Techn. Univ., Diss., 2009--Darmstadt.

Frankfurt (Main), Universiẗat, Diss., 2003.

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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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.

Mammalian neurons are anoxia-sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by calcium entry through N-methyl-D-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest increase in cytosolic [Ca2+] during anoxia. The aim of this study was to determine if activation of the mitochondrial permeability transition pore (mPTP) decreases NMDAR currents through release of mitochondrial Ca2+. The data indicate that mPTP opening is sufficient to cause a decrease in NMDAR currents during normoxia and the anoxia-mediated rise in cytosolic [Ca2+] and depolarization of the mitochondrial membrane potential is due to opening of the mPTP. Furthermore, since a mitochondrial uncoupler releases additional calcium during anoxia we speculate that the mitochondrial membrane potential decreases in a regulated fashion to a new set-point.

Mammalian neurons are anoxia-sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by calcium entry through N-methyl-D-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest increase in cytosolic [Ca2+] during anoxia. The aim of this study was to determine if activation of the mitochondrial permeability transition pore (mPTP) decreases NMDAR currents through release of mitochondrial Ca2+. The data indicate that mPTP opening is sufficient to cause a decrease in NMDAR currents during normoxia and the anoxia-mediated rise in cytosolic [Ca2+] and depolarization of the mitochondrial membrane potential is due to opening of the mPTP. Furthermore, since a mitochondrial uncoupler releases additional calcium during anoxia we speculate that the mitochondrial membrane potential decreases in a regulated fashion to a new set-point.

Working memory refers to the ability to retain information for short periods of time to guide future behavior. This type of short-term memory has been shown to play an important role in mental disorders such as schizophrenia and therefore further investigations into the neural basis of this cognitive function may aid in the study of disease states where this cognitive function is defective. A likely neural correlate of working memory has been identified in the persistent neural activity observed during the memory retention intervals of various behavioral tasks. Computational and cellular physiology has suggested that this persistent activity depends on NMDA receptor activation. Indeed, pharmacological studies on both human and animal subjects have reported a significant decrease in working memory task performance following the administration of NMDA-antagonists such as ketamine. However, the task and experimental design of these previous studies have not been ideal, and have therefore only shown equivocal evidence that NMDA-antagonists impair working memory, especially its capacity. Here we aimed to determine the effect of low-dose ketamine injection (0.25-mg/kg and 0.50-mg/kg IM) on the performance of macaque monkeys on a visual sequential comparison task, a task whose performance has minimal influence from other cognitive functions besides working memory. All monkeys showed a detrimental effect of ketamine administration on visual working memory performance, either at higher ketamine doses or with high memory loads. There was also an effect on performance in sessions without a memory component, indicating that the effect of ketamine was no limited to working memory maintenance. Although the effect of ketamine on memory load varied per animal, this study provides solid evidence in support of the hypothesis that working memory maintenance is dependent on NMDA receptor integrity. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2011-05-27 14:56:41.726

Neuronal vulnerability to ischemia is dependent on the balance between pro-survival and pro-death cellular signaling. In the latter, it is increasingly appreciated that toxic Ca2+ influx can occur not only via postsynaptic glutamate receptors, but also through other cation conductances. One such conductance, the Transient receptor potential melastatin type-2 (TRPM2) channel, is a non-specific cation channel having similar homology to TRPM7, a conductance reported to play a key role in anoxic neuronal death. The role of TRPM2 conductances in ischemic Ca2+ influx has been difficult to study due to the lack of specific modulators. Here we used TRPM2-null mice (TRPM2(-/-)) to study how TRPM2 may modulate neuronal vulnerability to ischemia. TRPM2(-/-) mice subjected to transient middle cerebral artery occlusion (tMCAO) exhibited smaller infarcts when compared to wild-type (WT) animals, suggesting the absence of TRPM2 to be protective. Surprisingly, field potentials (fEPSPs) recorded during oxidative stress in brain slices taken from TRPM2(-/-) mice revealed increased excitability, a phenomenon normally associated with ischemic vulnerability, whereas WT fEPSPs were unaffected. The upregulation in fEPSP in TRPM2(-/-) neurons was blocked selectively by an NR2A antagonist. This oxidative stress-induced increase in excitability of TRPM2(-/-) fEPSPs depended on the upregulation and downregulation of NR2A and NR2B-containing NMDARs, respectively, and augmented pro-survival signaling via Akt and ERK pathways culminating in the inhibition of the proapoptotic factor, GSK3β. Cultured hippocampal neurons from TRPM2(-/-) animals subjected to oxygen glucose deprivation had a reduction in cell death in comparison to WT neurons, demonstrating that absence of TRPM2 is protective at the neuronal level in vitro. Our results suggest that TRPM2 plays a role in downregulating pro-survival signals in central neurons and that TRPM2 channels may comprise a therapeutic target for preventing ischemic damage.