Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 105-113). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Restricted until October 2005. Bibliography: leaves 71-94.

Les rats GAERS (Genetic Absence Epilepsy Rats from Strasbourg) et NEC (Non-Epileptic-Control rats) dérivent de la lignée de rats Wistar-Hannover (WH). Chez les GAERS les premières crises d’épilepsies apparaissent vers l’âge de 25 jours puis leur nombre et leur durée augmentent pour se maintenir à l’âge adulte à raison d’une crise de 20 secondes en moyenne par minute lorsque l’animal est en veille calme. Ce développement spontané des crises d’épilepsies permet d’étudier la période silencieuse avant les premières crises ainsi que la survenue de ces premières crises (l’épileptogènese). De nombreuses études pharmacologiques et électrophysiologiques dans des modèles animaux d’épilepsie révèlent un rôle critique de la transmission dopaminergique dans la susceptibilité aux crises. Les GAERS adultes présentent une surexpression de l’ARNm des récepteurs dopaminergiques D3 comparé aux rats NEC. L’expression ainsi que la fonction du récepteur D3 et celles du transporteur de la dopamine (DAT) sont liées. Le but de ce travail a été de déterminer l’implication du RD3 et du DAT dans le développement de l’épilepsie en mesurant leur expression et fonction chez les GAERS avant l’apparition des premières crises. L’expression et la fonction du RD3 ont été mesurées par autoradiographie au [125I]-PIPAT et induction de bâillement par injection de quinpirole respectivement, dans les 3 lignées de rats (GAERS, NEC and WH) chez des rats adultes, à P14 et P21. L’expression du DAT a été analysée par imagerie SPECT à [123I]-Ioflupane chez les adultes et par autoradiographie au [3H]-GBR12935 chez les adultes, P14 et P21. Par ailleurs l’activité du DAT a été mesurée par capture de dopamine tritiée sur des synaptosomes de striatum, cortex et hippocampe de rats adultes des 3 lignées. La participation du RD3 dans les crises a été évaluée par EEG vidéo après l’injection d’agoniste D3 (quinpirole et PD128907) ou d’antagoniste (SB277011 et SR21502). Les expériences de radiographie ont révélé une surexpression des RD3 chez les GAERS dans les structures connues pour participer à l’initiation des crises (cortex somatosensoriel), à leur contrôle (noyaux accumbens) ainsi que dans d’autres structures (thalamus antérieur, tubercules olfactifs et ilots de calleja), à P14 et P21 comparé aux autres lignées. Ni le SPECT ni l’autoradiographie n’a relevé de modification d’expression du DAT chez les rats adultes, cependant à P14 et P21 le DAT est surexprimé au niveau du striatum. Ces résultats sont accompagnés d’une augmentation de la fonctionnalité du DAT aux 3 âges chez les GAERS compares aux autres lignées. Les agonistes D3 induisent une augmentation des décharges de pointes-ondes sans que les antagonistes aient un effet. De plus l’injection chronique d’aripiprazole (un neuroleptique connu pour sa capacité à stabiliser la libération de dopamine) chez les rats GAERS dès la naissance entraine une diminution des crises associée à une diminution d’expression des RD3. L’utilisation de lentivirus contenant une séquence shRNA anti-RD3 chez les GAERS adultes entraine aussi une diminution du nombre de crises d’épilepsie. Nos résultats suggèrent ainsi que l’augmentation du nombre de RD3 chez le GAERS est concomitante à une augmentation de la sensibilité du système dopaminergique, et cela avant l’apparition des premières crises épileptiques. Cette augmentation au cours de la maturation pourrait participer à l’aggravation progressive des crises d’épilepsie qui est la base de l’épileptogenèse et expliquer le déséquilibre de la tonicité dopaminergique que nous avons démontré chez l’adulte / Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and Non-Epileptic-Control rats (NEC) derive from an original Wistar-Hannover rat strain (WH). The onset age of spike-and-waves discharges in GAERS is about 25 days post-natal (P25). In adult GAERS with fully developed epilepsy, dopamine plays a modulatory role in seizure expression. Adult GAERS display an over-expression of dopaminergic D3-receptors (D3R) mRNA as compared to NEC. Expression and function of D3R and dopamine transporter (DAT) are closely related. The aim of this work was to investigate the putative involvement of D3R and DAT during epileptogenesis by measuring their expression and functionality in GAERS before the onset of epilepsy (P25). D3R expression and functionality was investigated by [125I]-PIPAT autoradiography and quinpirole-induced yawning, respectively, in the three strains of rats (GAERS, NEC and WH) in adults, P14 and P21. DAT expression was investigated in GAERS and NEC by [123I]-Ioflupane SPECT imaging in adults and [3H]-GBR12935 autoradiography in adults, P14 and P21 rats. Furthermore, DAT activity was assessed by 3H-dopamine reuptake in synaptosomal living fractions of striatum, cortex and hippocampus of adult rats in the three strains. The involvement of the D3R was further investigated by video-EEG recording following systemic injections of either D3R agonist (quinpirole and PD128907) or antagonists (SB277011 and SR21502). Autoradiography showed an over-expression of D3R in GAERS in structures known to be involved in seizure initiation (somato-sensory cortex), seizure control (nucleus accumbens,) as well as in other structures (anterior thalamus, olfactory tubercles and islands of Calleja) at P14 and P21, as compared to age-matched NEC and WH. As in adults, this over-expression was associated with a higher number of quinpirole-induced yawns in GAERS at P14 and P21. Neither SPECT imaging nor autoradiographic data revealed any modification in DAT expression between the three strains in adults, however at P14 and P21 DAT is overexpressed in the striatum of GAERS rats. However, we found a consistent increase in 3H-dopamine reuptake in adult GAERS as compared to NEC and WH in the functional assay supporting an increase in dopamine translocation velocity. Administrations of D3R agonists increased spike-and-wave discharges, whereas antagonists had no effect. Furthermore, the chronic injection of aripiprazole (an atypical neuroleptic known to stabilize dopamine release) to GAERS pups reduced the number of seizures in adults along with a decreased expression of D3R. Preliminary data using lentiviral infection with shRNA anti-D3R also support reduced seizure number in adult GAERS rats. Our results suggest that an over-expression of functional D3R already exists before the onset of seizures in GAERS and that, despite a lack of changes in DAT expression, functional changes in this transporter occur in adults. They further support that a profound modification in basal ganglia function together with changes in D3R could be a conditional factor for epileptogenesis. The dopaminergic system appears persistently altered in spontaneous epileptic rats, which could contribute to the development of the chronic epileptic state and may represent a potential new target for antiepileptic therapies and/or improvement of quality of life of epileptic patients.

L'objectif de cette thèse était l'investigation du fonctionnement et des dysfonctionnements des deux systèmes visuels principaux (magnocellulaire et parvocellulaire). Une synthèse théorique sur la dissociation de ces deux systèmes et une revue sur les procédures expérimentales permettant de les dissocier constituaient la première partie de cette thèse. Ensuite, des arguments neuropsychologiques concernant le vieillissement normal et pathologique du système visuel ont été synthétisés et différents résultats de la littérature ont été analysés pour étayer cette dissociation. La quinzaine d’expériences réalisées a mis en évidence deux résultats principaux. Il y a un déficit massif du système parvocellulaire dans le vieillissement normal dès les étapes précoces du traitement et qui perdure aux étapes plus tardives, ainsi qu’un déficit magnocellulaire sous certaines conditions seulement. L'étude des dysfonctionnements visuels dans les pathologies à syndromes extrapyramidaux a montré que ces patients présentaient un déficit spécifiquement magnocellulaire en lien avec la dénervation dopaminergique dans le système central, objectivée à l’aide d’une technique de scintigraphie cérébrale. / The objective was to investigate the normal functioning and the impairment of the two main visual systems, the parvocellular and the magnocellular systems. A theoretical review on the two systems and on the experimental procedures that have been used to dissociate them is proposed in the first chapter. In a second part, a synthesis of the neuropsychological studies focusing on the normal aging of the visual system and on the visual deficits found in various pathologies, that support the parvocellular / magnocellular dissociation is presented. The experimental chapter comprised about fifteen experiments and showed two main results: 1/ a major low-level parvocellular impairment with normal aging that was not compensated at the later stages, and a slighter magnocellular impairment under certain conditions: 2/ a specific deterioration of the magnocellular system in extrapyramidal patients, correlated with dopamine denervation in two structures of the central system.

In an attempt to determine the mechanism by which the tripeptide l-prolyl-l-leucyl-glycine amide (PLG, MIF-I) exerts its antiparkinsonian effect, the action of this substance on various postsynaptic components of striatal dopaminergic nerves was studied. It was shown that injection of rats with MIF-I (1 mg/kg, IP×5, 24 hr intervals) did not alter tyrosine hydroxylase, dopa decarboxylase, choline acetyltransferase and glutamic acid decarboxylase activities in the striatum under the conditions tested. The activities of adenylate cyclase, dopamine-stimulated adenylate cyclase, and guanylate cyclase were not altered in vitro by various concentrations of MIF-I (0.1 to 1000 μM), although VIP and neurotensin had some effect. Also the rate of uptake of 3H-dopamine by rat striatal synaptosomes was unchanged, as was the binding of 3H-dopamine and 3H-spiperone to beef caudate membranes. This series of studies indicates that MIF-I does not act directly on the striatal dopamine postsynaptic receptor under the conditions tested, although it is possible that MIF-I could act indirectly at this or another site in vivo by releasing or activating some other factor.

Methylphenidate (MPH) is the most commonly prescribed drug for the treatment of ADHD in males and females. However, a majority of previous studies investigated the effect of MPH in only males, and little is known regarding consequences of female exposure to MPH. This is unfortunate because the few studies that have been conducted indicate that females have a greater sensitivity to MPH. Previous research in male mice has shown that chronic exposure to MPH causes dopaminergic neurons within the nigrostriatal pathway to be more sensitive to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, estrogen has been shown to protect dopaminergic neurons from MPTP neurotoxicity. Therefore, in this study, we test the hypothesis that chronic MPH exposure in female mice will render dopaminergic neurons in the nigrostriatal pathway more sensitive to MPTP, and that estrogen may play a protective role. Interestingly, proestrus females exhibited greater sensitivity to MPTP, with significantly reduced dopaminergic neurons in the SN and significant increases in DA quinone production. Chronic MPH exposure contributed to GSH depletion, but surprisingly, it did not increase dopamine quinone levels or dopaminergic cell loss. There were no significant differences in anestrus animals, with the exception of a depletion in GSH seen when animals received chronic high-dose (10 mg/kg) MPH followed by MPTP. Thus, estrogen may actually sensitize neurons to MPTP in this model, and chronic MPH may contribute to GSH depletion within the striatum. This study provides insight into how chronic psychostimulant use may affect males and females differently.

Ce travail de thèse est consacré à l'étude en imagerie multimodale des bases neurobiologiques des pathologies psychiatriques, avec un intérêt particulier pour les voies dopaminergiques et le système de récompense. Son principal objectif est d'établir, par l’intermédiaire de la Tomographie à Emission de Positons (TEP) et l’Imagerie par Résonance Magnétique (IRM), un lien entre le système dopaminergique et le système de récompense d’un point de vue fonctionnel et structurel chez l’Homme, et en particulier chez des patients présentant des troubles psychiatriques tels que la schizophrénie, l’addiction à la cocaïne et la dépression. De nombreuses études ont démontré l’existence concomitante d’anomalies de la fonction dopaminergique et du système de récompense dans plusieurs troubles mentaux. Cependant, la connaissance des liens entre dysfonctions dopaminergiques et dysfonctions du circuit de la récompense dans les pathologies psychiatriques reste limitée. L’objectif de cette thèse est ainsi d’améliorer les connaissances sur la physiopathologie de plusieurs troubles mentaux comme la schizophrénie, l’addiction et la dépression, et de démontrer l’intérêt d’une approche dimensionnelle et de l’utilisation de l’imagerie multimodale pour l’exploration du niveau moléculaire de réseaux neuronaux fonctionnels dans la recherche en psychiatrie. En perspective, ce travail de thèse soutient l’intérêt de l’imagerie en pratique psychiatrique, car elle pourrait par la suite permettre de préciser le diagnostic, prédire les réponses aux traitements ou étudier l’évolution de la maladie au cours du temps. / This work focuses on the study of the neurobiological bases of psychiatric disorders using multimodal imaging, with a particular interest in the dopaminergic pathways and the reward system. Its primary objective is to establish, through Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI), a link between the dopaminergic system and the reward system from a functional and structural point of view in humans, and especially in patients with psychiatric disorders such as schizophrenia, cocaine addiction and depression. Numerous studies have demonstrated the concomitant existence of abnormalities affecting dopaminergic function and reward system in several mental disorders. However, understanding of the linkages between dopaminergic dysfunction and dysfunction of the reward circuit in psychiatric disorders remains limited. The main aims of this thesis are to improve knowledge about the pathophysiology of several mental disorders such as schizophrenia, addiction and depression, and to demonstrate the interest of both a dimensional approach and the use of multimodal imaging in psychiatric research, to explore the molecular level of functional neural networks. In perspective, this thesis supports the interest of brain imaging in clinical practice, as it could later clarify the diagnosis, predict response to treatments or follow the course of the disease.

To study the role of dopamine D1 receptors in enhanced oral activity effects of SKF 38393 ((±)-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol) in neonatal 6-hydroxydopamine-lesioned rats, SKF 38393 was compared to the full agonist, A77636 ((1R,3S)-3-(1′-adamantyl)-1-aminomethyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyran). At 3 days after birth rats were treated with 6-hydroxydopamine HBr (200 μg, salt form, i.c.v.; desipramine (20 mg/kg i.p.), 1 h) or vehicle. At 6-8 months a 0.01 mg/kg dose of A77636 HCl increased oral activity in 6-hydroxydopamine vs. control rats (P < 0.01). A77636 and SKF 38393 produced identical maximal responses of 35-36 oral movements at 0.1 and 1.0 mg/kg, respectively. SCH 23390 (R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) HCl (0.3 mg/kg i.p.) attenuated the response to A77636. Neither A77636 HCl (0.01-1.0 mg/kg i.p.) nor SKF 38393 HCl (0.03-3.0 mg/kg i.p.) induced oral activity in intact rats. The findings demonstrate that A77636 is more potent than SKF 38393, and that supersensitized dopamine D1 receptors are involved in the induction of oral behavior in neonatal 6-hydroxydopamine-lesioned rats.

Phosducin (Pdc) is a 28 kDa binding partner for the G protein beta gamma subunit dimer (G-beta-gamma) found abundantly in the photoreceptor cells of the retina and pineal gland. In the retina, light-dependent changes in cAMP and Ca2+ control the phosphorylation of Pdc at serine 73 and 54, respectively, which in turn controls the binding of Pdc to G protein beta gamma subunit dimer . G protein beta gamma subunit dimer binding has been proposed to facilitate light-driven transport of G protein beta gamma subunit dimer from the site of phototransduction in the outer segment of the photoreceptor cell to the inner segment, thereby decreasing light sensitivity and contributing to the process of light adaptation. Dopamine and melatonin are neuromodulators whose concentrations in the retina vary reciprocally during the circadian cycle, with dopamine high during the day and melatonin high during the night. Together, they control numerous aspects of light and dark adaptation in the retina. In this study, we have investigated the possible roles of dopamine and melatonin in regulating Pdc phosphorylation. Using phosphorylation-site specific antibodies to serines 54 and 73, we show that dopamine decreases the phosphorylation of both sites. This decrease is blocked by D4 receptor antagonists and pertussis toxin, indicating that dopamine causes a decrease in photoreceptor cell cAMP and Ca2+ concentration via the D4 receptor coupled to the Gi protein. Conversely, melatonin increases the phosphorylation of both S54 and S73, most likely via the inhibition of dopamine synthesis. These results demonstrate that dopamine and melatonin control the phosphorylation state of phosducin by changing the concentration of cAMP and Ca2+ in photoreceptor cells, and they suggest that dopamine and melatonin may contribute to the light-induced movement of the photoreceptor G protein by regulating Pdc phosphorylation.

No description available.