It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and eight psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition, we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (-30%) and middle (-41%) portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.

Alzheimer s disease is characterized by synaptic alterations and neurodegeneration. Histopathological hallmarks represent amyloidplaques composed of amyloid-beta (Abeta) and neurofibrillary tangles containing hyperphosphorylated tau. To determine whether synaptic changes and neurodegeneration share common pathways we established an ex vivo model using organotypic hippocampal slicecultures from amyloid precursor protein transgenic mice combined with virus-mediated expression of EGFP-tagged tau constructs. Confocal high-resolution imaging, algorithm-based evaluation of spines and live imaging was employed to determine spine changes and neurodegeneration. We report that Abeta but not tau induces spine loss and shifts spine shape from mushroom to stubby through a mechanism involving NMDA receptor (NMDAR), calcineurin and GSK-3beta activation. In contrast, Abeta alone does not cause neurodegeneration but induces toxicity by phosphorylation of wt tau in a NMDAR-dependent pathway. We show thatGSK-3beta levels are elevated in APP transgenic cultures and that inhibiting GSK-3beta activity or use of phosphorylation-blocking tau mutations prevent Abeta-induced toxicity of tau. FTDP-17 tau mutants are differentially affected by Abeta. While R406W tau shows increased toxicity in the presence of Abeta, no change is observed with P301L tau. While blocking NMDAR activity abolishes toxicity of both wt and R406W tau, the inhibition of GSK-3beta only protects against toxicity of wt tau but not of R406W tau induced by Abeta. Tau aggregation does not correlate with toxicity. We propose that Abeta-induced spine pathology and tau-dependent neurodegeneration are mediated by divergent pathways downstream of NMDA receptor activation and suggest that Abeta affects wt and R406W tau toxicity by different pathways downstream of NMDAR activity.

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Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / This thesis provides evidence to support the hypothesis that synaptic plasticity in the primary somatosensory cortex is a cellular correlate of associative learning, that the process depends upon acetylcholine and that only certain cortical neurons display this plasticity. In a first series of experiments, single-imit recordings were carried out in the barrel cortex of awake, adult rats subjected to whisker pairing, an associative learning paradigm where deflections of the recorded neuron's principle vibrissa were repeatedly paired with those of a non-adjacent one. On average, this form of sensory preconditioning increased the responses of a recorded unit to the stimulation of the non-adjacent vibrissa. In contrast, following explicitly unpaired control experiments, neuronal responsiveness decreased. The effect of pairing was further enhanced by local, microiontophoretic delivery of NMDA and the nitric oxide synthase inhibitor L-NAME and reduced by the NMDA receptor competitive antagonist AP5. These results and the fact that the influence of the pharmacological agents on neuronal excitability were either transient (liinited to the delivery period) or simply absent indicated that the somatosensory cerebral cortex is one site where plasticity emerges following whisker pairing. In subsequent experiments, using a similar conditioning paradigm that relied on evoked potential rather than single-unit recordings, increases in the responses of cortical neurons to the non-adjacent whisker were blocked by atropine sulfate, an antagonist of muscarinic cholinoreceptors. Administration of norn-ial saline or atropine methyl nitrate, a muscarinic antagonist that did not cross the blood-brain barrier, instead of atropine sulfate, did not affect plasticity. Analysis of the behavioral state of the animal showed that the changes observed in the evoked potential could not be attributed to fluctuations m the behavioral state of the animal. By combining the results described in this thesis with data foimd in related literature, the author hypothesizes that whisker pairing induces an acetylcholine-dependent form of plasticity within the somatosensory cortex through Hebbian mechanisms.

No / In the November issue (2001) of Neuroscience Letters, Holmin et al. (Neurosci. Lett. 314 (2001) 151) reported that the synthesis of the intermediate filament protein nestin was upregulated by potassium-induced depolarization in the rat cortex. In this letter, we provide supplementary evidence that repeated cortical spreading depression elicited by potassium induces a delayed upregulation of nestin. However, we argue against the authors' conclusion, Nestin expression was N-methyl-D-aspartate (NMDA)-receptor dependent since dizocilpine (MK-801) treatment abolished the response because spreading depression itself is very sensitive to NMDA-receptor block, and the drug treatment was initiated prior to potassium application to the cortex in Holmin et al.'s study.

Overexpression of the NR2B subunit of the NMDA receptor in the forebrain has been shown to improve learning and memory in mice (Tang et al 1999), which provides exciting implications for the enhancement of human cognition. However, it was first essential to establish replicability, and since the Tang et al (1999) study used only male mice we wished to investigate possible sex differences. On the hidden platform watermaze, we found a trend for male NR2BOE mice to learn the task more quickly than male wildtype mice (as observed by Tang et al. 1999), but the opposite trend in female mice; female NR2BOE mice were slower to reach the hidden platform than female wildtype mice. This pattern of results was also observed on the spatial reference Y memory task and open field task (for anxiety), although not on the spatial working memory T maze task (despite a sex difference). However, wildtype and NR2BOE mice performed at similar levels on the novel object recognition task, the spatial novelty preference task, visible platform watermaze and visual discrimination task. A battery of tests considering some species typical behaviours of mice demonstrated that wildtype and NR2BOE mice were comparable on tests of motor ability, strength, co-ordination, anxiety, burrowing and nesting. This suggests that our behavioural results are not due to a general impairment or enhancement of species typical behaviours. We considered the possibility that the difference between the results of Tang et al (1999) and those we observed may be caused by age differences; hence we attempted to replicate our results on the hidden platform watermaze, spatial reference Y maze and open field test in age matched mice. However, the second cohort of NR2BOE mice performed at similar levels to wildtype mice, and at significantly improved levels compared to the mice of the first cohort. We also considered the effects of knocking out the NR2B subunit on learning and memory, and NR1 subunit deletion within the hippocampus. On the spatial working memory T maze, these mouse strains performed similarly to their respective wildtype strains. Similarly, on a two beacon watermaze (with one indicating the platform position), mice lacking the NR2B subunit were able to locate the platform in a similar length of time. To ensure that the null results we had observed in the second cohort were not due to loss of the NR2B protein overexpression in the forebrain, we performed polymerase chain reactions (PCR), quantitative real-time PCR, and Western blots. We ascertained that the transgene was indeed present and that NR2B mRNA and protein levels were elevated in the hippocampi of the NR2BOE mice. In conclusion, it is unclear why the behaviours we observed in the NR2BOE mice are different to those published in the literature. It is possible that they may be due to differences in environmental enrichment, but the cause of the genotype by sex differences observed in the mice of cohort 1 is unclear. Nonetheless, we have advanced our knowledge of the effects of modifications in the levels of the NR2B subunit of the NMDA receptor on learning and behaviour.

Glutamate excitotoxicity is the predominant mechanism implicated in neuronal cell death associated with neurological disorders such as stroke, epilepsy, traumatic brain injury and ALS. Excessive stimulation of NMDA subtypes of glutamate receptors leads to protracted intracellular calcium elevations triggering calcium mediated neurotoxic mechanisms culminating in delayed neuronal cell death. In addition, glutamate excitotoxicity induces a NMDA dependent extended neuronal depolarization mediated by continuous calcium influx that correlates with delayed neuronal death. Attempts to prevent neuronal death by blocking calcium entry into the neurons using calcium channel blockers or NMDA receptor antagonists have failed to provide any beneficial effects in clinical trials. Thus, calcium continues to enter the neurons despite the presence of calcium entry blockers. This phenomenon is known as the "calcium paradox of stroke" and represents a major problem in developing effective therapies for treatment of stroke. Here employing a combination of patch clamp recordings, fluorescent calcium imaging and neuronal cell death assays in well-characterized in vivo and in vitro models of glutamate excitotoxicity, we report the discovery of a novel calcium permeable ion channel that is activated by excitotoxic glutamate injury and mediates a calcium current that is an early initiating step in causing neuronal death. Blocking this calcium permeable channel with high concentrations of Zn2+ or Gd3+ by removing extracellular calcium for a significant time period after the initial injury is effective in preventing calcium entry, apoptosis and neuronal death, thus accounting for the calcium paradox. This injury induced-calcium permeable channel provides a unique mechanism for calcium entry following stroke and offers a new target for extending the therapeutic window for preventing neuronal death after the initial excitotoxic (stroke) injury.

Nonspecific NMDA receptor antagonists induce hyperlocomotion in rats. The aim of this work is to determine whether the NMDA receptor antagonist specific for NR2 subunit exhibit similar negative effect as nonspecific antagonists. This subunit is predominant in the brain in the early postnatal period. The introduction summarizes the data on NMDA receptors and the development of rat. The experimental part deals with the action of a specific NMDA receptor antagonist Ro 25-6981 on motor performance of developing rats. Substance was repeatedly administered to rats at postnatal days 7 to 11. Spontaneous locomotion and motor performance of the animals were repeatedly tested up to adulthood by battery of tests appropriate for individual ages. Our research demonstrated that this substance does not have significant effect on motor system of laboratory rat and that it might be further tested as a possible age-bound antiepileptic drug.

Herein is reported the synthesis of molecular probes for action of neuroactive steroids in vitro and in living organisms. In the first part, preparation of enantiomeric pregnane steroids is investigated, ultimately resulting into the total synthesis of ent-progesterone. The chirality of the target molecule is introduced by a highly effective organocatalytic asymmetric Robinson annulation. A new method for the sequential construction of five-membered carbocyclic ring is introduced as the key step. This is composed of substrate-controlled copper-catalyzed conjugate addition followed by radical oxygenation and subsequent thermal cyclization employing the persistent radical effect. The synthesis of truncated neurosteroid analogs is described and their biological activity at the NMDA receptor is compared with the native hormone. In the second part, methodology for specific deuterium labeling of both angular methyls of the 5β-pregnane steroid core is explored. Special attention was paid to the Barton-McCombie deoxygenation as the tool for introduction of the last deuterium atom into the methyl group. Both positions were labelled with total of three deuterium atoms in high isotopic purity.

N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by agonist glutamate and co-agonist glycine. They play a key role in mediating the fast excitatory synaptic neurotransmission in the mammalian central nervous system. To create a functional heterotetrameric receptor, the presence of two GluN1 subunits combined with GluN2 or GluN3 subunits is necessary. Previous studies confirmed the importance of M3 transmembrane helix and extracellularly localized cysteines in regulation of surface expression of functional NMDA receptors. The aim of my thesis is to elucidate an influence of clinically relevant mutations in M3 transmembrane helix and the role of all known cysteines that form disulphide bonds on surface delivery of NMDA receptor expressed in heterologous monkey kidney fibroblasts cell culture (COS-7). Using molecular biology methods, immunocytochemistry and microscopy I found that the clinically relevant mutations M641I and Y647S in GluN1 subunit and also the mutations of particular cysteines forming disulphide bonds caused substantial decrease of surface expression of NMDA receptors. Furthermore, I discovered that the effect of mutated GluN1 subunits on decrease of surface expression depends on the subunit composition. The contribution of my results lies in elucidating the...