No description available.

<p>Patients suffering from different forms of neurodegenerative diseases, such as: Creutzfeldt Jacob Disease (CJD), Alzheimer disease (AD), mild cognitive impairment (MCI), frontotemporal dementia and Parkinson’s disease (PD) were examined with Positron Emission Tomography (PET) and the combination of different radiotracers: ¹⁵O-water, N-[¹¹C-methyl]-L-deuterodeprenyl (DED), [¹⁸F] 2-fluorodeoxyglucose: (FDG), N-methyl-[¹¹C]2-(4-methylaminophenyl)-6-hydroxybenzothiazole (PIB) and L-[¹¹C]-3,4-dihydroxiphenyl-alanine (DOPA). The radiotracers and the combinations of different radiotracers were selected with the intention to detect, in the brain, patterns of neuronal dysfunction, astrocytosis, axon degeneration or protein aggregation (amyloid), in the brain which are pathognomonic for specific diseases and may contribute to improve clinical differential diagnoses. Examinations in healthy volunteers were performed to allow comparisons with patients. In addition, animal studies were conducted to complement the information. In some cases, the PET findings could be compared with the results of autopsies.</p><p>In contrast to the micropathology, in which only a limited part of a tissue (obtained post-mortem or by biopsy) is inspected, one PET acquisition provides an image of the whole system (e.g.: the brain and the cerebellum). This form of imaging pathology is “<i>in vivo</i>”, where the examination is innocuous for the patient. </p><p>This thesis is an attempt to stimulate the development of new tracers, new tracer combinations and methods that directly or indirectly describe the anatomo-physiopathological changes produced in the brain in neurodegenerative diseases. A better description of different diseases can be obtained, confirming or questioning the clinical diagnoses and widening our understanding of the mechanisms underlying neurodegeneration. Different pathologies can produce similar symptoms and thus causing confusion regarding clinical diagnosis. The used PET combinations improved the accuracy of the diagnoses. The incipient knowledge emerging from a new neuroimaging pathology in combination with other disciplines may open the way to new classifications of dementias and neurodegenerative diseases based on an “<i>in vivo</i>” pathology. </p>

No description available.

Parkinson’s disease, the second most common neurodegenerative disorder,is characterized by loss of nigrostriatal dopaminergic neurons. To date,there is no defined cause and cure for the disease. An ideal treatmentstrategy is to replace the lost neurons by transplanting fetal dopaminergicneurons to the brain of parkinsonian patients. Clinical trials have beenperformed and the outcome was variable where one significant obstaclewas the limited graft reinnervation of the host brain. To study this issue,organotypic tissue culture can be utilized to monitor dopaminergic nervefiber outgrowth in vitro and their association with astrocytes. Using thisculture technique, dopaminergic nerve fibers appear in twomorphologically and temporally different types. The early appearing nervefibers are formed in the absence of astrocytes, reach long distances, andare called non-glial-associated tyrosine hydroxylase (TH) -positive nervefibers. After a few days, the second sequence of nerve fibers, the glialassociatedTH-positive nerve fibers, are formed, and their growth arelimited to the presence of astrocytes, that migrate and form a monolayersurrounding the plated tissue. The aim of this thesis was to study theinteraction between nerve fiber formation and astrocytes with a specialfocus on the long-distance growing nerve fibers. Ventral mesencephalic(VM) organotypic slice cultures from embryonic day (E) 12, E14, and E18were incubated for 14, 21, 28, and 35 days in vitro (DIV). The resultsrevealed that the two morphologically different processes were found incultures from the younger stages, while no non-glial-associated growthwas found in cultures of tissue from E18. Instead neurons had migratedonto the migrating astrocytes. Astrocytes migrated longer distances intissue from older stages, and the migration reached a plateau at 21 DIV.Co-cultures of E14 VM tissue pieces and cell suspension of matureastrocytes promoted migration of neurons, as seen in E18 cultures. Thus,9the maturity of the astrocytes was an important factor for nerve fiberoutgrowth. Hence, targeting molecules secreted by astrocytes might bebeneficial for regeneration. Chondroitin sulfate proteoglycan (CSPG), amember of proteoglycan family, is produced by the astrocytes and has adual role of being permissive during development and inhibitory afterbrain injury in adult brain. Cultures were treated with chondroitinase ABC(ChABC) or methyl-umbelliferyl-β-D-xyloside (β-xyloside) in twodifferent protocols, early and late treatments. The results from the earlytreated cultures showed that both compounds inhibited the outgrowth ofnerve fibers and astrocytic migration in cultures from E14 tissue, while β-xyloside but not ChABC promoted the non-glial-associated growth incultures derived from E18 fetuses. In addition, β-xyloside but not ChABCinhibited neuronal migration in E18 cultures. Taken together, β-xylosideappeared more effective than ChABC in promoting nerve fiber growth.Another potential candidate, integrin-associated protein CD47, was studiedbecause of its role in synaptogenesis, which is important for nerve fibergrowth. Cultures from E14 CD47 knockout (CD47-/-) mice were plated andcompared to their wildtypes. CD47-/- cultures displayed a massive and longnon-glial-associated TH-positive nerve fiber outgrowth despite theirnormal astrocytic migration. Blocking either signal regulatory protein-α(SIRPα) or thrombospondin-1 (TSP-1), which bind to CD47, had nogrowth promoting effect. In conclusion, to promote nerve growth, youngertissue can grow for longer distances than older tissue, and inhibiting CSPGproduction promotes nerve growth in older tissue, while gene deletion ofCD47 makes the astrocytes permissive for a robust nerve fiber growth.

Synaptic transmission in the nervous system involves the activation of receptor proteins that permit rapid transmembrane fluxes of ions. Ionic gradients across the membrane determine the direction and driving force for the flow of ions and are therefore crucial in setting the properties of synaptic transmission. These ionic gradients are established by a variety of mechanisms, including pump and transporter proteins. However, the gradients can be affected by periods of neural activity, which in turn, are predicted to influence the properties of ongoing synaptic transmission. In this thesis I have examined the concentration gradients of two ions that play a fundamental role in synaptic transmission: chloride ions (Cl-) and protons (H+). Type A γ-Aminobutyric acid receptors (GABAARs) are primarily permeable to Cl- and mediate the majority of fast post-synaptic inhibition in the brain. The transmembrane concentration gradient for Cl- is therefore a critical parameter in governing the strength of synaptic inhibition. In the first part of the Thesis I use a combination of experimental and theoretical approaches to demonstrate that influxes of Cl- via activated GABAARs can overwhelm a neurons ability to maintain a stable Cl- concentration gradient. The consequence is that subsequent activation of GABAARs results in weaker inhibition or even excitation, which alters how the neuron integrates synaptic inputs. This process is shown to be dependent upon the level of activity of the GABAAR, the post-synaptic cells membrane potential and the cellular compartment into which the Cl- flows. These principles were extended to demonstrate that popular optogenetic strategies for silencing neural activity have different effects upon GABAAR transmission. A light-activated Cl- pump was shown to cause substantial accumulations in intracellular Cl, which meant that the strength of synaptic inhibition was significantly reduced following light offset. In the second part of the Thesis I use electrophysiological and fluorescence imaging techniques to demonstrate that the activation of GABAARs during epileptiform activity results in pronounced changes to the transmembrane Cl- gradient. Indeed, these changes convert synaptic inhibition into synaptic excitation during the course of a seizure event. As part of this work I characterise a novel, genetically-encoded reporter for measuring intracellular Cl- dynamics in different cell types and subcellular compartments. A significant advantage of this reporter is that it permits the simultaneous quantification of H+ fluxes, which are also shown to change in an activity-dependent manner and which have been a confounding factor for previous Cl- reporters. In the third and final part of the Thesis I use genetically-encoded reporters to investigate activity-dependent changes in intracellular H+ concentration. I demonstrate that markedly different pH changes occur in neurons and astrocytes during epileptiform activity. Whereas neurons become acidic, astrocytes become alkaline and the dynamics of these pH shifts exhibit a very different temporal relationship with the seizure event. In conclusion, this thesis demonstrates that the intracellular concentrations of Cl- and H+ are dynamic variables that evolve across time and space, in an activity-dependent manner. Changes in the transmembrane gradients of these two ions influence ongoing synaptic transmission. Therefore this work has significant implications for our understanding of network activity and the balance of synaptic excitation and inhibition.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Electric fields, which are generated by the movement of charged ions across membranes, are found in all biological systems and can influence cellular components ranging from amino acids to biological macromolecules. Physiological field strengths range from 1 – 200 mV/mm, and these electric fields are especially elevated at sites of cellular growth during development and regeneration. It has previously been demonstrated that elevated electric fields induce alignment of astrocyte processes in vitro, enhancing the rate of neurite outgrowth. It is believed that electric fields of varying physiological strength affect other astrocytic responses associated with regeneration. To characterize the duration over which these changes emerge, cultured rat astrocytes were exposed to different direct-current electric field strengths. The resulting cellular behaviors were recorded every three minutes with an inverted microscope equipped with DIC optics and a stage incubator. Electric fields were found to induce astrocyte responses similar to those observed during periods of neurodevelopment and regeneration. Changes in astrocyte movement, proliferation, & morphology emerged within the first hour and persisted through the course of the electric field application, leading mammalian astrocytes to revert to an earlier maturation state resembling those seen in amphibian astrocytes associated with central nervous system regeneration. Collectively, these results suggest that applied electric fields lead to astrocyte dedifferentiation, with certain electric field strengths eliciting and enhancing specific cell responses.

This dissertation elucidates several independent molecular mechanisms that function in astrocytes and glial tumor cells, and suggest that developmental and inflammatory signals may contribute to the development of brain tumors. First, we analyzed the mechanism of TIMP-1 activation in astrocytes and glioblastoma cells. TIMP-1 expression is activated by IL-1, which is the major neuroinflammatory cytokine, via simultaneous activation of IKK/NF-kB and MEK3/6/p38/ATF-2 pathways in primary human astrocytes. In contrast to astrocytes, TIMP-1 is expressed at lower levels in glioblastomas, and is not regulated by IL-1 due to either dysfunctional IKK/NF-kB or MEK3/6/p38/ATF-2 activation. Thus, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after tissue injury to limit the ECM degradation. This mechanism does not operate in gliomas, and may in part explain the increased invasiveness of glioma cells.Inflammation has been associated with the development of several cancers, including glioblastoma multiforme. However, it has not been linked to other brain tumors. Here we show for the first time that inflammation is associated with oligodendroglioma tumors as pro-inflammatory cytokines, such as OSM, IL-6, MCP1, MIP1α, and MIP1β and inflammatory markers, such as ACT and COX-2, were expressed at higher levels in oligodendroglioma samples. In addition, cytokine-induced STAT3 signaling, but not NF-kB, is highly activated in the oligodendroglioma patients. Moreover, OSM promotes oligodendroglioma cell proliferation in vitro, and this effect is mediated through STAT3. In summary, oligodendroglioma tumors secrete and respond to inflammatory mediators, with OSM being the major cytokine that activates STAT3 to promote the growth of tumor cells, and express ACT and COX-2 as a hallmark of ongoing inflammation. Since STAT3 promotes the growth of oligodendroglioma, as well as glioblastoma cells, and also regulates gliogenesis, we studied molecular mechanisms of this process in an in vitro differentiation model. We turn our attention to the NFI family of transcription factors since they have recently emerged as novel regulators of the development of vertebral neocortex. We developed a stem cell-neural progenitor-astrocyte differentiation model, in which the generated astrocytes were characterized by proper morphology, increased glutamate uptake, and expression of early and late astrocyte markers. Moreover, we found that NFI-X and NFI-C but not NFI-A or NFI-B, control the expression of GFAP and SPARCL1, the markers of terminal differentiation of astrocytes.In summary, the three mechanisms of gene regulation we studied, provided new insights into astrocyte biology, with the important implications for understanding the basis leading to the development and progression of brain tumors.

Dans le présent travail, le rôle de la glie dans l’expression de la douleur cancéreuse et de la douleur neuropathique a été étudié de façon comparative. Le modèle animal de douleur cancéreuse a été obtenu par injection osseuse dans le tibia, chez la rate Sprague-Dawley, de cellules de carcinome mammaire de type MRMT-1. Le modèle de douleur neuropathique a été obtenu chez le rat Wistar par ligature des nerfs spinaux L5 et L6. Les données obtenues par l’analyse du comportement douloureux en réponse à la stimulation par des filaments de von Frey ont permis de quantifier l’allodynie et l’hyperalgésie mécaniques statiques. La douleur chronique, hors stimulation nociceptive, a été mesurée à l’aide d’un test d’impotence. Les agents pharmacologiques ont été administrés par voie intrapéritonéale ou par voie intrathécale, à l’aide d’un cathéter implanté de façon chronique. L’analyse des comportements nociceptifs après stimulation par filaments de von Frey montre que l’inhibition fonctionnelle transitoire de la glie spinale par le fluorocitrate est sans effet sur la douleur dans les deux modèles. Dans les deux modèles, l’expression des réponses douloureuses dépend de l’activation des récepteurs NMDA spinaux. L’administration par voie intrathécale d’une seule dose de D-aminoacide oxydase, qui dégrade la D-sérine, co-agoniste endogène du récepteur NMDA, réduit l’allodynie et l’hyperalgésie chez les rats neuropathiques et l’allodynie chez les rats cancéreux. Les effets d’un traitement chronique par le fluoroacétate chez les rats neuropathiques sont réversés par l’administration intrathécale de D-sérine. La D-sérine altère légèrement le seuil nociceptif chez les rats cancéreux. Aucun des agents pharmacologiques utilisés ne réverse la réduction d’appui du membre lésé chez les rats cancéreux ou neuropathiques. Ces résultats montrent que, chez le rat, la douleur neuropathique comme la douleur osseuse cancéreuse dépend de la co-activation des récepteurs NMDA spinaux par un de ses ligands endogènes, la D-sérine, mais que seule la douleur neuropathique requiert une glie spinale fonctionnelle. Ils suggèrent donc un rôle différentiel de la glie dans la physiopathologie de ces deux types de douleur chronique / The present work has investigated the role of glia upon pain symptoms in a well established peripheral neuropathic pain model and a bone cancer pain model. The neuropathic pain model was obtained by right L5-L6 spinal nerve ligation in male Wistar rats. Bone cancer pain was induced by injecting MRMT-1 rat mammary gland carcinoma cells into the right tibia of Sprague-Dawley female rats. Mechanical allodynia and hyperalgesia were quantified using von Frey hairs and ambulatory incapacitance using dynamic weight bearing. Drugs were administered either acutely or chronically using osmotic pumps, through intrathecal catheters chronically implanted in experimental animals. Using von Frey hair stimuli, we found that transient inhibition of glia metabolism by intrathecal injection of fluorocitrate was ineffective in both models. In both models, pain symptoms required spinal NMDA receptor activation. Intrathecal administration of a single dose of D-aminoacid oxidase, which degrades D-serine, a co-agonist of NMDA receptors, reduced mechanical allodynia and hyperalgesia in neuropathic rats and allodynia in cancer rats. The effect of chronic fluoroacetate in neuropathic rats was reversed by acutely administered intrathecal D-serine, which had only a slight effect in cancer rats. None of these compounds altered the functional disability shown by neuropathic or cancer animals and measured by the dynamic weight bearing apparatus. These results show that neuropathic pain and cancer pains depend upon D-serine co-activation of spinal NMDA receptors but only neuropathic pain requires functional spinal cord glia in the rat. Glia may thus play different roles in the development and maintenance of chronic pain in these two situations.

Les noyaux supraoptiques (NSO) de l’hypothalamus sont composés de neurones magnocellulaires (NMCs) synthétisant et sécrétant l’ocytocine (OT) ou la vasopressine (VP). L’OT est impliquée dans des fonctions de reproduction comme la parturition et la lactation, la VP quant à elle participe à l’homéostasie hydrominérale et vasculaire. La libération de VP et d’OT dans la neurohypophyse est contrôlée par l’activité électrique des NMCs, elle-même régulée par les principales afférences glutamatergiques et GABAergiques qu’ils reçoivent. Les récepteurs kaïnate (rKA) pré-synaptiques exercent une action modulatrice sur la libération de neurotransmetteur dans le système nerveux central (SNC). Cet effet peut basculer de facilitateur à inhibiteur en augmentant la concentration d’agonistes des rKA. Ils peuvent également être présents sur le compartiment post-synaptique et participer à la réponse synaptique. Nous avons démontré, pour la première fois que des rKA fonctionnels sont présents dans le NSO, à la fois sur les afférences GABAergiques et glutamatergiques mais également sur les NMCs eux-mêmes. Les rKA contenant la sous-unité GluR5 régulent différemment la transmission glutamatergique sur les neurones à OT et à VP. En effet, l’application d’agonistes exogènes pour ces récepteurs induit un effet diamétralement opposé sur ces neurones, un effet facilitateur sur les neurones à OT et inhibiteur sur les neurones à VP, dû à la présence de GluR5 uniquement sur ces derniers. En effet, l’activation de ce récepteur post-synaptique induit la libération d’un messager rétrograde, vraisemblablement la dynorphine, responsable de l’inhibition de la transmission glutamatergique. En ce qui concerne la régulation de la transmission GABAergique, nous avons pu démontrer que l’augmentation de glutamate ambiant générée par la rétraction des processus gliaux se produisant dans le NSO de rate allaitante était suffisante pour inverser l’effet des rKA de facilitateur à inhibiteur sur la libération de GABA. Cette inversion de l’effet des rKA est causée par une modification du mode d’action de ces récepteurs du type ionotropique au type métabotropique. Les résultats obtenus au cours de mes travaux de thèse montrent donc que les récepteurs kaïnate sont présents sur l’ensemble des sites de la synapse dans le NSO de l’hypothalamus de rats adultes. De plus, ces rKA régulent différemment les neurones à OT et les neurones à VP, ce qui suggère qu’ils pourraient jouer des rôles importants dans la régulation de leur activité et des processus physiologiques les impliquant. / Magnocellular neuroendocrine cells (MNCs) from the supraoptic nucleus (SON) of the hypothalamus synthesize and release the hormones oxytocin (OT) and vasopressin (VP). OT is involved principally in reproductive functions such as parturition and lactation, whereas VP plays a key role in body fluid and cardiovascular homeostasis. The release of OT and VP from the neurohypophysis is controlled by the electrical activity of hypothalamic MNCs, which is itself regulated by GABAergic and glutamatergic synaptic inputs. Presynaptic kainate receptors (KARs) exert a modulatory action on transmitter release in different structures of the central nervous system. This effect can be switched from facilitation to inhibition by an increased concentration of KAR agonists. KAR can also be present postsynaptically where they were shown to participate to the synaptic response in some brain regions. We have demonstrated for the first time that functional KARs were present on GABAergic and glutamatergic inputs as well as on SON neurons. GluR5-containing KARs differentially regulate glutamatergic transmission on OT and VP neurons. Indeed, applications of exogenous agonists of GluR5 induced opposite effects, a facilitatory effect on OT neurons and an inhibitory effect on VP neurons, the latter resulting from an indirect action mediated by postsynaptic GluR5-containing KARs on VP neurons. Thus, activation of these receptors induced the release of a retrograde messenger, probably dynorphin, which in turn act presynaptically to inhibit glutamate release. Regarding the modulation of GABAergic transmission in the SON, we here showed that the increased levels of ambient glutamate associated with the physiological withdrawal of astrocytic processes occuring during lactation could modify the activity of presynaptic KARs. We demonstrated for the first time that a physiological astrocytic plasticity modifies the mode of action of presynaptic KARs from ionotropic to metabotropic, thereby inversing their coupling with GABA release from facilitation into inhibition. The results obtained during my PhD have thus showed that KARs are present both pre-and post-synaptically on adult MNCs. Moreover, KARs differentially regulate OT and VP neurons, which suggest that KARs could play key roles in the regulation of their activity and in physiological processes in which MNCs are involved