Bibliography: pages 181-201. / A genomic library of the collagenolytic Vibrio alginolyticus strain was established in Escherichia coli HB101 employing the positive selection vector pEcoR251. A glutamine synthetase (GS) gene, glnA was identified by complementation of the glnA deletion in E. coli ET8051 glnA, glnL, glnG deletion strain. The glnA region of V. alginolyticus was cloned on a 5.7 kb insert in pRM210.

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Le récepteur du glutamate de type NMDA (NMDAR) est l'un des deux principaux récepteurs glutamatergiques, et donc un des principaux mediateurs de la neurotransmission excitatrice dans le système nerveux central. Les NMDARs sont impliqués dans la plasticité synaptique, le corrélat cellulaire de l'apprentissage et de la mémoire. Actuellement, la technologie de pointe permettant l'investigation des propriétés des récepteurs synaptiques dans leur environnement natif est la photolyse de composés chimiques cagés, mais cet outil a des limitations concernant sa capacité à stimuler des NMDAR de manière très précise spatiellement et temporellement, à cause de la limite de diffraction de la lumière, qui définit le volume minimal de décageage duquel les molécules diffusent, et à cause de la nature irréversible de la réaction de décageage. En revanche, les molécules photoréversibles peuvent être activées et désactivées rapidement et de manière repétée, ce qui permet d'éviter les limitations de la diffusion afin d'accomplir une stimulation plus précise au niveau spatial et temporel. J'ai établi une collaboration autour du premier composé chimique photoréversible spécifique pour les NMDAR, et de plus, le premier qui est inactif dans son état le plus stable : l'azobenzene triazole glutamate (ATG). J'ai caracterisé ce composé chimique par activation un-photon et bi-photon, par l'application en bain et l'application locale, et dans plusieurs paradigmes expérimentaux. Dans ma thèse, je décris le fonctionnement de ce composé chimique, ses avantages et ses inconvénients, et certaines modifications à considérer pour l'optimisation future des composés chimiques photoréversibles. / The NMDA-type glutamate receptor (NMDAR) is one of two principal glutamate receptors, the main mediators of excitatory neurotransmission in the central nervous system. NMDARs are critically implicated in synaptic plasticity, the cellular correlate of learning and memory. Although significant advances have been made in understanding the behavior of this receptor, many questions remain. Currently, the state-of-the-art technology for investigating receptor properties in the native environment is caged compounds, which are restricted in their ability to precisely control the spatial and temporal activation of NMDAR due to the diffraction limit of light, which defines the minimum volume of uncaging from whence uncaging molecules diffuse, and the irreversible nature of uncaging. Photoswitchable molecules, by contrast, can rapidly and repeatedly be switched on and off, circumventing the diffusion limitation to permit fine spatial and temporal control of receptor activation. With this in mind, I formed a collaboration with a team of chemists to characterize a novel compound, azobenzene triazole glutamate (ATG), the first photoswitchable compound specific for NMDAR and biologically inert in its thermally stable state. Such a tool holds great promise for finely probing receptor behavior in its native environment. I characterized this compound using one- and two-photon activation, through bath and local application, and through a variety of different experimental paradigms. I demonstrate in detail the properties of this novel compound, propose potential applications of ATG as a novel tool, and suggest possible modifications to optimize future photoswitchable compound design.

Degree awarded (2004): PhDBmS, Neuroscience Program, George Washington University / Glutamate is critically important as an excitatory neurotransmitter in the central nervous system. Increasing evidence suggests additional signaling roles for glutamate in cell proliferation and migration in normal and oncogenic states. Recently, glutamate release from glioma cells has been shown to increase tumor growth in vivo. To investigate the mechanism of glutamate enhancement of tumor growth, we investigated the effect of glutamate on tumor cell proliferation, invasion, and glioma-induced cell death. Here we demonstrate that glutamate enhances tumor growth via increasing tumor cell proliferation and inducing excitotoxic death of cells surrounding the solid tumor mass, thereby facilitating tumor expansion. The evidence that glutamate enhances tumor growth suggests that regulating extracellular levels of glutamate may restrict tumor growth. In the normal brain, extracellular glutamate levels are maintained by a family of glutamate transporters. To investigate the therapeutic potential of regulating extracellular glutamate concentrations on tumor growth, we utilized a transgenic mouse model of EAAT2 glutamate transporter overexpression. In this report, we demonstrate that increased glutamate transport limits tumor growth in vivo and provides protection against glioma-associated neuronal cell death. In addition, seizure activity, often associated with the presence of a CNS tumor, is attenuated in transgenic mice overexpressing the glutamate transporter, EAAT2. These findings suggest that glutamate transporters may provide a new therapeutic target for limiting tumor expansion and secondary epileptogenesis. / Advisory Committee: Dr. Margaret Sutherland (Chair), Dr. Steven Patierno (Chair), Dr. Tim Hales, Dr. Vincent Chiappinelli, Dr. Linda Werling, Dr. Frances Noonan

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 104-118).

Two studies were conducted to compare nitrogen (N) and glutamate metabolism in Polypay and percentage White Dorper lambs.First, a two-phase digestion/N metabolism trial was conducted with 18 wether lambs of three genetic types: Polypay (PP), 1/2 White Dorper 1/2 Polypay (1/2 D), and 3/4 White Dorper 1/4 Polypay (3/4 D). Six lambs of each genetic type were fed a high roughage diet (HR; Phase 1) or high concentrate diet (HC; Phase 2). DM and N digestion was higher for 1/2 D than PP or 3/4 D fed HC. N retention was highest for 1/2 D regardless of diet.The second study analyzed the effect of genetic type on glutamate transporter and metabolizing enzyme expression in liver, kidney, longissimus dorsi muscle (LD muscle), and subcutaneous fat (Sub Q Fat) tissue of 18 wether lambs of three genetic types: PP, 1/2 D, and 15 /16 White Dorper 1/16 Polypay (15 /16 D). Tissue samples were analyzed for protein and mRNA content of GS, GDH, ALT, EAAC1, and GLT-1. Glutamate transport and metabolism capacity was lowest for the heavier muscled 15 /16 D lambs.The results suggest genetic type has an effect on N metabolism due to differential expression of glutamate transporters and metabolizing enzymes.

Schizophrenia is a serious psychiatric disorder that affects 1% of the population. Current theories implicate NMDA receptor hypofunction as a contributor to the symptomology and pathological alterations in schizophrenia. Cognitive impairments are increasingly recognized as not only fundamental to schizophrenia, but the strongest predictor of patient functional outcomes. Current antipsychotics do not improve the cognitive symptoms of the disorder; however, recent efforts have resulted in the identification of novel drug targets. One target is metabotropic glutamate receptors as they interact with and modulate NMDA receptors. Another approach focuses on dopamine, the neurotransmitter system targeted by traditional antipsychotics. Tetrahydroprotoberberines, such as D- and L-govadine, are synthetic compounds derived from traditional medicine that have demonstrated efficacy in treating schizophrenic symptoms. The present study assessed the effects of CDPPB (a metabotropic glutamate receptor 5 positive allosteric modulator), D- and L-govadine, and the typical antipsychotic haloperidol on the Paired Associates Learning (PAL) task in rats. The PAL task is impaired in patients with schizophrenia, has been adapted for use with rodents using touchscreen-equipped operant chambers, and has been promoted by MATRICS as a promising behavioural task with the potential to further translational health research in schizophrenia. The objectives of this study were: 1) examine the effects of acute NMDA receptor antagonism with MK-801 as a model for schizophrenia on performance of the PAL task; 2) test the effects of the putative antipsychotics, CDPPB and D- and L-govadine on reversing the effects of NMDA receptor antagonism on the task; and 3) to compare these novel therapeutics to a classic antipsychotic. Two squads of male Long-Evans rats were trained to perform the PAL task in touchscreen-equipped operant chambers. After the rats reached criterion the following treatment schedules were divided between the two squads: 1) vehicle (10% cyclodextrin; i.p.), and CDPPB (1.0, 3.0, and 10.0 mg/kg, i.p.); or 2) vehicle (10% cyclodextrin; i.p.), CDPPB (3.0 mg/kg, i.p.), the NMDA receptor antagonist MK-801 (0.15 mg/kg, i.p.), and CDPPB with MK-801; or 3) vehicle (50% DMSO; s.c.), MK-801, D-govadine (1.0 mg/kg; s.c.), L-govadine (1.0 mg/kg; s.c.) and MK-801 with each isomer of govadine; or 4) vehicle (sodium acetate and acetic acid, pH 5.0, s.c.), and haloperidol (0.05 and 0.1 mg/kg, s.c.). Acute MK-801 significantly reduced the number of trials completed, impaired accuracy, and increased the number of errors in the PAL task. CDPPB had no effect on the PAL task and did not improve the MK-801 induced impairments. Administration of L-govadine, but not D-govadine, prior to MK-801 improved accuracy and reduced errors compared to MK-801 alone. L-govadine alone, but not D-govadine, reduced total responding compared to vehicle. Haloperidol caused a dose-dependent decrease in all activity in the task confounding interpretation of the results in regard to cognition. These data establish disruptive effects of acute MK-801 treatment on PAL task performance and demonstrate that L-govadine is capable of cognitive enhancement in a rodent model of schizophrenia.

Thèse (M.Sc.) -- Université Laval, 1997. / Bibliogr.: f. 131-155. Publ. aussi en version électronique.

L'épilepsie affecte des millions de patients dans le monde. Les traitements disponibles sont symptomatiques, ils per récepteur mGlu7 du glutamate dans la modulation non seulement de l’excitabilité, mais également de l'hypersynchronisation des réseaux de neurones, deux facteurs cruciaux dans les crises d'épilepsie. J'ai parachevé ces découvertes qui ont été à l’origine d’une première publication (Tassin, Girard et al. 2016).A l’aide d’un nouvel agoniste du récepteur mGlu7, le LSP2-9166, lors de ma thèse j'ai ensuite étudié l’impact de ce récepteur dans différents modèles d’épilepsie chez la souris. Deux modèles complémentaires ont été utilisés : le kindling (embrasement), modèle chimique induit par le pentylènetétrazol (PTZ) qui sensibilise le cerveau jusqu’à générer des crises tonico-cloniques généralisées, et l’injection intra-hippocampique de kainate, mimant l’épilepsie mésiale du lobe temporal chez l'homme.Dans un premier temps j'ai observé une atténuation de la progression de la sévérité des crises dans le modèle de kindling au PTZ, sous l’activation du récepteur mGlu7. Cet effet a été corrélé à une inflammation, et une activation microgliale et astrocytaire plus faibles. Dans le modèle d’injection intra-hippocampique de KA, considéré comme pharmaco-résistant, l’activation du récepteur mGlu7 pendant la période d’épileptogenèse a augmenté la durée des périodes interictales et diminué la durée des crises ainsi que la réorganisation neuronale. Une fois les crises chroniques installées, l’activation aigu du récepteur mGlu7 a diminué le nombre de crises aussi fortement que le diazépam couramment utilisé dans le cadre clinique. Pour finir, des injections chroniques de LSP2-9166 chez des animaux naïfs (non épileptiques) n’engendrent pas de déficit cognitif ou comportemental détectables, ni de modification du niveau d’ARNm du récepteur mGlu7. L’activation du récepteur mGlu7 offre donc une cible stratégique dans nos deux modèles.Ces travaux permettent une meilleurmettent de traiter les crises sans pour autant éviter la progression de la maladie et présentent de lourds effets secondaires. La découverte de nouvelles cibles thérapeutiques et de nouveaux composés reste primordiale pour dépasser les limites des stratégies thérapeutiques actuelles. Au début de ma thèse, des études précédentes avaient montré une implication due compréhension du rôle du récepteur mGlu7 dans l’épileptogenèse. Ils participent ainsi à la recherche de futurs traitements. antiépileptiques adéquats. / Epilepsy affects millions of patients worldwide. The available treatments are symptomatic, they treat seizures without preventing the progression of the disease and have heavy side effects. The discovery of new therapeutic targets and new compounds is therefore essential to overcome the limitations of current therapeutic strategies. Previous studies have demonstrated substantial involvement of the mGlu7 receptor in modulating not only excitability but also hypersynchronization of neural networks, two crucial factors affecting epileptic seizures. These discoveries were at the origin of a first publication that I completed at the beginning of my thesis (Tassin, Girard et al., 2016).Using a new mGlu7 receptor agonist, LSP2-9166, in my thesis I then studied the impact of this receptor in different epilepsy models in mice. Two complementary models were used: kindling, a chemical model induced by pentylenetetrazol (PTZ) which sensitizes the brain to induce generalized tonic-clonic seizures, and intra-hippocampal injection of kainate, mimicking mesial temporal lobe epilepsy in humans.At first, I observed an attenuation of the progression of the seizures severity in the PTZ kindling model, under the activation of the mGlu7 receptor. This effect was correlated with weaker inflammation, and microglial and astrocytic activation. In the intra-hippocampal injection model of KA, considered as drug-resistant, activation of the mGlu7 receptor during the epileptogenesis period increased the duration of interictal periods and decreased the duration of seizures as well as neuronal reorganization. Once chronic seizures were established, acute activation of the mGlu7 receptor decreased the number of seizures as strongly as diazepam, commonly used in clinical settings. Finally, chronic injections of LSP2-9166 into naive (non epileptic) animals do not generate any detectable cognitive or behavioral deficits or changes in mGlu7 receptor mRNA level. The activation of the mGlu7 receptor thus presents a strategic target in our two models.This work provides a better understanding of the role of the mGlu7 receptor in epileptogenesis. It participates in the search for future more adequate treatments.

Immunohistochemical studies were conducted to assess the subunits of ionotropic and metabotropic glutamate receptor present in the rostral ventrolateral medulla (RVLM) of the rat. Double labeling the medullary sections with polyclonal GluR1, GluR2/3, GluR4, NMDAR1, NMDAR2A/B, mGluR1α, and mGluR2/3 antiserum and monoclonal tyrosine hydroxylase (TH) antiserum revealed nearly all TH immunoreactive (irTH) cells and many TH-negative neurons were immunoreactive to GluR2/3 (irGluR2/3), NMDAR1 (irNMDAR1), and NMDAR2A/B (irNMDAR2A/B). A few RVLM neurons were immunoreactive to GluR1 (irGluR1) and GluR4 (irGluR4), but they were generally TH-negative. Immunoreactivity to mGluR1α (irmGluR1α) appeared to be localized exclusively to fiber-like elements in the RVLM area. Our results show that neurons in the RVLM, including irTH, are endowed mainly with GluR2/3 and NMDAR1 or NMDAR2A/B ionotropic receptor subunits, and that irmGluR1α splice variant appears to be located on nerve fibers ramifying within the RVLM. Moreover, TH-negative neurons in the RVLM appear to bear similar subunits of ionotropic glutamate receptors.