Dissertations / Theses on the topic 'NMDA'
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Garside, Molly. "Interactions of NMDA receptor subunits." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414089.
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De, Jesus Domingues António Miguel. "Novel NMDA receptor splice variants." Thesis, University of Leicester, 2010. http://hdl.handle.net/2381/7884.
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Injury to white matter oligodendrocytes is central to several important disorders including cerebral palsy, stroke and multiple sclerosis. Dr. Fern’s group and others have recently shown that NMDA receptors are present on oligodendrocyte processes and mediate injury of these myelinating processes. The pharmacological profile of NMDA receptors present in white matter is quite unique. NMDA receptors are composed of the subunits NR1, NR2A-D and NR3A-B that assemble to form a heterotetrameric complex. Importantly, the subunit composition determines the properties of the receptor complex. Two possibilities were proposed to explain the unusual profile of NMDA receptor-mediated currents in white matter: (1) novel splice variants are expressed in glia and/or (2) the major NMDA receptor complex present is composed of an uncharacterized NMDA receptor subunit stoichiometry. In this PhD project I explored these two hypotheses. NR1, NR2C and NR3A, which are thought to be the major components of NMDA receptors in oligodendrocytes, were cloned from the myelinating rat optic nerve. In addition, all known NMDA receptor subunits were cloned from the neonate rat brain. This analysis revealed that only a subset of NR1 splice variants, those lacking exon 5, is expressed in white matter. I have also cloned NR3B and identified several putative novel splice variants of this subunit in both the optic nerve and the brain. Novel splice variants of NR2B-D were also cloned. Four of these novel NR3B variant were characterized by single cell Ca2+ imaging revealing that the novel variants form function receptors. Furthermore, NR3 subunits influence NMDA receptor glutamate sensitivity and Mg2+ in an NR2-dependent manner. The results here presented reveal a previous uncharacterized wealth of NMDA receptor splice variants which modify NMDA receptor physiology.
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McClean, Mercedes. "NMDA antagonists as antinociceptive agents." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311427.
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Fedele, Laura. "Dysfunctional NMDA receptors in neurological disorders." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10043277/.
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N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that together with a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and kainate receptors mediate the vast majority of the fast excitatory neurotransmission in the central nervous system. Given this role, any dysfunction in neurotransmission is likely to have a severe impact on brain physiology. Recent mutations have been reported in NMDAR subunits that cause patients to suffer with a variety of neurodevelopmental disorders. Here, we use multidisciplinary structural modelling, site-directed mutagenesis, electrophysiology and kinetic modelling techniques to investigate how de novo missense mutations in distinct regions of the GluN2B subunit, affect NMDAR function. We predicted that these mutations would have pathophysiological implications and we sought to examine their effects on the cellular and molecular function of NMDARs. We developed a virtually complete 3D model of the human GluN1-GluN2B receptor based on the recently solved crystal structures of the frog and rat NMDARs. The human NMDAR structure locates the positions of the residues of interest, allows deductions about their potential impact on the protein as well as provides insight into the binding sites for Mg2+ and memantine using molecular docking. The functional effects of the missense mutations were first analysed in recombinant NMDARs and revealed gain-of-function and loss-of-function phenotypes, with some lacking an overt phenotype. We selected four most profound phenotypes for study in hippocampal cultured neurons revealing how these mutations can compromise excitatory neurotransmission. In addition, we also explored the therapeutic potential of the FDA-approved channel blocker memantine both in heterologous system as well as on excitatory neurotransmission as a potential therapeutic. Overall, the results suggest strong correlations between the effects of the missense mutations with patient phenotypes. Moreover, the study indicates which pharmacotherapeutic interventions are most likely to be successful as targeted therapies.
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Tang, Tina Tze-Tsang. "Molecular mechanisms regulating NMDA receptor trafficking." Thesis, University of Bristol, 2009. http://hdl.handle.net/1983/684cfaf8-a402-4518-b710-ebc237a3e170.
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Tanguay, Jeffrey Joseph. "The effects of redox agents on NMDA receptor-mediated glutamate release from rat hippocampal slices and on NMDA-mediated toxicity in murine fibroblasts expressing human NMDA receptors." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape9/PQDD_0005/MQ42695.pdf.
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Marwick, Katherine Freda McEwan. "Functional consequences of mutations in GRIN2A and GRIN2B associated with mental disorders." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/23549.
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GRIN2A and GRIN2B encode the GluN2A and GluN2B subunits of the NMDA receptor, a subtype of ionotropic glutamate receptor that displays voltage-dependent block by Mg2+ and a high permeability to Ca2+. These receptors play important roles in synaptogenesis, synaptic transmission and synaptic plasticity, as well as contributing to neuronal loss and dysfunction in several neurological disorders. Recently, individuals with a range of childhood onset epilepsies, intellectual disability and other neurodevelopmental abnormalities have been found to carry heterozygous gene-disrupting or protein-altering point mutations in GRIN2A and GRIN2B. This thesis addresses the hypothesis that these point mutations cause key functional disturbances to NMDA receptor properties that contribute to neurodevelopmental disorders. To test this hypothesis, a group of related mutations were selected for functional assessment in heterologous systems: four missense mutations affecting residues in or near the subunit pore regions, all of which are associated with epilepsy and intellectual disability. To model the impact of gene disrupting mutations in GRIN2A, a preliminary analysis of the functional consequences of GluN2A haploinsufficiency in a genetically modified rat was also performed. Three of the four missense mutations were found to be associated with profound alterations in fundamental NMDA receptor properties: compared to wild type, GluN2AN615K was found to reduce Mg2+ block, GluN2BN615I and GluN2BV618G to cause potentiation by Mg2+, and GluN2AN615K and GluN2BN615I showed reduced conductance. GluN2AR586K was not found to influence the parameters assessed. When GluN2AN615K was expressed alongside wild type subunits in the same NMDA receptor, it was found to have a dominant negative effect. Finally, I established successful gene targeting in a new rat Grin2A knock-out model, and observed that heterozygous neurons had lower GluN2A protein expression and current density, making a good model to study human epilepsies associated with loss of a GRIN2A allele. This thesis provides evidence that three missense mutations in GRIN2A and GRIN2B affect physiologically important properties of the NMDA receptor, and that GluN2A haploinsufficiency influences important neural properties in vitro. This is consistent with these mutations causing disease and highlights these and related mutations as potential therapeutic targets in the future.
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Begg, Alison Jane. "Analysis of NMDA receptor regulated gene expression." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/25053.
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Mutation of two NMDA receptor complex proteins, PSD95 and SynGAP, results in altered phenotypes in NMDA dependent phenomena. As components of the receptor complex, necessary for NMDA mediated signalling, mutation of these proteins may alter pathways that regulate gene expression. Affymetrix microarray analysis of RNA from PSD95-/- forebrain and SynGAP-/- hippocampi, compared to wildtype samples, revealed significant changes, greater than 1.5 fold, in a limited numbers of genes. Of the 12000 transcripts analysed 0.22% were significantly altered in PSD95 mutant tissue and 0.35% were changed in SynGAP mutant tissue. The genes altered in each genotype were distinct, apart from an overlap of 3 genes that were found similarly down regulated in PSD95-/- forebrain and SynGAP-/- hippocampi. These 3 genes, c-fos, nur77 and egr2, are activity dependent and are regulated, in part, through the NMDA receptor. It is possible that changes in gene expression may underlie the electrophysiological and behavioural phenotypes seen PSD95-/- and SynGAP+/- animals. It is likely that the genes altered in each of the mutants represent a subset of the genes regulated by NMDA receptor signalling. To get an understanding of the complete set of genes regulated by the NMDA receptor complex and in vitro method of NMDA receptor stimulation was sought. A primary cultured neuron system was used, allowing NMDA receptor activity to be manipulated by the pharmacological treatment of the culture medium. NMDA and bicuculline treatment of primary cultured cortical neurons proved ineffective methods of inducing activity dependent genes as measured by cfos expression. However, AP5 treatment of primary cultured neurons decreased activity dependent gene expression. Electrophysiological analysis of the cultures revealed that bicuculline treatment had no significant effect on culture activity, where as AP5 treatment caused a significant decrease in neuronal activity.
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Sokolovski, Alexandra. "Sigma-1 Receptors Modulate NMDA Receptor Function." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23652.
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The sigma-1 receptor (σ-1R) is an endoplasmic reticulum (ER) protein that modulates a number of ion channels. It is hypothesized that σ-1Rs activated with agonist translocate to the plasma membrane. The σ-1R potentiates N-methyl-D-aspartate Receptors (NMDARs), important constituents of synaptic plasticity. NMDARs are anchored in the plasma membrane by Postsynaptic Density Protein-95 (PSD-95). The mechanism behind σ-1R modulation of NMDARs is not known. The results of my investigation confirm that σ-1Rs localize extrasomatically. Following σ-1R activation, σ-1R localization to dendrites and postsynaptic densities (PSDs) is upregulated. Unpublished work from our lab has shown that σ-1Rs associate with PSD-95 and NMDARs. Furthermore, immunocytochemistry (ICC) showed σ-1R colocalization with PSD-95 and NMDAR subunits. After σ-1R activation there was significantly increased colocalization between σ-1R, PSD-95, and GluN2B. Overall, this study may have provided insight into the molecular mechanism behind σ-1R modulation of NMDARs, which could have implications in the understanding of synaptic plasticity.
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Bakshi, Deeksha. "The role of NMDA receptors in excitotoxicity." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/43907.
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NMDA receptors are glutamate-gated cation channels named after their prototypical selective agonist NMDA. The channels occur as multiple subtypes, which are formed from interactions between different receptor subunits. NMDA receptor subunits are classified into three families: NR1, NR2A-D, and NR3A, B.
NMDA receptors are implicated in HD pathology. During HD, a subset of medium-sized aspiny interneurons in the striatum that co-localize SST, NPY, and the enzyme NOS are selectively spared. In contrast, medium-sized spiny cells that constitute 80 % of all striatal neurons undergo selective neurodegeneration. While it was suggested that the interneurons survive because they lack NMDA receptors, studies including from our lab have shown the presence of NR1 in SST-positive striatal neurons. The finding of NR1 expression and co-localization with SST-positive neurons indicates that NMDA receptor-induced toxicity may be regulated in a receptor-specific manner. Therefore, the present study was conducted to investigate whether NMDA application leads to toxicity that is receptor-specific in HEK293 cells stably transfected with NR1, NR2A, or NR2B.
The main findings of this study indicate that NMDA application causes cell death, which varies in intensity and nature, depending upon the NMDA concentration applied, and the receptor-type expressed by the cells. Cells expressing NR1 were found to undergo apoptosis but not necrosis, while cells expressing NR2A/NR2B underwent both apoptosis and necrosis in a receptor-specific manner. In cells expressing NR2A/NR2B, exposure to low concentrations of NMDA resulted in cell death that was predominantly apoptotic. In contrast, exposure to high concentrations of NMDA produced mostly necrosis. In cells expressing NR1, NMDA application caused apoptosis, which exhibited a gradual increase in response to greater concentrations of NMDA. In addition, cell death through apoptosis and/or necrosis was determined to be the greatest at all NMDA concentrations in cells expressing NR2B, followed by those expressing NR2A, and then NR1. Taken together, these results indicate that the activation of receptors formed by NR1, NR2A, or NR2B have different toxic consequences. Thus, the selective neurodegeneration observed during HD may be due to the variation in expression levels of NR1, NR2A, and NR2B between medium-sized aspiny interneurons and medium-sized spiny projection neurons.
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Kaye, Samantha Louise. "Modelling and simulation studies of NMDA receptors." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442836.
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Mallon, Andrew Peter. "An investigation of NMDA receptor subunit pharmacology." Thesis, University of Glasgow, 2004. http://theses.gla.ac.uk/3127/.
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N-Methyl-D-aspartate (NMDA) receptors are critically involved in synaptic transmission, neural development and various forms of neuronal plasticity including long-term potentiation (LTP) and long-term depression (LTD). They are also involved in the production of neuronal damage following excessive activation by glutamate released as a result of hypoxia or ischaemia. Each heteromeric receptor includes one or two NRl subunits, at least two of the four NR2A-D subunits and less usually the NR3AJB subunits. This study demonstrates that the putative NR2B subunit-containing NMDA receptor antagonist Ro 25-6981 potentiates the effects ofNMDA on rat hippocampal slices. The NR2A subunit antagonist PEAQX blocks the effects of NMDA alone and the potentiated response following Ro 25-6981 application. Furthermore, Ro 25-6981 was not neuroprotective as reported previously but unexpectedly precipitated excitotoxicity. The potentiating effect of Ro 25-6981 required around 20 minutes to become apparent, took a further 30 minutes to reach its maximum effect and was irreversible. It was not prevented by staurosporine (a broad-spectrum protein kinase inhibitor), okadaic acid (a potent inhibitor of the serine/threonine protein phosphatases types 1 and 2A) or anisomycin (a protein synthesis inhibitor). However, the potentiation was prevented by cyclosporin A (an inhibitor of Ca2+/calmodulin-dependent phosphatase 2B [calcineurin]). The results indicate that in an intact neuronal network, NR2B subunits tonically gate NR2A subunit-containing receptor function by a negative coupling mechanism involving ca1cineurin activation. NMDA receptor-dependent LTP induced by high frequency stimulation was prevented by PEAQX, an NR2A antagonist. Ro 25-6981 was unable to prevent L TP induction but was associated with a marginal reduction in the magnitude of LTP induced. There is evidence for the binding of homoquinolinic acid to an NMDAinsensitive novel binding site in the brain. This study investigated the pharmacology of homoquinolinate on the evoked field excitatory synaptic potential (fEPSP) recorded from the CAl area of rat hippocampal slices. Two NMDA receptor agonists, quinolinic acid 150/lM and homoquinolinic acid 2.5/lM, caused an approximately 50% inhibition of fEPSP slope. Paired-pulse studies suggested there might be a presynaptic component to this action that is independent of presynaptic adenosine Al receptor activation. The broad-spectrum EAA antagonist kynurenic acid and the NMDA receptor blockers 2-amino-5-phosphonopentanoic acid and dizocilpine could prevent the inhibition of fEPSP slope. None of these antagonists revealed any other NMDA-insensitive activity of homoquinolinic acid. The use of 2-carboxy-3-carboxymethylquinoline (CCMQ) to displace the reported NMDA-insensitive binding had no effect on either baseline fEPSP slope or the depression caused by homoquinolinic acid. It was also apparent that responses to homoquinolinic acid were blocked completely by the NR2A subunit-selective antagonist PEAQX, but not by the NR2B subunit-selective blocker Ro 25-6981. It was concluded that the novel binding site for homoquinolinic acid does not affect synaptic potentials in the hippocampus and that homoquinolinic acid appears to be a selective agonist at NMDA receptors that include the NR2A subunit. Although the NR2B agonist site may be maximally activated under normal conditions and therefore it is not possible to observe any additional effects upon fEPSP slope. This study next investigated the negative coupling between NR2B and NR2A subunit-containing receptors, combining the NR2A1B subunit selective agonist HQA with the NR2B and NR2A selective antagonists Ro 25-6981 and PEAQX. The negative coupling observed previously with applications of NMDA was also seen using HQA and QA. The potentiation of responses to HQA by Ro 25-6981 application was also associated with an enhancement of paired-pulse interactions. The subsequent application of PEAQX was able to block both the depression of fEPSP slope and the associated enhancement of paired-pulse interactions. The presence of a presynaptic element during applications of HQA alone and potentiated responses alike and the blockade of these effects by PEAQX suggests the NR2A subunit-containing NMDA receptor is responsible for the presynaptic effects acting either directly at presynaptic sites or indirectly at postsynaptic sites leading to the raising of a retrograde signal. The NR2B subunit in both its activated and antagonised state was associated with enhancements in paired-pulse interactions which suggest that it is not able to modulate directly the presynaptic element. However, whilst paired-pulse interactions are generally accepted to he presynaptic phenomena, it does not follow that postsynaptic effects cannot influence the appearance of changes in these interactions in field recordings. The absence of any observable difference between HQA, QA and NMDA results suggests that the NR2D subunit is not obviously involved in these processes.
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Hitchcock, Ian Stuart. "Functional activity of NMDA receptors on megakaryocytes." Thesis, University of York, 2003. http://etheses.whiterose.ac.uk/9856/.
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Ryan, Tomás John. "Functional investigation of NMDA receptor molecular evolution." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608544.
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Esmenjaud, Jean-Baptiste. "Dynamique structurale et allostérie des récepteurs NMDA." Thesis, Paris Sciences et Lettres (ComUE), 2018. http://www.theses.fr/2018PSLEE016/document.
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Les récepteurs ionotropiques du glutamate sont responsables de la vaste majorité de la neurotransmission excitatrice rapide dans le système nerveux central. Parmi eux, les récepteurs NMDA (rNMDA) sont les médiateurs de la plasticité synaptique, fondement cellulaire des processus d’apprentissage et de mémoire. Leurs dysfonctionnements sont impliqués dans de nombreuses pathologies neurologiques et psychiatriques comme les maladies d’Alzheimer et de Parkinson, l’épilepsie et la schizophrénie. Les rNMDA forment des complexes hétérotétramériques massifs (>500 kDa) dotés de propriétés allostériques uniques grâce à un ensemble de 8 domaines extracellulaires bilobés organisé en deux strates superposées : la couche de domaines N-terminaux (NTD) et la couche de domaines de liaison de l’agoniste (ABD). Malgré un nombre croissant de structures complètes de rNMDA, le mécanisme de transduction permettant aux interactions entre ces domaines de contrôler l’activité du récepteur restait inconnu. En combinant analyse expérimentale et computationnelle, nous montrons qu’un mouvement de roulis à l’interface entre les deux dimères de la couche d’ABD est un déterminant clé du processus d’activation et de modulation des rNMDA. Cette rotation des deux dimères d’ABD constitue un commutateur conformationnel qui règle l’ouverture du canal en fonction de la conformation des NTD situés à l’opposé. Ce travail révèle comment des changements conformationnels concertés entre couches de domaines gouvernent l’activité des rNMDA. Il illumine notre compréhension d’un récepteur synaptique majeur du système nerveux central et ouvre la voie à la conception de nouveaux agents pharmacologiques ciblant le mécanisme allostérique élucidéIonotropic glutamate receptors are responsible for the vast majority of fast excitatory neurotransmission in the central nervous system. Among them, NMDA receptors (NMDARs) are key mediators of synaptic plasticity, which is considered as the cellular basis of learning and memory. NMDAR dysfunction is implicated in numerous neurological and psychiatric brain disorders such as Alzheimer and Parkinson’s disease, epilepsy and schizophrenia. NMDAR form massive hetero tetrameric complexes (>500 kDa) endowed with unique allosteric capacity provided by a cluster of eight extracellular clamshell-like domains arranged as two superimposed layers: the Nterminal domain (NTD) layer and the agonist binding domain (ABD) layer. Despite an increasing number of full-length NMDAR structures, the transduction mechanism by which these domains interact in an intact receptor to control its activity remained poorly understood. Combining experimental and in silico analysis, we identify a rolling motion at an interface between the two constitute dimers in the ABD layer as a key determinant in NMDAR activation and modulation pathways. This rotation of the two ABD dimers acts as a conformational switch that tunes channel opening depending on the conformation of the membrane-distal NTD layer. This work unveils how NMDAR domains move and operate in a concerted manner to transduce conformational changes between layers and command receptor activity. It illuminates our understanding of a major synaptic receptor of the central nervous system and paves the way for the development of new pharmacological tools targeting the elucidated allosteric mechanism
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Pöhler, Thomas. "Synthese kleiner Substanzbibliotheken zur Untersuchung von Polyamin-Bindungsstellen des NMDA-Rezeptors." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969360959.
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Atlason, Palmi por. "The folding and assembly of NMDA receptor subunits." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487137.
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The NMDA receptor is a member of the ionotropic glutamate receptor family, which includes the AMPA and kainate receptors. Functional NMDA receptors are heteromeric and made up from three receptor subunit families; NR1, NR2 and NR3. Currently, the receptor is thought to form in a dimer-of-dimer assembly with two NRI subunits and two NR2 subunits. The NR3 can associate with both NRI and NR2 to form a unique receptor or NRI alone to form an excitatory glycine receptor. The exact assembly pathway and the nature of assembly intermediates are currently unknown. The folding status of NR1, NR2A and NR3A was examined in heterologous systems, both individually expressed and co-expressed. It was found that while NRI appeared to fold well when expressed on its own and contain relatively few free cysteine residues, the NR3A subunit appeared to form aggregates when analysed under non-reducing conditions. BothNR2A and NR3A aggregated when treated with the sulfhydryl crosslinker BMH, indicating mUltiple free cysteine residues and a degree of misfolding. NRI migrated as a dimer when treated with BMH. A small pool of fast degrading NRI was detected but the majority of the protein was very stable in cells. NR2A and NR3A were turned over rapidly when expressed on their own with NR2A showing stabilisation in the presence of NRI. FRAP analysis indicated a degree of misfolding of NR2A and NR3A. NRI readily associated with both NR2A and NR3A but no association of NR2A and NR3A was detected in the absence ofNRl. The analysis of receptor assembly using BiFC confirmed the homodimerization of NRI but failed to give any evidence for homodimerization of either NR2A or NR3A. The BiFC analysis further indicates that the homodimer of NRI is readily disassociable. The robust complementation seen between NRI and NR2A, coupled with the absence of complementation ofNR2A and NR2A in the presence ofNRl suggest the preferential assembly of heterodimers. Possibly, steric constraints prevent complementation in the tetramer, suggesting a 1-2-1-2 symmetry around the pore. A similar constraint due to a coiled-coil domain in NR3A might explain the absence of complementation between NRI and NR3A. . Taken together, the results suggest the central role of NRI in the folding and assembly of the other subunit families. The probable assembly pathway suggests recruitment of NRI from an intracellular pool to associate with newly synthesised NR2 or NR3 to form heterodimers which then assemble to form the functional tetrameric receptor.
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White, Lynn H. "NMDA receptor blockade and spatial learning : a reinvestigation." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69672.
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N-methyl-D-aspartate (NMDA) receptor activation is believed necessary for certain types of learning. The present experiments investigated the effects of the NMDA antagonist, MK-801, on spatial learning and memory in rats. Experiment 1 tested the effects of MK-801 on the acquisition and retention of a water maze task. MK-801 produced a performance, but not a spatial learning deficit. Experiment 2 tested the effects of MK-801 on the acquisition and retention of a radial arm maze task (RAM). MK-801 had no effect on initial acquisition and retention, but impaired subsequent reversal learning when the pattern of rewarded and unrewarded arms was reversed. Experiment 3 investigated the effects of MK-801 on RAM reversal learning in rats previously trained on the initial task in the absence of drugs. MK-801 produced a dose dependent impairment on reversal learning. These results are consistent with one interpretation that MK-801 impairs the ability to suppress interference from previously learned information.
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Gutnikov, Sergei A. "Behavioural studies of the NMDA system in rats." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294382.
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McClymont, David W. "Exploring open channel block of the NMDA receptor." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12996/.
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The G1uN3 subunits of the NMDA receptor are thought to reduce the Ca 2+ permeability and Mg2+ sensitivity of NMDA receptors. cRNA for rat NMDA receptor subunits were injected into Xenopus oocytes and responses were recorded using two electrode voltage clamp at -100, -75 and -50 mV. G1uN1-1a/2A, GluN1-1a/2A/3A and G1uN1-1a/2A/3B containing receptors were characterised using Mgz+, memantine, philanthotoxin-343, methoctramine and MK-801. IC50 values were calculated and generally showed significant increases between those containing G1uN1-1a/2A/3 subunits and G1uN1-1a/2A, while those with G1uN3B were found to be significantly higher than G1uN3A. Activity was also typically shown to be partially restored with mutations at the N and N+1 site asparagines of G1uN3A. As the ICS0 was only partially restored the changes cannot be attributed to the loss of the N-site alone. Further differences may be due to a constricted threonine ring within the M3 vestibule region, or due to continued reduced flux through the channel. Another possibility is that to restore block it may require both the double N and N+1 mutation at the N-site. Multi-target-directed ligands combine two pharmacophores to produce drugs which retain the properties of the constituents. Memantine has been approved for use in Alzheimer's disease and there is a search for drugs that have similar actions. A range of multi-target compounds were tested to determine if NMDA receptor blockade activity was obtained. The pharmacophores explored were tacrine, donepezil, lipoic acid carvedilol and dimebon. The most promising compounds were carbacrine(3) (tacrine and carvedilol) and lipocrine (lipoic acid and tacrine), and it was found that the former was equipotent and the latter more potent than memantine. Potency was likely due to the tacrine moiety. These compounds should be further categorised to determine if they retain the kinetics that gives memantine its favourable side effect profile.
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Lin, Raozhou, and 林饒洲. "Kif5b interaction with NMDA receptors regulates neuronal function." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/208429.
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Intracellular transportation is an essential cellular event controlling neuronal development, morphology, function and survival. Kinesin-1 is the molecular motor conveying cargo along microtubule by utilizing the chemical energy from ATP hydrolysis. This motor consists of two heavy chains and two light chains. Both heavy and light chains are responsible for cargo bindings. There are three kinesin-1 heavy chains in eukaryotic cells. Kif5a and Kif5c are neuronal specific, while Kif5b is ubiquitously expressed. Kif5b carries various cargos essential for neuronal functions, and the early embryonic death of Kif5b null mice suggests the importance of Kif5b in vivo.
N-methyl-d-aspartate receptors (NMDARs) are glutamate elicited channel, which is permeable to calcium and crucial for synaptic plasticity in the central nervous system. NMDARs are heteromeric assemblies consisting of NR1, NR2 and NR3 subunits. These transmembrane subunits contain three parts. Other than the transmembrane domain, the extracellular domain serves as the ligand binding site while the intracellular domain interacts with various partners regulating downstream signaling and receptor trafficking. Synaptic NMDAR activation regulates synaptic plasticity, while extrasynaptic NMDAR activation leads to excitotoxicity.
In this project, I find that kinesin-1 directly interacts with NMDAR subunit, NR1, NR2A and NR2B in vivo. NMDAR colocalizes with kinesin-1 in the cell body and neurites. By GST-pull-down assays with different Kif5b fragments, the cytoplasmic domains of NR1, NR2A and NR2B are found to directly bind with Kif5b via a Kif5b C-terminal region independent of kinesin light chains.
To examine the role of Kif5b in NMDAR trafficking, dominant negative Kif5b fragments are expressed in cell lines together with NR1-1a and GFP-NR2B. Overexpression of dominant negative Kif5b significantly disrupts GFP-NR2B forward trafficking and prevents it from entering into Golgi apparatus. Furthermore, the surface NR1 and NR2B levels are significantly reduced whilst the NR2A levels are not affected in Kif5b+/- mice in which the Kif5b protein level is reduced by 50% compared with the wild-type littermates. Consistent with this observation, the NR1 and NR2B levels are decreased in fractions containing synaptosomal membrane but not the one containing only postsynaptic densities, suggesting that the extrasynaptic NMDAR levels are affected in Kif5b+/- mice.
NMDARs are highly permeable to calcium while activated, thereby activating neuronal nitric oxide synthases (nNOS) to produce nitric oxide (NO). It is found that NMDA triggered calcium influx is perturbed in Kif5b+/- neurons, while the synaptic NMDA receptor mediated calcium influx is normal. In Kif5b+/- slices, the production of NO reduces significantly. Calcium ionophore, A23187, rescue this NO defect, indicating insufficient supply of calcium as the main contribution to this defect. Therefore, Kif5b-dependent extrasynaptic localization of NMDA receptors mediates calcium influx upon NMDA stimulation and controls NO production.
In the summary, above results suggest kinesin-1 as a novel motor involving in NMDA receptor trafficking. This interaction may contribute to the extrasynaptic distribution of NMDARs. By regulating NO production through interaction with NMDARs, Kif5b may mediate neuronal survival in cerebral ischemia and certain aggressive behaviors. This provides a novel target for therapy development against stroke and schizophrenia.published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy
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Sladek, Meik. "Peripheral NMDA receptors in inflammatory and visceral pain." Thesis, University of Bristol, 2007. http://hdl.handle.net/1983/3ba7218b-74bf-4734-8433-4582e49d576c.
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Substantial evidence has accumulated for the involvement of peripheral N-methyl-D-aspartate (NMDA) receptors in visceral/chronic pain. Notably, poorly brain-penetrating glycinceB NMDA antagonists were particularly potent in in vivo models of inflammatory visceral pain. The aim of this thesis was to further investigate the pharmacology of peripheral, compared to central, NMDA receptors.
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Giertler, Christian. "Die Rolle des Nucleus accumbens bei der Akquisition und Expression von instrumentellem Verhalten der Ratte." [S.l. : s.n.], 2003. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10790765.
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Johansson, Tobias. "Neurosteroids Induce Allosteric Effects on the NMDA Receptor : Nanomolar Concentrations of Neurosteroids Exert Non-Genomic Effects on the NMDA Receptor Complex." Doctoral thesis, Uppsala University, Department of Pharmaceutical Biosciences, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8503.
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The neurosteroids constitute a group of powerful hormones synthesized and acting in the central nervous system. They participate in a number of important central processes, such as memory and learning, mood and neuroprotection. Their effects emerge from rapid interactions with membrane bound receptors, such as the N-methyl-D-aspartate (NMDA) receptor, the gamma-amino-butyric acid receptor and the sigma 1 receptor. The mechanisms of action are separate from classical genomic interactions.
The aims of this thesis were to identify and characterize the molecular mechanisms underlying the effects of nanomolar concentrations of neurosteroids at the NMDA receptor.
The results show that the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate 3α5βS) differently modulate the NMDA receptor, changing the kinetics for the NMDA receptor antagonist ifenprodil, through unique and separate targets at the NR2B subunit. The effects that appear to be temperature independent were further confirmed in a calcium imagining functional assay. A second functional study demonstrated that PS and 3α5βS affect glutamate-stimulated neurite outgrowth in NG108-15 cells.
Misuse of anabolic androgenic steroids (AAS) has powerful effects on emotional states. Since neurosteroids regulate processes involved in mood it can be hypothesised that AAS can interact with the action of neurosteroids in the brain. However, chronic administration of the AAS nandrolone decanoate did not alter the allosteric effects of PS or 3α5βS at the NMDA receptor, but changed the affinity for PS, 3α5βS and dehydroepiandrosterone sulfate to the sigma 1 receptor. The results also showed that the neurosteroids displace 3H-ifenprodil from the sigma 1 and 2 receptors without directly sharing the binding site for 3H-ifenprodil at the sigma 1 receptor. The decreased affinity for the neurosteroids at the sigma 1 receptor may be involved in the depressive symptoms associated with AAS misuse.
The NMDA receptor system is deeply involved in neurodegeneration and the NMDA receptor antagonist ifenprodil exert neuroprotective actions. The findings that neurosteroids interact with ifenprodil at the NMDA receptor may be an opportunity to obtain synergistic effects in neuroprotective treatment.
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Дзюбенко, Н. В. "Роль глутаманатних рецепторів NMDA-типу в регуляції шлункової секреції." Diss. of Candidate of Biological Sciences, КНУТШ, 2008.
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AL, Saidi Waleed Hamdan Khalfan. "Theoretical Investigation of NMDA Effect on the Cerebral Cortex." The University of Waikato, 2008. http://hdl.handle.net/10289/2465.
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This thesis examines the dynamical behaviour of incorporating NMDA (an excitatory neurotransmitter) for the electrodynamic model of the cerebral cortex. The model used is the mean-field model developed by Steyn-Ross et al. (2005) which describes the behaviour of the cortex in terms of parameters averaged over spatially localised populations. The behaviour of the model is determined by the four control parameters: inhibitory effect li, subcortical drive s, and NMDA neurotransmitter e ect set by an excitatory factor le and the magnesium concentration C. Adopting this model could give a better understanding of the cortex functionality and the anaesthetic mechanism. The model predicts that there are either one or three stationary states available to the cortex. We identify two of these with highly activated state and a quiescent state and focus on the transition between the two. Theoretical stability predictions (eigenvalue analysis) verified by a numerical simulation show that the system is unstable between the two Hopf bifurcations. In addition, in the stable region the steady states remains stable under a small perturbation, while in the unstable region either a transition between states or a limit cycle (oscillation) occurs depending on the position of the steady state.
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Brasier, Daniel James. "Novel roles of NMDA receptors in release and plasticity." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3275082.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed October 3, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Yılmaz, Nigar Vural Hüseyin. "Ratlarda kalori kısıtlamasının NMDA reseptör subünit konsantrasyonları üzerine etkisi /." Isparta : SDÜ Tıp Fakültesi, 2007. http://tez.sdu.edu.tr/Tezler/TT00347.pdf.
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Turnock, Julia Jane. "NMDA receptor subunit alterations in the isolation-reared rat." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613288.
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Timony, Paula Anne. "Functional over expression of mammalian non-NMDA glutamate receptors." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368365.
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Misra, Charu. "Properties of NMDA receptors in identified cerebellar cell types." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324977.
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Papadakis, Michalis. "Assembly and trafficking of NMDA receptors : a biochemical approach." Thesis, University College London (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415341.
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Cho, Kathleen K. A. (Kathleen Kyung-Ah). "Functional role of NMDA receptor subunit composition in metaplasticity." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/47890.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Brain and Cognitive Sciences, 2009."June 2009."
Includes bibliographical references (leaves 127-150).
Modification of synapses by neural activity has been proposed to be the substrate for experience-dependent brain development, learning, and recovery of function after brain damage. In the visual cortex, the strength of cortical synapses can be bidiredionally modified, where in response to a critical level of postsynaptic activation, synapses are strengthened (long-term potentiation; LTP) and below this level, synapses are weakened (long-term depression; LTD). Previous work in visual cortex has suggested that the threshold for synaptic modifications is dependent on the recent history of visual experience, a phenomenon called metaplaticity. Recent mechanistic studies have shown that experience-dependent adjustments of the modification threshold correlate with changes in the subunit composition and function of NMDA-type glutamate receptors (NMDARs). However, causality has not been conclusively established. Here we examined the mechanistic basis of metaplaticity, and specifically how this process is mediated by a switch in NMDAR subunit composition by focusing on the NR2A subunit of the NMDA receptor in visual cortex. We provide evidence for the functional significance of the NR2A subunit in metaplastic changes both in synaptic platicity elicited in vitro and in naturally-occurring platicity in vivo. We also performed a comparison of in vitro methods of inducing plasticity and those which subserve in vivo experience-dependent changes in synaptic strength. These findings represent an important step forward in understanding how plasticity thresholds are regulated in the brain.
by Kathleen K. A. Cho.
Ph.D.
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Huang, Zhuo. "Properties of NMDA receptors in the rat basal ganglia." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1443978/.
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Native subtypes of NMDA receptors with distinct developmental and anatomical distribution are present in the brain. Knowledge of their function and pharmacology greatly helps in understanding the involvement of NMDA receptors in physiological and pathological processes in the central nervous system. Multiple patch-clamping methods were carried out in my experiments. First, the single-channel properties of native NMDA receptors were studied in outside-out patches excised from neuronal cell bodies in substantia nigra pars compacta (SNc) and subthalamic nucleus (STN) from 7-day old rats. The steady state channel activations produced by NMDA (100 nM or 200 uM) and glycine (10 uM) were studied at -60 mV. The results showed that both large and small conductance channels are present on STN and SNc neurons. These large conductance NMDA channels from SNc and STN display a high ifenprodil sensitivity, suggesting NR2B-containing NMDA receptors are present on SNc and STN neurons. In addition, direct transition analysis suggests that the small conductance channels may be due to NR2D-containing NMDA receptors. Second, whole-cell patch-clamp recording of bath application-induced NMDA receptor-mediated currents (Inmda) in dopaminergic neurons of SNc in brain slices from P7 rats was used to characterize the ifenprodil inhibition and voltage-dependence of Mg2+ block of NMDA receptors. The NMDA-induced whole-cell currents were evoked by NMDA (10 uM or 200 uM) and glycine (10 pM). There are two main findings from the whole-cell experiments. (1) The NMDA-induced whole-cell currents display a high ifenprodil sensitivity, suggesting the presence of NR2B-containing NMDA receptors on P7 rat SNc neurons. (2) The combined application of Mg2+ and ifenprodil reduced the voltage-dependent Mg2+ block, which is consistent with recombinant NR2D-containing NMDA receptors that have a lower affinity for Mg2+. This suggests that NR2D-containing NMDA receptors are present in P7 rat SNc. Third, in order to evaluate the deactivation kinetics of extrasynaptic NMDARs, outside-out patches containing multiple channels were obtained from P7 rat dopaminergic neurons of SNc and stimulated with a brief synaptic-like (l-4ms) pulse of ImM glutamate. The results suggested that the triheteromeric NR1-NR2B-NR2D rather than diheteromeric NR1-NR2D NMDA receptors are present on extrasynaptic sites of SNc neurons. In addition, in order to investigate voltage-dependent Mg2+ block and memantine block, several channel block models were developed to evaluate multiple Mg2+ effects on native NMDA receptors and competition between Mg2+ and memantine for block of NMDAR channels.
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Gielen, Marc. "Mécanismes moléculaires du contrôle de l'activité des récepteurs NMDA." Paris 6, 2009. http://www.theses.fr/2009PA066438.
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Kochli, Daniel Edward. "NMDA and dopaminergic contributions to context fear memory reconsolidation." Miami University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=miami150065280374774.
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Veras, Lea. "NMDA Receptor Transmembrane Domain: Structure and Divalent Ion Selectivity." Research Showcase @ CMU, 2014. http://repository.cmu.edu/dissertations/1036.
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During a synaptic signal, NMDA receptors are the only ionotropic glutamate receptor subfamily that besides glutamate require glycine and membrane depolarization to allow ion permeation. The depolarization is necessary to release Mg2+ of the channel of NMDA receptors. Of the ions that permeate these ion channels, Ca2+ is of importance because it is essential for learning and memory. Furthermore, NMDA receptor dysfunction has been associated with several nervous system disorders, and thus understanding NMDA receptor functions and dysfunctions are relevant for rational drug design. The mechanisms by which NMDA receptors select Ca2+ for permeation over all other physiological ions, while binding Mg2+ and restricting other ions’ permeation, are not well understood. We hypothesize that the slightly different atomic properties of Mg2+ and Ca2+ result in different mechanisms for how each divalent ion moves across the channel. To create a more complete picture of the permeation mechanism and prove our hypothesis, we performed a multi-level computational chemistry approach. Our research methods consisted of three main steps. The first step was to perform quantum chemical and molecular dynamic calculations to quantitatively predict ion interactions with solvents that mimic the heterogeneous environment of the protein. The second step consisted of modeling, refining, and equilibrating a homology model of the NMDA receptor transmembrane domain. The final step consisted of using the equilibrated transmembrane domain NMDA receptor model to study the actual ionic environment in the protein and simulate the energy involved in the permeation process. For the first step, we found that the solvents mimic the behavior of the residues in the core of our NMDA receptor model because in both set of systems Ca2+ is more permissive than Mg2+ to exchange ligands. As the conclusions in second and third steps, we also observed that the aspargines in the NMDAR model provide the ideal cage environment, that functions like branches and capture the each divalent ion. Hence, an equilibrated TMD NMDAR model was built, the presence of each divalent ion in the protein was simulated, and the permeation mechanism was better understood.
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Goebel, Susan Michelle. "Phospho-regulation of hippocampal NMDA receptor localization and function /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
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Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 200-233). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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Honse, Yumiko. "Effects of prenatal ethenol treatment on native NMDA receptors /." Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3026206.
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Bohn, Ines. "Die Rolle des orbitalen präfrontalen Kortex bei instrumentellen Lernvorgängen der Ratte." [S.l. : s.n.], 2003. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10370444.
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Dziuganowska, Zofia. "Preparation and biological evaluation of new Selfotel structural analogues to treat tinnitus." Thesis, Montpellier, Ecole nationale supérieure de chimie, 2013. http://www.theses.fr/2013ENCM0004.
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Dans le cadre de la recherche d'antagonistes du NMDA, nous avons synthétisé une librairie de nouveaux composés dérivés du Selfotel (CGS 19755). Ces phosphonates aromatiques ont été obtenus par réaction de couplage croisé utilisant une catalyse au palladium (couplage d'Hirao). Leur hydrolyse a conduit aux acides phosphoniques correspondants. Dans une second temps, la série pipéridine a été obtenue par hydrogénation complète des motifs pyridiniques en utilisant de l'oxyde de platine comme catalyseur.Lors de l'analyse de ces composés, des propriétés spectroscopiques intéressantes en RMN ont été mises en évidence. Des monocristaux ont été obtenus sur certains des acides, ce qui a permis de confirmer leurs structures par diffraction des rayons X. Ces experiences ont clairement montré que l'hydrogénation du cycle aromatique est un processus de cis-hydrogénation.Les études biologiques préliminaires effectuées sur des cultures de neurones ont indiqué une activité certaine sur les récepteurs NMDA. Une partie d'entre eux agissait comme antagonistes du NMDA tandis que l'autre montrait des activités de neuroprotection. Des expériences complémentaires par un test de fluorescence, ont été mises en place. Dans ce contexte, les outils de génie génétique ont été utilisés pour créer une lignée cellulaire de mammifère exprimant de façon stable le récepteur NMDA. Toutefois, ces expériences n'ont pas conduit à des résultats permettant d'expliquer les résultats biologiques obtenusThe library of novel compounds being derivatives of NMDA antagonists Selfotel (CGS 19755) was synthesized. The series of aromatic esters were obtained in palladium catalyzed cross-coupling reaction (Hirao coupling), followed by their hydrolysis and then the series of aliphatic acids was obtained upon their hydrogenation over PtO2. Majority of designed compounds have not been synthesized before. What is worth emphasizing, none of this compound was studied as potential NMDA receptor antagonist. During the analysis of compounds, interesting spectroscopic properties were observed as well as few of compounds were obtained as monocrystals, which enabled to obtain their crystal structures. Crystal structures of aliphatic acids unequivocally identified that hydrogenation of aromatic ring is a cis process and yields predominantly only one diastereomeric mixture.Initial biological studies performed on cultures of neurons indicated that the compounds posses activity towards NMDA receptors – part of them acting as antagonists and part as protectants or facilitators.More advanced investigation of biological activity by means of fluorescence screening assay, was planned to be performed. Consequently, genetic engineering tools were used in order to create mammalian cell line stably expressing NMDA receptor what would allow to perform these studies. However, these experiments were not successful. Therefore, further investigation should be performed in order to confirm the obtained biological results
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Holtkamp, Johanna. "Die neuroprotektive Wirkung der NMDA-Rezeptorantagonisten CGS, Memantin und Ifenprodil, sowie Roscovitin und NMDA auf die hypoxiebedingte Zellschädigung an embryonalen kortikalen Zellen von Ratten." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-161530.
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Die vorliegende Arbeit beschäftigt sich mit dem Einfluss der NMDA-Rezeptorantagonisten, Memantin, MK-801, CGS und Ifenprodil auf die hypoxieinduzierte Zellschädigung an kortikalen Zellen der Ratte. Außerdem wurde der Einfluss von subtoxischen Konzentrationen von NMDA sowie von Roscovitin, einem Hemmer Cyclin-abhängiger Kinasen, auf die hypoxiebedingte Zellschädigung untersucht. Ziel dieser Arbeit war es, die neuroprotektive Wirkung dieser Substanzen zu erfassen.
Zur Untersuchung der hypoxischen Schädigung wurden zwei 48-Well-Zellkulturplatten mit 15 Tage alten kortikalen Zellen der Ratte verwendet. Eine Kulturplatte wurde für vier Stunden mit HEPES(N-2-Hydroxyethylpiperazine-N’-2-Ethansulfonsäure)-Puffer (ohne Glucose) unter hypoxischen Bedingungen inkubiert. Die zweite Platte, mit glukorisiertem HEPES-Puffer, wurde für vier Stunden unter normoxischen Bedingungen inkubiert. Der HEPES-Puffer wurde nach vier Stunden entfernt, die Kulturplatten mit Dulbecco’s Modified Eagle Medium (DMEM) gewaschen und mit diesem Medium für 24 Stunden unter normoxischen Bedingungen inkubiert. Anschließend wurde das Medium ent¬fernt, durch NMDA, Memantin, Roscovitin, CGS und Ifenprodil ersetzt und die Ansätze für weitere 24 Stunden unter normoxischen Bedingungen inkubiert.
Zur Beurteilung der Zellschädigung wurden der Aktivitätsanstieg der Laktat-Dehydrogenase (LDH), die Freisetzung freier Sauerstoffradikale und die Steigerung der Caspase-Aktivität bestimmt. Während die Bestimmung der LDH-Aktivität und die Freisetzung der freien Sauer¬stoff¬radikale nekrotische Veränderungen der Zellen charakterisiert, zeigt eine Zunahme der Caspase-Aktivität apoptotische Vorgänge an.
LDH ist ein stabiles zytoplasmatisches Enzym, das in fast allen Körperzellen vorkommt. Beim Absterben der Zelle wird das Enzym durch die Schädigung der Plasmamembran aus der Zelle freigesetzt, so dass es zu einem Anstieg der LDH-Aktivität proportional zur Anzahl der toten Zellen kommt. Diese Aktivität wurde spektrophotometrisch mit einem Mikrotiterplatten-Lesegerät bestimmt. Die Ergebnisse des LDH-Tests zeigen, dass nach der 24-stündigen Behandlung der Zellen mit MK-801 die LDH-Aktivität um 11%, bei Roscovitin um 13%, bei Memantin (5 µM) um 56%, bei Memantin (0,5 µM) um 52% und mit NMDA (5 µM) um 44% signifikant vermindert wurde.
Bei einer hypoxiebedingten Schädigung kortikaler Zellen kommt es auch zur Bildung freier Sauer¬stoff¬radikale. 2’,7’-Dichlorfluorescein Diacetat (2’,7’-H2DCF-DA) wird von den Zellen auf¬ge¬nommen und intrazellulär mit Sauerstoff- und Stickstoffspezies zum Fluoreszenz¬farb-stoff 2’,7’-Dichlorodihydrofluorescein (DCF) deacetyliert. DCF verbleibt dabei in den Zellen, so dass die Messung der Fluoreszenz der Zellen als Maß für intrazelluläre Oxidationsprozesse verwendet werden kann. Die DCF-Fluoreszenz-Änderung wurde mittels eines Fluorimeters gemessen und die daraus resultierenden Daten mit einer im Fluorimeter integrierten Software bearbeitet. Die Ergebnisse zeigen, dass die Freisetzung der freien Sauerstoffradikale, der hypoxiegeschädigten Zellen, signifikant durch Ifenprodil (10 µM) um 119%, Memantin (50 µM) um 88% und NMDA (5 µM) um 134% reduziert wurde.
Die hypoxieinduzierte Zellmembranschädigung führt desweiteren zu einem Anstieg der Caspase-Aktivität. Mit Hilfe des Apo-One Homogeneous Caspase-3/7-Assays (Promega) wurde die Aktivität der Caspasen 3 und 7 fluorimetrisch bestimmt. Um die unterschiedliche Zelldichte in den Kulturschalen zu berücksichtigen, wurde eine Proteinbestimmung nach der Bicinchoninsäure-Methode (Smith et al. 1985) durchgeführt.
Einen protektiven Effekt auf die Zellschädigung zeigen Memantin und NMDA in Bezug auf die Beeinflussung dieser Caspase-Aktivität. Der hypoxiebedingte Anstieg der Caspase-3-Aktivität konnte nach 24-stündiger Inkubation mit Memantin (5 µM) um 24%, mit Memantin (0,5 µM) um 28% und mit NMDA (5 µM) um 24% vermindert werden.
CGS hat in diesen Versuchen keinen protektiven Einfluss auf die hypoxie¬induzierte Zellschädigung.
Diese Arbeit zeigt, dass die Applikation niedriger NMDA-Konzentrationen neuroprotektive Effekte auf die Entwicklung der hypoxischen Schädigung von kortikalen Zellen der Ratte hat. Darüber hinaus wird vermutet, dass NMDA sogar einen trophischen Effekt auf das Über-leben der kortikalen Neurone ausübt. Dieser schützende Mechanismus von NMDA scheint denselben, wenn nicht sogar einen größeren protektiven Effekt wie Memantin zu induzieren.
Um die Therapiemöglichkeiten der zerebralen Hypoxie durch neuroprotektive Medikamente zu optimieren, wären jedoch weitergehende Untersuchungen besonders als In-vivo-Modelle wünschenswert.
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Kellermayer, Blanka. "Super-resolution imaging reveals differential organization and regulation of NMDA receptor subtypes." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0005/document.
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Résumé: Les récepteurs du glutamate de type NMDA (NMDAR) sont des canaux ioniques impliqués dans les phénomènes de plasticité de la transmission synaptique dans le système nerveux central, des mécanismes supposés être à la base du développement neuronal, de l’apprentissage et de la formation de la mémoire. Les NMDAR forment des tétramères à la membrane plasmique, constitués de deux sous-unités obligatoires GluN1 et deux sous-unités variables GluN2 (GluN2A-D) ou GluN3. Dans le prosencéphale, les récepteurs comportant les sous-unités GluN2A (GluN2A-NMDAR) et GluN2B (GluN2B-NMDAR) sont les plus abondants et présentent des profils d’expression différents au cours du développement, les GluN2B-NMDAR étant fortement exprimés aux stades précoces tandis que l’expression des GluN2A-NMDAR augmente progressivement au cours du développement postnatal. Des contributions relatives de ces deux sous-types majoritaires de NMDAR aux propriétés de signalisation distinctes dépendent directement les phénomènes de plasticité neuronale, tels que l’adaptation des synapses glutamatergiques et des circuits neuronaux excitateurs. Bien que la régulation moléculaire des NMDAR ait fait l’objet d’intenses recherches ces dernières décennies, la localisation précise de ces deux sous-types de récepteurs dans la membrane postsynaptique demeurait méconnue. Pour répondre à cette question, nous avons étudié la distribution des NMDAR à la surface de neurones d’hippocampe de rats en combinant deux techniques de microscopie de super-résolution - la microscopie de reconstruction optique stochastique directe (dSTORM) et la déplétion d’émission stimulée (STED) - permettant de dépasser la limite de résolution inhérente à la diffraction de la lumière. Ces techniques nous ont permis de mettre en évidence que les sous-types de récepteurs GluN2A- et GluN2B-NMDAR présentent une nano-organisation différente à la surface neuronale. En effet, ils sont organisés en structures nanoscopiques (nanodomaines) qui diffèrent en nombre, en surface et en morphologie, notamment au niveau des synapses. Au cours du développement, l’organisation membranaire des deux sous-types de NMDAR évolue, avec en particulier de profonds changements de distribution des GluN2A-NMDAR. De plus, cette organisation nanoscopique est impactée différemment par des modulations de l’interaction avec les protéines d’échafaudage à domaine PDZ ou de l’activité de la kinase CaMKII suivant le sous-type de NMDAR considéré. En effet, la réorganisation des GluN2A-NMDAR implique principalement des changements de nombre de récepteurs dans les nanodomaines sans modification de leur localisation, tandis que la réorganisation des GluN2B-NMDAR passe essentiellement par des modifications de localisation des nanodomaines sans changements du nombre de récepteurs qu’ils contiennent. Ainsi, les GluN2A- et GluN2B-NMDAR présentent des nano-organisations différentes dans la membrane postsynaptique, reposant vraisemblablement sur des voies de régulation et des complexes de signalisation distinctsNMDA-type glutamate receptors (NMDARs) are a type of ion permeable channels playing critical roles in excitatory neurotransmission in the central nervous system by mediating different forms of synaptic plasticity, a mechanism thought to be the molecular basis of neuronal development, learning and memory formation. NMDARs form tetramers in the postsynaptic membrane, most generally associating two obligatory GluN1 subunits and two modulatory GluN2 (GluN2A-D) or GluN3 (GluN3A-B) subunits. In the hippocampus, the dominant GluN2 subunits are GluN2A and GluN2B, displaying different expression patterns, with GluN2B being highly expressed in early development while GluN2A levels increase gradually during postnatal development. In the forebrain, the plastic processes mediated by NMDARs, such as the adaptation of glutamate synapses and excitatory neuronal networks, mostly rely on the relative implication of GluN2A- and GluN2B-containing NMDARs that have different signaling properties. Although the molecular regulation of synaptic NMDARs has been under intense investigation over the last decades, the exact topology of these two subtypes within the postsynaptic membrane has remained elusive. Here we used a combination of super-resolution microscopy techniques such as direct stochastic optical reconstruction microscopy (dSTORM) and stimulated emission depletion (STED) microscopy to characterize the surface distribution of GluN2A- or GluN2B-containing NMDARs. Both dSTORM and STED microscopy, based on different principles, enable to overcome the resolution barrier due to the diffraction limit of light. Using these techniques, we here unveil a differential nanoscale organization of native GluN2A- and GluN2B-NMDARs in rat hippocampal neurons. Both NMDAR subtypes are organized in nanoscale structures (termed nanodomains) that differ in their number, area, and shape. These observed differences are also maintained in synaptic structures. During development of hippocampal cultures, the membrane organization of both NMDAR subtypes evolves, with marked changes for the topology of GluN2A-NMDARs. Furthermore, GluN2A- and GluN2B-NMDAR nanoscale organizations are differentially affected by alterations of either interactions with PDZ scaffold proteins or CaMKII activity. The regulation of GluN2A-NMDARs mostly implicates changes in the number of receptors in fixed nanodomains, whereas the regulation of GluN2B-NMDARs mostly implicates changes in the nanodomain topography with fixed numbers of receptors. Thus, GluN2A- and GluN2B-NMDARs have distinct organizations in the postsynaptic membrane, likely implicating different regulatory pathways and signaling complexes
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Jeromin, Monika. "Functional studies of NMDA receptor concatemers in Xenopus laevis oocytes." [S.l.] : [s.n.], 2007. http://deposit.ddb.de/cgi-bin/dokserv?idn=98457591X.
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Alvestad, Rachel Marie. "Phosphorylation and subcellular localization of NMDA receptors : modulation by ethanol /." Connect to full text via ProQuest. IP filtered, 2005.
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Thesis (Ph.D. in Pharmacology) -- University of Colorado, 2005.Typescript. Includes bibliographical references (leaves 145-170). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Kronenberg, Ute [Verfasser]. "Synthesis and radiofluorination of putative NMDA receptor ligands / Ute Kronenberg." Köln : Universitäts- und Stadtbibliothek Köln, 2011. http://d-nb.info/1013740130/34.
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Li, Victor. "Functional characterization of a novel NMDA receptor positive allosteric modulator." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62144.
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The NMDA receptor is a glutamatergic ionotropic receptor key in mediating neuronal
plasticity across virtually all synaptic circuits in the brain. An increasing list of neurological
disorders have implicated NMDA receptor hypofunction as an integral part of pathogenesis,
necessitating the production of NMDA receptor potentiators as therapeutics. To date, most of
these attempts have used increased co-agonism at the glycine binding site of NMDA receptors,
but this strategy has been plagued by low specificity and efficacy. Specific allosteric modulation
of NMDA receptors is an ideal solution, but until recently, no known drugs were capable of
doing so. Building off previous work in our lab that discovered a novel family of compounds
capable of modulating NMDA receptor activity through its apical N-terminal domain, we
identified and characterized a drug candidate, Npam59, predicted to potentiate both GluN2A- and 2B-containing NMDA receptors. Npam59 was shown to potentiate NMDA currents
mediated by both subtypes with EC50 in the low-micromolar range. Npam59 also potentiated d-amphetamine-induced dopamine release in the ventral striatum in an NMDA receptor-dependent manner, but had no observable effect when administered alone. Finally, Npam59 potentiated d-amphetamine-induced hyperlocomotion in Sprague-Dawley rats. These results demonstrate that Npam59 can potentiate the function of NMDA receptors, including both GluN2A- and 2B-containing ones, suggesting its potential as a research tool and drug candidate for further development. Npam59 is the first known NMDA receptor allosteric potentiator with specificity for both GluN2A and GluN2B. Its characterization provides the foundation for therapeutic development and novel insights into the interaction of dopamine-glutamate signaling in the ventral striatum.Medicine, Faculty of
Graduate
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Sawyer, Dale C. "The interactions of putative neuroprotectant compounds with NMDA ion channels." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq25152.pdf.
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Bradford, Andrea Marie. "Molecular pharmacology of a novel NR2B-selective NMDA receptor antagonist." Thesis, Durham University, 2006. http://etheses.dur.ac.uk/2736/.
Full textAbstract:
The NMDA receptor is a heteromeric ligand-gated ion channel in the central nervous system (CNS). There are three families of NMDA receptor subunits with various combinations of NRl, NR2A-D and NR3A֊B subunits producing unique receptors with distinctive pharmacological and biochemical properties. Pharmacological and functional properties of the NMDA receptor are highly dependent on the composition of the receptor complex. The NMDA receptor is the focus of drug development for therapy and prevention of numerous neurological and psychiatric disorders. The focus of this thesis was to investigate NMDA receptor subtype selectivity of NMDA antagonists, in particular, RGH-896, a novel NR2B֊selective antagonist. The study has utilised radioligand binding competition binding assays with RGH-896 in native, recombinant and immunopurified NMDA receptor preparations. In addition, ligand autoradiography has been employed to quantify and delineate the regional distribution of [^3H] RGH-896 binding sites in rodent and human brain tissue. This study provides the first evidence that [3H] RGH-896 binds to a distinct binding site which displays a significantly lower affinity for spermidine compared to the [^3H] Ro 25,6981 binding site. In addition, the low sensitivity of [^3H] MK-801 for unlabelled RGH-896 compared to prototypical NR2B ligand ifenprodil is further evidence for the difference in binding sites. Novel immunopurification studies using [3H] RGH-896, in contrast to [3H] CP 101,606, binds to NR2B-containing receptors irrespective of NMDA receptor subunit combinations. Ligand autoradiography in human brain has shown a surprising overall preservation of NR2B receptors in dementia with Lewy bodies (DLB) patients compared to age matched controls in the anterior cingulate cortex (ACC). However, this study has revealed the first evidence of an upregulation of NR2B receptors in ACC of DLB cases related to severity of auditory, but not visual hallucinations.
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Zeller, Kathrin [Verfasser]. "Untersuchungen zur Hemmwirkung von Diphenhydramin an NMDA-Rezeptoren / Kathrin Zeller." Ulm : Universität Ulm. Medizinische Fakultät, 2013. http://d-nb.info/1044957255/34.
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