This thesis tests the hypothesis that increased neuronal cell death in SIDS infants is related to the ability of risk factors, such as prone sleeping, to expose infants to intermittent hypercapnic hypoxia (IHH). Based on the hypothesis that the NMDA system is linked to neuronal death, by way of excitotoxicity, correlations were also sought between cell death and changes in NMDA receptor (NR1) expression in brainstem nuclei controlling cardiorespiratory function. The first aim of this study was to verify that increased neuronal cell death occurs in SIDS infants. To verify a piglet model of SIDS risk factors, brainstem changes were examined in piglets exposed to IHH, and comparisons were made to changes seen in SIDS infants. The NMDA receptor was characterised in controls for both the human infant and the piglet groups. Comparisons of neuronal changes were made with SIDS infants, and piglets exposed to IHH. Non-radioactive in-situ hybridisation and immunohistochemistry were performed on formalin fixed and paraffin embedded brainstem tissue to identify markers of cell death (caspase-3, active caspase-3, and TUNEL), and to examine NR1 mRNA and protein expressions. Staining was quantified using computerised image analysis software. Eight nuclei from the brainstem medulla (caudal in piglets, and mid in infants), and two nuclei from the rostral pons (infants) were studied. The first dataset included human infants aged 1-6 months with a diagnosis of SIDS (n=15) or non-SIDS (n=10). The second dataset comprised developing piglets aged 13-14 days, with controls (n=6), against those exposed to IHH for 2 (n=6) or 4 (n=5) days. Increased neuronal cell death was not verified in the SIDS infants, but abnormalities in NR1 expression were present in selected nuclei of the medulla. Piglets exposed to IHH had increased neuronal cell death and changes in NR1 in selected nuclei of the medulla. There was also a positive correlation between increased cell death and high NR1 levels. Preliminary data showed that SIDS infants who usually slept prone had some differences in NR1 compared to those who did not usually sleep prone. From these findings, it was concluded that IHH may underlie the abnormalities in NMDA receptor expression that are present in the brainstem of SIDS infants. Although IHH can induce an increase in neuronal cell death, its significance in the aetiology of SIDS is not known. In piglets, IHH induced cell death correlated with high NMDA expression in some brainstem nuclei, supporting the hypothesis that excitotoxicity may be involved in the mechanism for cell death. Moreover, this thesis presents for the first time, �preliminary pathological proof� of an association between prone sleeping and abnormal NMDA receptor expression in SIDS infants.

Le cervelet est une structure cérébrale nécessaire au maintien de la posture, à la coordination motrice ainsi qu'aux apprentissages moteurs. Son implication dans des fonctions cognitives ou émotionnelles, ainsi que dans des pathologies, comme l'autisme ou la schizophrénie, est également de plus en plus avancée. C'est enfin un modèle de choix pour comprendre le fonctionnement des neurones.<br />La cellule de Purkinje est un élément clef de la fonction cérébelleuse. Elle reçoit 2 grands types d'afférences glutamatergiques: les fibres parallèles (FP), et une fibre grimpante (FG), unique à partir du 21e jour postnatal chez la souris. La nature et le rôle des différents récepteurs du glutamate exprimés à ces synapses ont été largement étudiés et sont désormais assez bien connus. De manière surprenante, le récepteur N-methyl-D-aspartate (R-NMDA), qui joue un rôle clef dans la plupart des neurones intégrateurs du cerveau, en agissant comme détecteur de coïncidence et en permettant une signalisation calcique cruciale dans de nombreux processus cellulaires, a gardé quant à lui une fonction méconnue dans les cellules de Purkinje néonatales, et sa présence est demeurée quasiment inconnue chez les cellules adultes. Le but de cette thèse était donc, d'une part, d'étudier le rôle de la dépolarisation, via notamment les R-NMDA, dans les cellules de Purkinje néonatales, et d'autre part de clarifier la présence, puis le rôle des R-NMDA chez l'adulte. <br />Par le biais d'une étude menée en collaboration, nous pouvons proposer un rôle des R-NMDA dans l'effet neuroprotecteur de la dépolarisation, lors de la mort cellulaire développementale intervenant aux alentours du 3e jour post-natal. Dans des co-cultures olive-cervelet, la survie des cellules de Purkinje de cet âge est significativement favorisée par le voisinage de FGs. La synaptogénèse avec ces fibres aurait donc un effet neuroprotecteur sur les cellules de Purkinje et ce, via leurs R-NMDA juvéniles. Ces récepteurs, en détectant le glutamate libéré par les FGs voisines, pourraient ainsi favoriser la survie des seules cellules contactées par ces afférences à cette étape critique du développement.<br />Parce que les R-NMDA ne sont pas détectés dans les cellules de Purkinje entre la 2e et la 3e semaine postnatales, ces cellules étaient considérées comme un rare exemple de neurone intégrateur n'exprimant pas de R-NMDA chez l'adulte. Cependant, nous avons montré qu'après cette période transitoire d'absence, un nouveau type de R-NMDA apparaît aux synapses avec les FGs et reste exprimé à l'âge adulte. Nous avons mis en évidence la participation des R-NMDA dans la transmission excitatrice de FG, ce qui suggère leur rôle dans de nombreux mécanismes cellulaires au sein de la cellule de Purkinje, notamment dans la plasticité synaptique. En effet, nos résultats préliminaires présentés dans cette thèse sont en faveur d'un rôle des R-NMDA dans l'aiguillage de la plasticité à long terme des synapses FPs - cellules de Purkinje.<br />Nous avons également étudié la possibilité d'une compétition entre ces R-NMDA et un autre type de récepteur du glutamate spécifiquement exprimé dans les cellules de Purkinje, le récepteur GluRdelta2. Ce dernier joue un rôle essentiel dans la régression des FGs surnuméraires et la limitation de leur territoire dendritique d'innervation. Bien que nos résultats ne mettent pas en évidence une interaction entre ces récepteurs, ils montrent que chez la souris adulte n'exprimant pas GluRdelta2, lorsque l'innervation des cellules de Purkinje par de multiples FGs persiste, les R-NMDA ne participent qu'à la transmission synaptique de la plus forte FG. Ceci suggère également un rôle des R-NMDA dans le choix et la stabilisation d'une seule FG lors de la maturation finale de ces afférences.

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2008.

Anesthesia-induced spatial learning impairments in aged rats model postoperative cognitive dysfunction (POCD) in the elderly surgical population. Mechanisms underlying both normal age-related cognitive decline and anesthesia-induced spatial learning deficits in aged rats were investigated. With respect to the involvement of inflammasome activation and age-related cognitive decline, I hypothesized that the aged hippocampus exhibits an elevated activation of inflammasome components contributing to elevated levels of IL-1β in the aged brain. Age-related cognitive decline was identified in a subpopulation of male Fischer 344 rats. Activation of the NLRP1 inflammasome was elevated in the aged brain, contributing to spatial learning deficits in aged rats. With respect to anesthesia-induced spatial learning impairment in aged rats, I hypothesized that an increase in NR2B subunit in the hippocampus and cortex during and following isoflurane anesthesia exposure resulting in spatial learning impairment in aged rats via disruption of downstream signaling molecule, extracellular-signal regulated protein kinase (ERK). Anesthesia exposure resulted in chronic spatial learning impairment in aged rats that were previously unimpaired in spatial learning tasks. Additionally, anesthesia induced elevated levels of N-methyl-D-aspartate (NMDA) receptor NR2B subunit protein expression in aged. It was concluded that various factors contribute to age-related spatial impairment including: NLRP1 inflammasome activation and NMDA receptor NR2B protein expression elevation. It was also concluded that anesthesia exposure exacerbates the elevation in NR2B protein expression in the aged brain, with subsequent disruption of ERK activation leading to chronic spatial learning deficits in aged rats. In the final chapter, a relationship for the interplay between inflammation and NMDA receptor function in the aged brain is discussed. In addition, a novel mechanism for anesthesia-induced cognitive deficits is presented. Therapeutic treatments for cognitive decline and anesthesia-induced cognitive deficits are explored. Finally, future lines of research are proposed.

Research in the field of molecular neuroscience contributes a better perception of the events that trigger neurodegeneration. At the forefront of this work is the study of intracellular calcium as a consequence of mitochondrial dumping and NMDA receptor activation by glutamate. Increased intracellular calcium presages excitotoxicity with ultimate apoptosis of the cell. Among the many disorders involving this sequence is depression, a disorder that in and of itself is a risk factor for neurodegenerative disorders such as Alzheimer disease. The successful use of anti-depressants to alleviate the depressive state leads to the question about whether these pharmacological agents, as part of their effect to ameliorate depression, might have an effect on intracellular calcium. Until now, this has not been explored directly but such exploration was initiated with this thesis. As a model for astrocytes and neurons, two cell lines, C6 and SH-SY5Y were used. These were differentiated with all-trans retinoic acid into astrocyte-like and neuron-like cells. Graphic imaging of intracellular calcium by ratiometrics is not new, but what is new is using this technique to evaluate the effect of the antidepressants mirtazapine and citalopram on intracellular calcium fluxes induced by glutamate. Furthermore, comparing the ratiometric intracellular calcium flux in the presence of mirtazapine and citalopram to that of known NMDA blockers was also done for the first time. Also studied were the acute and chronic effects of mirtazapine and citalopram on cell viability. The antidepressant agents, mirtazapine and citalopram, were chosen for this study. Mirtazapine blocks the adrenergic and serotonergic inhibitory autoreceptors which results in the increased release of these neurotransmitters and increases their concentration in the synapse. And it also has been shown to have an anti-oxidant and a calcium modulatory effect. Citalopram has the highest degree of serotonin reuptake selectivity of all the selective serotonin reuptake inhibitors. The ratiometric studies found that mirtazapine and citalopram reduce the effect of glutamate-induced increase in relative [Ca2+]i by either a direct or indirect action on NMDA receptors. This effect is not similar to the NMDA blockers memantine and AP5. The supporting evidence is that CCCP, which normally releases calcium from mitochondria, has no effect in cells treated with acute mirtazapine or citalopram. This indicates that no calcium entered the cell – and subsequently none was taken up by mitochondria – in response to glutamate. However, these observations were with a limited number of cells and, therefore, these results will have to be verified by different techniques by different laboratories. In microplate studies, all drugs studied reduced cell viability but the mechanism behind this reduced viability remains to be determined. This may be due to mutations in enzymatic expression, uptake of drug through the cell membrane, or other perturbations. The reduction in cell viability induced by acute glutamate was attenuated by pretreatment with mirtazapine or citalopram. Moreover, chronic treatment of the cells with mirtazapine or citalopram for 10 weeks before acute treatment with glutamate either attenuated the effect on viability or reversed it. Based on this present study, mirtazapine and citalopram may be useful as neuroprotective agents to alleviate not only depression but also to reduce cell death in neurodegenerative diseases, trauma and stroke.

NMDA (N-methyl D-aspartate) Receptor is a ligand and voltage gated ion channel and involved in many processes such as synaptic plasticity, memory formation, behavioral responses and cell survival. In the sense of functional activity, cellular localization of NMDAR is important since this receptor shows its activity on the membrane. Although NMDA receptor is intensely studied there are no satisfying study showing its localization with microscobic methods. Besides, the effect of florescent protein labelling of NMDA receptor on its distribution is not shown. It is expected to provide basis for further interaction and distribution studies with this comparison. Contrary to literature, in this study it is shown that NMDA receptor does not localize only in ER and membrane instead has a cytosolic pattern and this pattern is compatible with the distribution of wild type NMDA receptor. In addition, florescent protein labelling of NMDA receptor does not interrupt cellular distribution of NMDAR. Moreover, this study shows that N-terminal domain of NR1 subunit is sufficient to prevent degradation of NR2B in the cell. In consideration of this study it can be concluded that EGFP and mCherry labelled NMDA receptors can be used in interaction studies such as FRET and other studies, making use of fluorescent labelling of NMDA receptors, in terms of cellular distribution.

No description available.

Στον αμφιβληστροειδή επιτελείται η μετατροπή δηλαδή της φωτεινής ενέργειας σε ηλεκτρικό ερέθισμα. Κύριος διεγερτικός διαβιβαστής στον αμφιβληστροειδή είναι το γλουταμινικό οξύ του οποίου η δράση επιτελείται μέσω ιοντοτρόπων, NMDA και μη NMDA, και μεταβοτρόπων υποδοχέων. Οι υποδοχείς ΝΜDA παρουσιάζουν ιδιαίτερα χαρακτηριστικά, όπως μεγάλη αγωγιμότητα ασβεστίου και τασεοεξαρτώμενη αναστολή από το Μg, ενώ φαίνεται να παίζουν ιδιαίτερο ρόλο σε διαδικασίες συναπτικής πλαστικότητας, στην ανάπτυξη του νευρικού συστήματος καθώς και στην διεγερσιτοξικότητα του γλουταμινικού. Στον αμφιβληστροειδή εντοπίζονται κυρίως στα γαγγλιακά και βραχύϊνα κύτταρα αλλά και σε διάμεσους νευρώνες. Οι υποδοχείς NMDA είναι ετερομερή που αποτελούνται από τις υπομονάδες NR1, NR2 και NR3. Το γονίδιο της βασικής λειτουργικής υπομονάδας NR1 περιέχει τρία εξόνια τα οποία υφίστανται εναλλακτικό μάτισμα προς δημιουργία οχτώ ισομορφών. Οι ισομορφές που περιλαμβάνουν το εξόνιο 21 ή C1 στο καρβοξυτελικό άκρο έχουν την χαρακτηριστική ιδιότητα φωσφορυλίωσης στην θρεονίνη 879 και στις σερίνες 890, 896 και 897. Η φωσφορυλίωση αποτελεί έναν από τους κυριότερους μηχανισμούς ρύθμισης των υποδοχέων του γλουταμινικού οξέος επηρεάζοντας τις ιδιότητες τους, την μεταφορά τους προς την κυτταρική μεμβράνη, αλλά και την υποκυτταρική κατανομή των υπομονάδων τους. Στόχος της εργασίας είναι η μελέτη της φωσφορυλιωμένης υπομονάδας NR1 του υποδοχέα NMDA στις θέσεις σερίνης 896 και 897 (NR1-Ser896 και NR1-Ser897) κατά την ανάπτυξη στον αμφιβληστροειδή. Για τον σκοπό αυτό χρησιμοποιήσαμε επίμυες Wistar ηλικιών 9, 14, 21, 35 και 60 ημερών. Με την μέθοδο ανοσοαποτύπωσης κατά Western μελετήσαμε τα επίπεδα της φωσφορυλιωμένης πρωτείνης. Η ΝR1-Ser897 παρουσιάζει ένα πρότυπο αύξησης μέχρι και την ηλικία των 35 ημερών με επακόλουθη πτώση σε αυτήν των 60 ημερών. Η NR1-Ser896 αυξάνεται μέχρι την ηλικία των 14 ημερών όπου και παρουσιάζει μια σταθερή πορεία μέχρι την ηλικία των 60 ημερών. Συμπερασματικά, οι φωσφορυλιώσεις στις διαφορετικές σερίνες της υπομονάδας NR1 του υποδοχέα NMDA παρουσιάζουν διαφορετικό αναπτυξιακό προφίλ και γενικότερα η φωσφορυλίωση αυτής της υπομονάδας φαίνεται ότι ρυθμίζεται αναπτυξιακά στον αμφιβληστροειδή. / The retina is responsible for the light conversion into nerve signals. Glutamate is the major excitatory neurotransmitter in the retina. Its actions are mediated by glutamate ionotropic (NMDA and non NMDA) and metabotropic receptors. The NMDA receptors (NMDARs) are permeable to Ca++ and are unique among glutamate receptors in that they are blocked by Mg++ in a voltage dependent manner. These receptors also seem to play an important role in the development of the nervous system, in synaptic plasticity as well as in glutamate neurotoxicity. In the retina they are expressed in many ganglion and amacrine cells and occasionally in horizontal and glial cells. Functional NMDARs are heteromers composed of the NR1 NR2 and NR3 subunits. The gene of the NR1 subunit has three exons which undergo alternative splicing to generate theoretically eight NR1 splice variants. Half of them include the exon 21 or C1 in carboxy-terminus domain which can be phosphorylated in the following residues: threonine 879 and serines 890,896 and 897. Protein phosphorylation has been recognized as a major mechanism for the regulation of glutamate receptor function, changing their properties and because of its proposed role in trafficking and targeting of the NMDARs, as well as in clustering NR1 subunits into receptor-rich domains. The aim of the present work is to study the phosphorylated subunit NR1 at the serine residues 896 and 897 of the NMDAR (NR1-Ser896 and NR1-Ser897) during retinal development. Wistar rats at postnatal days 9, 14, 21, 35 and 60 are used for the developmental studies. The protein levels of the phosphorylated NR1 subunit were evaluated in Western blots. NR1-Ser897 increased gradually with a peak value observed at postnatal day 35, followed by a decrease at P60. NR1-Ser 896 was also increased to its peak level at the age of 14 and its levels sustained until the age of 60.These data reveal that the developmental profiles of the phoshorylated NR1 subunits at the serine residues 896 and 897 are different, and that the phosphorylation of the NR1 subunit is, in general, subject to regulation during development of the retina

Reduced activity of the N-methyl-D-aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine site on the NR1 subunit that may be a promising therapeutic target for psychiatric illness. Recently, D-serine has been discovered to be a high-affinity endogenous activator of the NMDAR glycine site. Levels of D-serine in the brain are controlled by its synthesis enzyme serine racemase (Srr) and its catabolic enzyme D-amino acid oxidase (DAO). This work investigates the NMDAR glycine site, D-serine, and D-serine-regulatory enzymes Srr and DAO in the pathophysiology and treatment of symptomatology relevant to schizophrenia and other psychiatric disorders. Pharmacological and genetic mouse models were used to alter glycine site function and D-serine availability. Behavioral responses in these models were assessed. Administration of exogenous D-serine and the glycine transporter 1 (GlyT-1) inhibitor ALX-5407 improved performance of C57BL/6J mice in behavioral tests examining prepulse inhibition (PPI) or latent inhibition (LI). These compounds also reversed impairments induced by the NMDAR antagonist MK-801, and produced similar beneficial effects to the classical atypical antipsychotic clozapine. Mice carrying a point mutation that leads to diminished NMDAR glycine site function demonstrated abnormally persistent LI and deficits in social approach and spatial recognition that were reversible by D-serine or clozapine administration. Similarly, mutant mice that lacked Srr function and had a severe reduction in D-serine displayed impairments in sociability, PPI, spatial recognition and memory. Behavioral deficits in mice without Srr were exacerbated by MK-801 and rescued by treatment with D-serine or clozapine. A genetically-induced loss of DAO function in mice resulted in the elevation of brain D-serine levels, and produced improvements in spatial reversal memory and extinction of a learned response in the Morris water maze, consistent with the effects of exogenous D-serine application in wild-type mice. Thus, deficiencies in NMDAR glycine site function and D-serine availability produce behavioral disturbances that are relevant to the negative and cognitive symptoms of schizophrenia. Activation of the NMDAR glycine site by D-serine, GlyT-1 inhibition, or diminished DAO activity may be beneficial for the treatment of schizophrenia and other psychopathologies involving cognitive dysfunction and persistent repetitive behaviors.

D-Serine is regarded as an obligatory co-agonist required for the activation of NMDA-type glutamate receptors (NMDARs). In the retina D-serine and a second NMDAR coagonist, glycine, are present at similar concentration and the cells that produce and release them are in close apposition. This arrangement allows for an abundant supply of coagonists and under certain conditions the NMDAR coagonist binding site could be saturated. There is also evidence suggesting that D-serine can act in an inhibitory manner at AMPA/kainate-type glutamate receptors (GluRs). Glutamate receptor activation can lead to direct and indirect elevation of intracellular calcium (Ca2+) concentration ([Ca2+]i). Therefore, in this thesis, I predominantly used Ca2+ imaging techniques to study the effect of D-serine on GluR activation in the mammalian retina. I first describe a novel method I developed to load retinal cells with Ca2+ indicator dye using electroporation and show that retinas remain viable and responsive following electroporation. This technique was used to explore the excitatory role of D-serine at NMDARs and its potential inhibition of AMPA/kainate receptors using cultured retinal ganglion cells (RGCs) and isolated retina preparations. Using cultured RGCs I demonstrated that D-serine and glycine enhance NMDAR-mediated Ca2+ responses in a concentration-dependent manner and are equally effective as coagonists. In isolated retinas I showed that D-serine application enhanced NMDA-induced responses consistent with sub-saturating endogenous coagonist concentration. Degradation of endogenous D-serine reduced NMDAR-mediated Ca2+ responses supporting the contribution of this coagonist to NMDAR activation in the retina. Using imaging and two different electrophysiological approaches, I found that D-serine reduced AMPA/kainate receptor-mediated responses in cultured RGCs and isolated retinas at concentrations that are saturating at NMDARs. Antagonist experiments suggest that the majority of inhibition is due to D-serine acting on AMPA receptor activity. Degradation of endogenous D-serine enhanced AMPA/kainate-induced responses of some cells in isolated retina suggesting that, under these conditions, D-serine concentration may be sufficient to inhibit AMPA receptor activity. Overall, the work in this thesis illustrates the utility of electroporation as a method to load Ca2+-sensitive fluorescent dyes into retinal cells and highlights the potential role for D-serine as a modulator of ionotropic GluRs in the CNS.