-D-aspartate (NMDA) mediated robust concentration-dependent increases in ERK phosphorylation in the SH-SY5Y cells, exhibiting EC50 values in good agreement with those reported for the agonists at recombinant α7 nAChRs and NMDARs, respectively. Importantly, the responses evoked by choline (10 mM) and by NMDA (50 μM) were significantly inhibited by the α7-selective antagonist α-bungarotoxin (100 nM) and by the NMDAR-selective antagonist MK-801 (50 μM), respectively. The increased ERK phosphorylation levels observed upon co-application of choline (1, 3, 10 mM) and NMDA (50 μM) compared to those produced by the two agonists on their own were fully reconcilable with additive effects and did not reveal substantial synergy between α7 nAChR and NMDAR signaling. Interestingly, however, the responses evoked by the “choline (10 mM) – NMDA (50 μM)” combination were almost completely inhibited by α-bungarotoxin (100 nM) as well as by MK-801 (50 μM), suggesting some sort of a link between α7 nAChR- and NMDAR-mediated ERK phosphorylation. Finally, oligomeric amyloid-β1–42 peptide (1000 nM) mediated robust inhibition of the ERK phosphorylation induced by choline (10 mM), NMDA (50 μM) and the “choline (10 mM) - NMDA (50 μM)” combination. In conclusion, ERK phosphorylation measurements in SH-SY5Y cells provides a robust assay for studies of α7 nAChR- and NMDAR-mediating signaling and putative functional interactions between the receptors.

Altered avalanche dynamics in a developmental NMDAR hypofunction model of cognitive impairment

Disturbed activity patterns in cortical networks contribute to the pathophysiology of schizophrenia (SZ). Several lines of evidence implicate NMDA receptor hypofunction in SZ, and blocking NMDA receptor signaling during early neurodevelopment produces cognitive deficits in rodent models that resemble those seen in schizophrenic patients. However, the altered network dynamics underlying these cognitive impairments largely remain to be characterized, especially at the cellular level. Here, we use in vivo two-photon calcium imaging to describe pathological dynamics, occurring in parallel with cognitive dysfunction, in a developmental NMDA receptor hypofunction model. We observed increased synchrony and specific alterations in spatiotemporal activity propagation, which could be causally linked to a previously unidentified persistent bursting phenotype. This phenotype was rescued by acute treatment with the NMDA receptor co-agonist D-serine or the GABAB receptor agonist baclofen, which similarly rescued working memory performance. It was not reproduced by optogenetic inhibition of fast-spiking interneurons. These results provide novel insight into network-level abnormalities mediating the cognitive impairment induced by NMDA receptor hypofunction.

Neurogranin in the nucleus accumbens regulates NMDA receptor tolerance and motivation for ethanol seeking

Dysfunction of N-methyl-d-aspartate receptor (NMDAR) signaling in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng), a calmodulin-binding protein, is exclusively expressed in the post-synapse, and mediates NMDAR driven synaptic plasticity by regulating the calcium-calmodulin (Ca2+-CaM) pathway. To study the functional role of Ng in AUD, we administrated behavior tests including Pavlovian instrument transfer (PIT), operant conditioning, and rotarod test using Ng null mice (Ng–/– mice). We used adeno-associated virus (AAV)-mediated Ng expression and pharmacological manipulation to validate behavioral responses in Ng–/– mice. The results from our multidisciplinary approaches demonstrated that deficit of Ng increases tolerance to NMDAR inhibition and elicit faster cue reactivity during PIT without changes in ethanol reward. Operant conditioning results demonstrated that Ng–/– mice self-administered significantly more ethanol and displayed reduced sensitivity to aversive motivation. We identified that ethanol exposure decreases mGluR5 (metabotropic glutamate receptor 5) expression in the NAc of Ng–/– mice and pharmacological inhibition of mGluR5 reverses NMDAR desensitization in Ng–/– mice. Together these findings specifically suggest that accumbal Ng plays an essential role in the counterbalance between NMDAR and mGluR5 signaling; which alters NMDAR resistance, and thereby altering aversive motivation for ethanol and may ultimately contribute to susceptibility for alcohol addiction.

Autocrine, paracrine and necrotic NMDA receptor signalling in mouse pancreatic neuroendocrine tumour cells.

N-Methyl-d-aspartate receptor (NMDAR) activation is implicated in the malignant progression of many cancer types, as previously shown by the growth-inhibitory effects of NMDAR antagonists. NMDAR-mediated calcium influx and its downstream signalling depend critically, however, on the dynamics of membrane potential and ambient glutamate concentration, which are poorly characterized in cancer cells. Here, we have used low-noise whole-cell patch-clamp recording to investigate the electrophysiology of glutamate signalling in pancreatic neuroendocrine tumour (PanNET) cells derived from a genetically-engineered mouse model (GEMM) of PanNET, in which NMDAR signalling is known to promote cancer progression. Activating NMDARs caused excitation and intracellular calcium elevation, and intracellular perfusion with physiological levels of glutamate led to VGLUT-dependent autocrine NMDAR activation. Necrotic cells, which are often present in rapidly-growing tumours, were shown to release endogenous cytoplasmic glutamate, and necrosis induced by mechanical rupture of the plasma membrane produced intense NMDAR activation in nearby cells. Computational modelling, based on these results, predicts that NMDARs in cancer cells can be strongly activated in the tumour microenvironment by both autocrine glutamate release and necrosis.

The effect of postnatal manganese exposure on the NMDA receptor signaling pathway in rat hippocampus.

Overexposure to manganese (Mn) is associated with neurological disorders in children. Evidence indicated that N-methyl-d-aspartate (NMDA) receptor signaling pathway was critical for neurobehavioral function. However, whether NMDA receptor signaling pathway contributes to Mn-induced neurotoxicity remains unknown. In this study, newborn Sprague-Dawley rats were randomly assigned to four groups exposed to 0, 10, 20, and 30mg/kg of Mn2+ by intraperitoneal injection (n=10/group: five males and five females). After 3weeks of Mn exposure, messenger RNA (mRNA) and protein expression of NMDA receptor subunits (NR1, NR2A, and NR2B), cAMP-response element binding protein (CREB), and brain-derived neurotrophic factor (BDNF) in hippocampus were measured by real-time quantitative RT-PCR and Western blot. In Mn-exposed rats, decreased mRNA and protein expression of NR1, NR2A, and NR2B, CREB, and BDNF was observed. The results imply that downregulated NMDA receptor signaling pathway may be of vital importance in the neuropathological process of Mn-induced neurotoxicity.

Altered NMDA receptor-evoked intracellular Ca2+ dynamics in magnocellular neurosecretory neurons of hypertensive rats.

NMDA receptor (NMDAR)-mediated Ca2+ signalling plays a critical role in modulating hypothalamic neurosecretory function. However, whether an altered NMDAR-evoked changes in Ca2+ (NMDAR-ΔCa2+ ) signalling in magnocellular neurosecretory cells (MNCs) may contribute to neurohumoral activation during disease states is unknown. We show that activation of NMDARs evoked similar inward currents in MNCs of sham and renovascular hypertensive (RVH) rats. Despite this, a prolonged and larger NMDAR-ΔCa2+ response was observed in the latter. The exacerbated NMDAR-ΔCa2+ responses in MNCs of RVH rats affected both somatic and dendritic compartments. Inhibition of the endoplasmic reticulum sarcoendoplasmic reticulum calcium trasport ATPase (SERCA) pump prolonged NMDAR-ΔCa2+ responses in sham rats, but not in RVH rats. Our study supports an altered spatiotemporal dynamic of NMDAR-ΔCa2+ signalling in MNCs from RVH rats, partly due to blunted endoplasmic reticulum Ca2+ buffering capacity. A growing body of evidence supports an elevated NMDA receptor (NMDAR)-mediated glutamate excitatory function in the supraoptic nucleus and paraventricular nucleus of hypertensive rats that contributes to neurohumoral activation in this disease. However, the precise mechanisms underlying altered NMDAR signalling in hypertension remain to be elucidated. In this study, we performed simultaneous electrophysiology and fast confocal Ca2+ imaging to determine whether altered NMDAR-mediated changes in intracellular Ca2+ levels (NMDAR-ΔCa2+ ) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in renovascular hypertensive (RVH) rats. We found that despite evoking a similar excitatory inward current, activation of NMDARs resulted in a larger and prolonged ΔCa2+ in MNCs from RVH rats. Changes in NMDAR-ΔCa2+ dynamics were observed both in somatic and dendritic compartments. Inhibition of the sarcoendoplasmic reticulum calcium trasport ATPase (SERCA) pump activity with thapsigargin prolonged NMDAR-ΔCa2+ responses in MNCs of sham rats, but this effect was occluded in RVH rats, thus equalizing the magnitude and time course of the NMDA-ΔCa2+ responses between the two experimental groups. Taken together, our results support (1) an exacerbated NMDAR-ΔCa2+ response in somatodendritic compartments of MNCs of RVH rats, and (2) that a blunted ER Ca2+ buffering capacity contributes to the altered NMDAR-ΔCa2+ dynamics in this condition. Thus, altered spatiotemporal dynamics of the NMDAR-ΔCa2+ response stands as an underlying mechanism contributing to neurohumoral activation in neurogenic hypertension.

Magnesium boosts the memory restorative effect of environmental enrichment in Alzheimer's disease mice.

Environmental enrichment (EE) has been shown to enhance cognitive function in mouse models of Alzheimer's disease (AD). Magnesium-L-threonate (MgT) is a compound with a newly discovered effect to rescue learning and memory function in aging and AD mice. To study the additive therapeutic effect of EE combined with MgT (EM) and the potential mechanism underlying the effects. APP/PS1 mice were treated with EE, MgT, or combination of EE and MgT (EM) and compared for restored memory function. EM was more effective in improving cognition and spatial memory than either treatment alone in either long-term (12months, started at 3months old, which was before disease manifestation) or short-term (3months, started at 6months old, which was after disease manifestation) treatment. The behavioral improvement has coincided with rescue of synaptic contacts in the hippocampal region of the AD mouse brain. Immunoblots also showed that EM but neither single treatment rescued the activity reduction in CaMKII and CREB, two important downstream molecules in the N-methyl-D-aspartate receptor (NMDAR) pathway. Environmental enrichment and MgT may synergistically improve recognition and spatial memory by reducing synaptic loss and restoring the NMDAR signaling pathway in AD mice, which suggests that combination of EE and MgT may be a novel therapeutic strategy for AD.

Neuroprotective Mechanisms of Lycium barbarum Polysaccharides Against Ischemic Insults by Regulating NR2B and NR2A Containing NMDA Receptor Signaling Pathways

Glutamate excitotoxicity plays an important role in neuronal death after ischemia. However, all clinical trials using glutamate receptor inhibitors have failed. This may be related to the evidence that activation of different subunit of NMDA receptor will induce different effects. Many studies have shown that activation of the intrasynaptic NR2A subunit will stimulate survival signaling pathways, whereas upregulation of extrasynaptic NR2B will trigger apoptotic pathways. A Lycium barbarum polysaccharide (LBP) is a mixed compound extracted from Lycium barbarum fruit. Recent studies have shown that LBP protects neurons against ischemic injury by anti-oxidative effects. Here we first reported that the effect of LBP against ischemic injury can be achieved by regulating NR2B and NR2A signaling pathways. By in vivo study, we found LBP substantially reduced CA1 neurons from death after transient global ischemia and ameliorated memory deficit in ischemic rats. By in vitro study, we further confirmed that LBP increased the viability of primary cultured cortical neurons when exposed to oxygen-glucose deprivation (OGD) for 4 h. Importantly, we found that LBP antagonized increase in expression of major proteins in the NR2B signal pathway including NR2B, nNOS, Bcl-2-associated death promoter (BAD), cytochrome C (cytC) and cleaved caspase-3, and also reduced ROS level, calcium influx and mitochondrial permeability after 4 h OGD. In addition, LBP prevented the downregulation in the expression of NR2A, pAkt and pCREB, which are important cell survival pathway components. Furthermore, LBP attenuated the effects of a NR2B co-agonist and NR2A inhibitor on cell mortality under OGD conditions. Taken together, our results demonstrated that LBP is neuroprotective against ischemic injury by its dual roles in activation of NR2A and inhibition of NR2B signaling pathways, which suggests that LBP may be a superior therapeutic candidate for targeting glutamate excitotoxicity for the treatment of ischemic stroke.

Vitamin B6 prevents isocarbophos-induced vascular dementia in rats through N-methyl-D-aspartate receptor signaling

ABSTRACTBackground: We have previously reported that the long-term exposure of organophosphorus induces vascular dementia (VD) in rats. As a coenzyme, vitamin B6 is mainly involved in the regulation of metabolisms. Whether vitamin B6 improves VD remains unknown. Methods: The model of VD was induced by feeding rats with isocarbophos (0.5 mg/kg per two day, 12 weeks). The blood flow of the posterior cerebral artery (PCA) in rat was assessed by transcranial Doppler (TCD). The learning and memory were evaluated by the Morris Water Maze (MWM) test. Results: Administration of vitamin B6 increased the blood flow in the right and left posterior cerebral arteries and improved the functions of learning and memory in isocarbophos-treated rats. Vitamin B6 increased the protein levels of N-methyl-D-aspartate receptor (NMDAR) 2B, postsynaptic densities (PSDs) protein 95, and calmodulin-dependent protein kinase II (CaMK-II) in the hippocampus, which were decreased by isocarbophos in rats. Morphological analysis by light microscope and electronic microscope indicated disruptions of the hippocampus caused by isocarbophos were normalized by vitamin B6. Importantly, the antagonist of NMDAR signaling by eliprodil abolished these beneficial effects produced by vitamin B6 on PCA blood flow, learning, memory, and hippocampus structure in rats, as well as the protein expression of NMDAR 2B, PSDs protein 95, and CaMK-II in the hippocampus. Conclusion: Vitamin B6 activates NMDAR signaling to prevent isocarbophos-induced VD in rats.

Stress hormone rapidly tunes synaptic NMDA receptor through membrane dynamics and mineralocorticoid signalling

Stress hormones, such as corticosteroids, modulate the transmission of hippocampal glutamatergic synapses and NMDA receptor (NMDAR)-dependent synaptic plasticity, favouring salient behavioural responses to the environment. The corticosterone-induced synaptic adaptations partly rely on changes in NMDAR signalling, although the cellular pathway underlying this effect remains elusive. Here, we demonstrate, using single molecule imaging and electrophysiological approaches in hippocampal neurons, that corticosterone specifically controls GluN2B-NMDAR surface dynamics and synaptic content through mineralocorticoid signalling. Strikingly, extracellular corticosterone was sufficient to increase the trapping of GluN2B-NMDAR within synapses. Functionally, corticosterone-induced potentiation of AMPA receptor content in synapses required the changes in NMDAR surface dynamics. These high-resolution imaging data unveiled that, in hippocampal networks, corticosterone is a natural, potent, fast and specific regulator of GluN2B-NMDAR membrane trafficking, tuning NMDAR-dependent synaptic adaptations.

Afferent specific role of NMDA receptors for the circuit integration of hippocampal neurogliaform cells

Appropriate integration of GABAergic interneurons into nascent cortical circuits is critical for ensuring normal information processing within the brain. Network and cognitive deficits associated with neurological disorders, such as schizophrenia, that result from NMDA receptor-hypofunction have been mainly attributed to dysfunction of parvalbumin-expressing interneurons that paradoxically express low levels of synaptic NMDA receptors. Here, we reveal that throughout postnatal development, thalamic, and entorhinal cortical inputs onto hippocampal neurogliaform cells are characterized by a large NMDA receptor-mediated component. This NMDA receptor-signaling is prerequisite for developmental programs ultimately responsible for the appropriate long-range AMPAR-mediated recruitment of neurogliaform cells. In contrast, AMPAR-mediated input at local Schaffer-collateral synapses on neurogliaform cells remains normal following NMDA receptor-ablation. These afferent specific deficits potentially impact neurogliaform cell mediated inhibition within the hippocampus and our findings reveal circuit loci implicating this relatively understudied interneuron subtype in the etiology of neurodevelopmental disorders characterized by NMDA receptor-hypofunction.

Involvement of NMDA receptors in the antidepressant-like effect of tramadol in the mouse forced swimming test.

Tramadol is an analgesic agent that is mainly used to treat moderate to severe pain. There is evidence that tramadol may have antidepressant property. However, the mechanisms underlying the antidepressant effects of tramadol have not been elucidated yet. Considering that fact that N-methyl-d-aspartate (NMDA) receptor signaling may play an important role in the pathophysiology of depression, the aim of the present study was to investigate the role of NMDA receptor signaling in the possible antidepressant-like effects of tramadol in the mouse forced swimming test (mFST). We found that tramadol exerted antidepressant-like effects at high dose (40mg/kg, intraperitoneally [i.p.]) in the mFST. Co-administration of non-effective doses of NMDA receptor antagonists (ketamine [1mg/kg, i.p.], MK-801 [0.05mg/kg, i.p.], or magnesium sulfate [10mg/kg, i.p.]) with sub-effective dose of tramadol (20mg/kg, i.p.) exerted significant antidepressant-like effects in the mFST. The antidepressant-like effects of tramadol (40mg/kg) was also inhibited by pre-treatment with non-effective dose of the NMDA receptor agonist NMDA (75mg/kg, i.p.). Our data suggest a role for NMDA receptor signaling in the antidepressant-like effects of tramadol in the mFST.

Pro-death NMDA receptor signaling is promoted by the GluN2B C-terminus independently of Dapk1.

Aberrant NMDA receptor (NMDAR) activity contributes to several neurological disorders, but direct antagonism is poorly tolerated therapeutically. The GluN2B cytoplasmic C-terminal domain (CTD) represents an alternative therapeutic target since it potentiates excitotoxic signaling. The key GluN2B CTD-centred event in excitotoxicity is proposed to involve its phosphorylation at Ser-1303 by Dapk1, that is blocked by a neuroprotective cell-permeable peptide mimetic of the region. Contrary to this model, we find that excitotoxicity can proceed without increased Ser-1303 phosphorylation, and is unaffected by Dapk1 deficiency in vitro or following ischemia in vivo. Pharmacological analysis of the aforementioned neuroprotective peptide revealed that it acts in a sequence-independent manner as an open-channel NMDAR antagonist at or near the Mg2+ site, due to its high net positive charge. Thus, GluN2B-driven excitotoxic signaling can proceed independently of Dapk1 or altered Ser-1303 phosphorylation.

Mechanism and properties of positive allosteric modulation of N-methyl-d-aspartate receptors by 6-alkyl 2-naphthoic acid derivatives

The theory that N-methyl-d-aspartate receptor (NMDAR) hypofunction is responsible for the symptoms of schizophrenia is well supported by many pharmacological and genetic studies. Accordingly, positive allosteric modulators (PAMs) that augment NMDAR signaling may be useful for treating schizophrenia. Previously we have identified several NMDAR PAMs containing a carboxylic acid attached to naphthalene, phenanthrene, or coumarin ring systems. In this study, we describe several functional and mechanistic properties of UBP684, a 2-naphthoic acid derivative, which robustly potentiates agonist responses at each of the four GluN1a/GluN2 receptors and at neuronal NMDARs. UBP684 increases the maximal l-glutamate/glycine response while having minor subunit-specific effects on agonist potency. PAM binding is independent of agonist binding, and PAM activity is independent of membrane voltage, redox state, and the GluN1 exon 5 N-terminal insert. UBP684 activity is, however, markedly pH-dependent, with greater potentiation occurring at lower pHs and inhibitory activity at pH 8.4. UBP684 increases channel open probability (Po) and slows receptor deactivation time upon removal of l-glutamate, but not glycine. The structurally related PAM, UBP753, reproduced most of these findings, but did not prolong agonist removal deactivation time. Studies using cysteine mutants to lock the GluN1 and GluN2 ligand-binding domains (LBDs) in the agonist-bound states indicate that PAM potentiation requires GluN2 LBD conformational flexibility. Together, these findings suggest that UBP684 and UBP753 stabilize the GluN2 LBD in an active conformation and thereby increase Po. Thus, UBP684 and UBP753 may serve as lead compounds for developing agents to enhance NMDAR activity in disorders associated with NMDAR hypofunction.