As average life expectancy increases, understanding the pathogenesis of age related degenerative brain disease such as Alzheimer's is of paramount importance. The brain is the most cholesterol dense human organ and normal neurophysiology is highly dependant upon this sterol. Sterol dys-regulation has been widely implicated in the pathogenesis of neurodegeneration, yet investigative efforts have failed to determine how these essential disturbances drive disease progression. The heme oxygenases (HOs) are dynamic sensors of cellular oxidative stress and likely mediators of tissue redox homeostasis across the phylogenetic spectrum. However, the heme catabolic process has proven to be one of janus face! Up-regulation of heme oxygenase-1 and altered cholesterol metabolism are characteristic of Alzheimer-diseased (AD) neural tissues. Our lab has previously demonstrated the propensity for HO-1 overexpression to impact sterol homeostasis. Herein, we query the mechanisms behind these HO-1/sterol interactions in vitro (cultured rat astroglia) and examine this relationship in post-mortem human AD brain (Religious Orders Study) and in vivo by comparing two mouse models; 1) a novel HO-1 transgenic mouse (GFAP.HMOX1) engineered to selectively express human HO-1 in the astrocytic compartment and 2) the previously described triple transgenic AD mouse (3xTg-AD). Data garnered from our experimentation indicate that HO-1 is a profound effecter of sterol regulation in the central nervous system and suggests that, under various conditions of cellular stress, HO-1 differentially impacts patterns of brain sterols and oxysterols. We present a novel model of brain sterol regulation in AD across the pathological spectrum and delineate the importance of HO-1 in this process. Aberrancy of HO-1/sterol dynamics akin to those reviewed herein constitute a ubiquitous pathway of pathological aging and, hence, a robust target for therapeutic intervention in AD and similar degenerative diseases. / Comprendre la pathogenèse d'une maladie dégénérative du cerveau telle la maladie d'Alzheimer est d'une importance primordiale pour notre population vieillissante. Le cerveau est l'organe du corps humain avec le plus haut taux de cholestérol, et la neurophysiologie normale dépend fortement de ce stérol. La dysrégulation du stérol a été largement impliquée dans la pathogenèse de la neurodégénérescence, mais les efforts de recherche n'ont pas réussi à déterminer comment ces perturbations nécessaires causent la maladie. Les oxygénases hème sont des capteurs dynamiques de stress oxydatif cellulaire et des médiateurs probables de l'homéostasie redox tissulaire à travers le spectre phylogénétique. Toutefois, le processus catabolique hème s'est avéré être l'un des visages de janus! La surexpression de l'hème oxygénase-1 et le métabolisme du cholestérol altéré est caractéristique des tissues neurologiques affectés par la maladie d'Alzheimer. Notre laboratoire a précédemment démontré que la propension de HO-1 surexpression peut avoir un impacte sur l'homéostasie des stérols. Dans les présentes, nous enquêtons les mécanismes derrière ces interactions HO-1/sterol in vitro (astrocytes de rat en culture) et examinons cette relation dans le cerveau atteint d'Alzheimer post-mortem (Religious Order Study) et in vivo en comparant deux modèles de souris; 1) une souris HO-1 transgénique (GFAP.HMOX1) conçue pour faire ressortir de façon sélective de la HO-1 humain dans le compartiment astrocytic et 2) une souris transgénique triple d'Alzheimer (3xTg-AD), décrite précédemment. Les données résultantes de notre expérimentation indiquent que la HO-1 est un profond effecteur de la régulation des stérols dans le système nerveux central et suggère que, dans diverses conditions de stress cellulaire, la HO-1 impact de façon différentielle les modes des stérols et oxystérols du cerveau. Nous présentons un nouveau modèle de régulation neurosterol d'Alzheimer's à travers le spectre pathologique et délimitons l'importance de la HO-1 dans ce processus. Mauvais dynamiques HO-1/stérol semblables à celles examinées dans les présentes constitue une voie omniprésente du vieillissement pathologique et, par conséquent, une cible solide pour l'intervention thérapeutique de la maladie d'Alzheimer et de maladies dégénératives similaires.

Neuronal growth and synaptic function are key determinants of nervous system behaviour. Understanding the molecular mechanisms that regulate these processes is central to comprehending nervous system function, both in health and during disease states. My thesis has focused on trying to better understand the mechanisms controlling glutamatergic neuronal growth, synaptic function and synaptic plasticity using the Drosophila melanogaster larval neuromuscular junction (NMJ) as a model system. This work has concentrated on the role that protein synthesis regulators play in modulating NMJ function and plasticity, and has revealed important roles for multiple regulators of cap-dependent translation in these processes. Importantly, genetic manipulations predicted to enhance cap-dependent translation in postsynaptic muscle can induce retrograde enhancements in synaptic function. In each case, these manipulations are without effect on neuronal growth, the number of synaptic sites or glutamate receptor levels. Instead, enhanced postsynaptic cap-dependent translation leads to increased presynaptic release probability. This retrograde pathway plays a key role in the expression of homeostatic synaptic plasticity at NMJ synapses. Glutamate receptor subunit IIA (GluRIIA) mutants exhibit reduced postsynaptic receptor function which is compensated for by enhanced presynaptic release; cap-dependent translation, under control of the TOR (target of rapamycin) pathway, is essential for this retrograde homeostatic response. Furthermore, we find that Parkinson's disease related protein LRRK2 (leucine rich repeat kinase 2), and its Drosophila orthologue dLRRK, can regulate synaptic function at the NMJ via cap-dependent translation. Importantly, however, mutant forms of LRRK2 are defective in their ability to regulate synaptic strength. Together, these findings further our understanding of the molecular mechanisms regulating glutamatergic synaptic function and plasticity. / La croissance neuronale et la fonction synaptique sont des constituants clés dans le comportement du système nerveux. Il est donc essentiel de comprendre les mécanismes moléculaires régulant ces processus à l'état normal ainsi qu'à l'état pathologique. Ma thèse a pour objectif une meilleure compréhension des mécanismes contrôlant la croissance neuronale, ainsi que la fonction et la plasticité synaptiques engendrés par la transmission glutamatergique, en utilisant comme modèle la jonction neuromusculaire (JNM) de la drosophile, Drosophila melanogaster. Cet ouvrage porte spécifiquement sur l'impact de régulateurs de la synthèse protéique sur la fonction et la plasticité synaptiques dans la JNM, et a identifié des rôles importants pour de multiples régulateurs de traduction coiffe-dépendante dans ces processus. Des manipulations génétiques menant à une augmentation de la traduction coiffe-dépendante dans le muscle post-synaptique induisent une augmentation rétrograde de la fonction synaptique. Dans chaque cas, ces manipulations n'affectent ni la croissance neuronale, ni le nombre de synapses, ni le niveau de récepteurs glutamatergiques. Plutôt, une traduction coiffe-dépendante post-synaptique conduit vers une augmentation de la probabilité de relâche pré-synaptique. Cette voie signalétique joue un rôle important dans l'expression de la plasticité homéostatique des synapses neuromusculaires. Des mutants du récepteur glutamatergique IIA (GluRIIA) montrent une réduction dans la fonction post-synaptique, qui est compensée par une augmentation de la probabilité de relâche pré-synaptique. La traduction coiffe-dépendante, sous le contrôle de la voie signalétique TOR (target of rapamycin), est essentielle pour cette réponse homéostatique rétrograde. De plus, la protéine reliée à la maladie de Parkinson, LRRK2 (leucine rich repeat kinase 2), ainsi que son orthologue chez la drosophile, dLRRK, peuvent réguler la fonction synaptique de la JNM via la traduction coiffe-dépendante. Cependant, les formes mutantes de LRRK2 sont défectueuses dans leur habileté à réguler la fonction synaptique. Globalement, ces trouvailles nous aident à comprendre les mécanismes moléculaires régulant la fonction et la plasticité synaptiques glutamatergiques.

The insula is an important cortical processing area for thin fiber somatosensory input, including nociceptive and thermal input. This thesis desribes a series of studies focusing on the human insular cortex, in which structural and functional MRI, together with psychophysical testing, were used to explore the relationship between brain changes and alterations in somatosensory processing and regulation. In Study 1, we reported insular gray matter (GM) changes in a unique patient lacking large-fiber somatosensory input (proprioception, discriminative touch), but with intact thin-fiber input projecting into insular cortex. The patient had increased cortical thickness and resting state connectivity of the insula compared to matched controls. In Study 2, we reported increases in insular GM volume, and white matter (WM) integrity and connectivity in long-term yoga practitioners, who had heightened pain tolerance compared to matched controls. In addition, we observed a positive correlation between insular GM and individual pain tolerance across yoga practitioners and controls. In Study 3, which was part of a larger investigation of age-related GM changes in chronic pain (fibromyalgia) patients, we observed insular GM increase in younger patients compared to matched controls, and this GM increase was inversely related to patients' experimental pain sensitivity. Further, the anterior insula of younger patients had relative to matched controls decreased resting state connectivity to a cortical area involved in processing of the emotional salience of painful stimuli. This thesis provides three novel contributions to our understanding of the insula. Study 1 revealed insular structural and functional correlates of loss of specific somatosensory fibers in humans, Study 2 provided the first evidence for the effects of yoga practice on brain structure in general and on insular GM in particular, and related these effects to pain tolerance, and Study 3 was the first study to directly investigate age-related effects of chronic pain on brain GM, and in particular on insular GM structure and functional connectivity. We interpret the observed insular GM enhancements across all three studies as being suggestive of adaptive plasticity related to a) compensatory use of thin-fiber input – most notably temperature – in lieu of abolished large-fiber sensations b) pain regulation, likely via increased intra-insular processing and c) increased pain regulation, likely via functional disengagement from a cortical salience processing network. This work has improved our understanding of the insula in somatosensory and notably pain processing, and could thus help guide future studies aimed at developing treatments for chronic pain. / L'insula est une aire corticale importante impliquée dans le traitement de l'input des fines fibres somato-sensorielles incluant l'input nociceptive et thermal. Cette thèse décrit une série d'études centrées sur le cortex insulaire humain dans lesquelles l'IRM structural et fonctionnel et l'évaluation psychophysique ont été utilisées pour explorer la relation entre les changements du cerveau et ceux liés au traitement somato-sensoriel et à sa régulation. Dans la première étude, nous décrivons les changements dans la matière grise (MG) de l'insula chez une patiente n'ayant pas d'input somato-sensoriel provenant des larges fibres (proprioception, touché discriminatif), mais ayant un input intact des fines fibres projetant au cortex insulaire. Lorsque comparée à un groupe apparié de sujets contrôle, cette patiente présentait une augmentation de l'épaisseur du cortex et de la connectivité insulaire à l'état de repos. Dans la deuxième étude, nous observons une augmentation du volume de MG insulaire ainsi que de l'intégrité et de la connectivité de la matière blanche (MB) insulaire chez des adeptes du yoga expérimentés présentant une augmentation de la tolérance à la douleur lorsque comparés au sujets d'un groupe contrôle apparié. Nous avons de plus observé une corrélation positive entre la MG insulaire et les résultats de tolérance à la douleur de l'ensemble des sujets (adeptes du yoga et groupe contrôle). Dans la troisième étude, qui représente l'examen des changements de MG liés à l'âge chez des patients souffrant de douleurs chronique (fibromyalgie), nous observons une augmentation de la MG insulaire chez les jeunes patientes comparativement aux sujets du groupe contrôle apparié. Cette augmentation de MG est inversement corrélée à la sensibilité des patientes à la douleur expérimentale. De plus, l'insula antérieure des jeunes patientes montre, lorsque comparée à celle des sujets du groupe contrôle, une diminution de la connectivité à l'état de repos à une aire corticale impliquée dans le traitement de l'aspect émotionnel des stimuli douloureux. Cette thèse apporte trois contributions nouvelles à notre compréhension de l'insula. L'étude 1 révèle les conséquences structurale et fonctionnelle liées à la perte de fibres nerveuses somato-sensorielles spécifiques chez l'humain. L'étude 2 apporte la première démonstration des effets de la pratique du yoga sur la MG insulaire et de sa relation avec la tolérance à la douleur et l'étude 3 est la première étude qui recherche directement les effets liés à l'âge de la douleur chronique sur la structure et la fonction de l'insula. Nous interprétons les augmentations observées de MG insulaire dans les trois études comme reflétant une plasticité d'adaptation liée a) à l'utilisation compensatoire de l'input des fines fibres nerveuses – notamment celles liées à la perception de la température – en remplacement des fibres de plus gros calibre; b) au contrôle de la douleur, probablement par l'augmentation du traitement intra-insulaire; et c) à l'augmentation du contrôle de la douleur, probablement via un désengagement fonctionnel d'un réseau cortical impliqué dans le traitement de la salliance. Ce travail a amélioré notre compréhension de l'implication de l'insula dans le traitement de l'information somato-sensorielle et douloureuse et pourrait aider à éclairer de futures études visant à développer des traitements contre la douleur chronique.

Movement, the ultimate or final output of most nervous system activity, is enabled by the coordinated action of motor neurons, which induce muscle contractions in the periphery by propagating electrical signals from neurons in the brain and spinal cord of the central nervous system. The assembly of neurons during development into precisely interconnected circuits is dependent on the diversification of motor neurons into genetically and topologically discrete subpopulations, each of which develop unique patterns of peripheral and central innervation. Motor neurons' diversification is regulated, in part, by networks of transcription factors that impose particular profiles of gene expression, and thus axonal projection and somatic migration behaviours, on populations of motor neurons. The studies outlined here address the mechanisms regulating motor neurons' diversification in the chick hypoglossal nucleus and their innervation of the lingual muscles. We show that the transcription factor Runx1 is expressed in a subset of lingual muscle-innervating motor neurons and provide an anatomical and molecular characterization of the topography and gene expression patterns of the developing hypoglossal nucleus. Implications for Runx1's possible role in mammalian hypoglossal motor neuron development and the generation of neural circuits are also discussed. / Le mouvement, la finalité de l'activité du système nerveux, est rendu possible par l'action coordonnée des neurones moteurs, qui induisent des contractions musculaires dans la périphérie en propageant des signaux électriques à partir les neurones du cerveau et de la moelle épinière du système nerveux central. L'assemblage précise des neurones au cours du développement en des circuits interconnectés dépend de la diversification des neurones moteurs en sous-populations génétiquement et topologiquement distinctes, dont chacun développent des innervations périphériques et centrales uniques. La diversification des motoneurones est réglementé, en partie, par des réseaux de facteurs de transcription qui imposent des profils particuliers de l'expression des gènes, et ainsi les projections axonales et les comportements migratoires somatiques, sur les populations de neurones moteurs. Les études présentées ici s'interessent aux mécanismes de régulation de la diversification des motoneurones dans le noyau hypoglosse de l'embryon de poulet et leur innervation des muscles linguaux. Nous montrons que le facteur de transcription Runx1 est exprimé par un sous-population de neurones moteurs qui innervent les muscle linguaux et nous fournissons une caractérisation structurale et moléculaire de la topographie et de l'expression des gènes du noyau hypoglosse au cours du développement. Les implications possibles de Runx1 dans le développement hypoglosse motoneurones mammifères dans la génération de circuits neuronaux sont également discutés.

This dissertation addresses the question of whether increases in neural activity caused by sensory stimulation require a significant acceleration of aerobic metabolism in the brain. A new technique for measuring cerebral oxygen consumption with Positron Emission Tomography (PET) was used to assess focal changes in oxidative metabolism in different conditions of visual stimulation. Specifically, positron emission tomography was used to determine whether statistically significant increases in regional cerebral oxygen metabolism are produced by different lengths and types of visual stimulation. These experiments employed a single-bolus inhalation of [15O]O2 to measure oxygen uptake into cortical tissue. / In one experiment, oxygen consumption during a monocular presentation of a stimulus restricted to a single visual hemi-field (after 3 minutes and 8 minutes of stimulation) was compared to oxygen consumption measured during a simple fixation baseline. Oxygen consumption increased by 20.5% after 3 minutes. In a second experiment similar procedure was used to examine the changes in oxygen metabolism produced by two different visual stimuli. Regional oxygen metabolism increased by 23% for both stimulus conditions, a reversing yellow-blue checkerboard and a rapidly blinking white-disk. In conclusion, focal activation of human visual cortex is accompanied by significant increases in oxygen metabolism.

After optic nerve transection in the adult hamster (Carter et al., 1989) and rat (Vidal-Sanz et al., 1987), axons of retinal ganglion cells (RGCs) can regrow long distances through peripheral nerve (PN) grafts and form connections in the superior colliculus, a normal target of RGC axons. This thesis further investigated synaptogenesis in the injured mammalian CNS: regenerating RGC axons of adult hamsters were guided through PN grafts into two novel targets, the cerebellum (Cb) and the inferior colliculus (IC). When examined 2 to 9 months later, RGC axons were found to have extended into the grey matter of both targets for distances of up to 650 $ mu$m. Growth into white matter was rare and limited to 50 $ mu$m. RGC axons formed terminals and synapses in both targets. Within the Cb, there was a marked preference for growth and synapse formation in the granule cell layer (GCL) which could not be explained by: the position of the PN graft in the Cb, a selective denervation of the GCL, local damage to other neurons, or the distribution of reactive gliosis. / These anatomical studies indicate that regenerating CNS axons can form connections with novel targets in the adult hamster. The preferential synaptogenesis in the GCL of the Cb suggests that such connections are not formed randomly.

Stereoanalogues of choline (R and S, $ alpha$- or $ beta$-methylcholine), hemicholinium-3 and choline mustard aziridinium ion were used as tools to investigate the structure and function of the high-affinity choline transporter. Three studies are presented. In the first, the methylcholines were used to study the stereoselective properties of the high-affinity choline transporter. The recognition of the methylcholines by the choline carrier was stereoselective, while their transport was stereospecific. This difference was interpreted as indicating that recognition and transport might represent distinct and dissociable components of the choline transporter. In a second study, the methylcholines were used to examine the stereoselectivity of ($ sp3$H) hemicholinium-3 binding to determine how this compared to the interaction of the methylcholines with choline uptake. The ability of these compounds to inhibit stereoselectively ($ sp3$H) hemicholinium-3 binding indicates that binding is to the substrate recognition site of the choline carrier. However, this site can assume two configurations, as defined by changes in its stereoselectivity for the methylcholines and affinity for hemicholinium-3. The conformational change of the substrate recognition site likely plays a role in initiating the substrate translocation process, since locking it in its high-affinity ($ sp3$H) hemicholinium-3 binding state reduces the rate of choline transport. In a third study, choline mustard aziridinium ion and hemicholinium-3 were used to investigate the mechanism by which activity regulates the velocity of choline transport. These studies indicate that the mechanism involves the changed activity of transport sites that are available to the mustard, and presumably choline, but not hemicholinium-3 prior to stimulation. In addition, two pools of basal transport sites have been identified on the basis of their sensitivity to choline mustard, as well as their ability to transport choline and b

The peptide cholecystokinin (CCK) is co-localized within oxytocin- and vasopressin-secreting magnocellular neurons in supraoptic, paraventricular, and accessory nuclei of the hypothalamus. These magnocellular nuclei are also prominent binding sites for CCK. Although the site of action remains to be determined, intracerebroventricular injection of CCK promotes a rise in plasma vasopressin. The present study evaluates the effect of CCK on the function of rat supraoptic magnocellular neurosecretory neurons. Extracellular recordings in vivo demonstrate that locally applied CCK excites both oxytocin- and vasopressin-secreting neurons. Intracellular recordings in superfused hypothalamic explants, reveal that CCK depolarizes a majority of magnocellular neurons. This response persists after synaptic blockade, indicating a direct activation of postsynaptic CCK receptors. Pharmacological tests identify the presence of CCK-B receptors. Voltage clamp analyses reveal that CCK evokes an inward current, accompanied by an increase in membrane conductance. E$ sb{ rm CCK}$ approximates $-$15 mV, suggesting activation of a non-selective cationic conductance. Activation of this conductance may also modulate the expression of other intrinsic currents. In perfused hypothalamic-neurohypophyseal explants, CCK was found to evoke the secretion of oxytocin and vasopressin from the neurointermediate lobe, through an action at the supraoptic nucleus. These results suggest that CCK may function as an excitatory neurotransmitter/neuromodulator in the hypothalamic-neurohypophyseal system.

This thesis focuses on the functional roles of adenosine on neuronal excitability and synaptic transmission in the rat hippocampal CA1 region in vitro. Hippocampal slices were placed in an interface recording chamber and constantly superfused with oxygenated artificial cerebral fluid at $30 sp circ$C. Intracellular (current-clamp) recording techniques were used to measure the spontaneous and evoked inhibitory and excitatory synaptic potentials as well as resting membrane potential and input resistance. / Our findings indicate that adenosine induces the opening of potassium channels in the postsynaptic membrane of CA1 neurones, including $ rm K sb{ATP}$ channels. Adenosine-induced attenuation of EPSPs and poly-synaptic IPSPs may be due to the activation of adenosine $ rm A sb1$ receptors at both the pre- and postsynaptic sites of excitatory synapses onto the pyramidal cells and interneurones. On interneurones, adenosine appears to have two different effects: one is a hyperpolarization and decrease in input resistance, similar to the inhibitory effect on pyramidal cells, and the other is a depression of the amplitude and duration of afterhyperpolarization which characterizes the action potential of these interneurones. A scheme is proposed in which adenosine $ rm A sb1$ receptors may be present at pre- and postsynaptic sites of excitatory synapses but not at presynaptic sites of inhibitory synapses. Adenosine was also found to tonically inhibit spikes associated with NMDA receptor activation and that at least some of these NMDA receptors may be present in the dendritic trees of the pyramidal cells. The slow excitatory effects of adenosine may be due to activation of adenosine $ rm A sb2$ receptors at both pre- and postsynaptic sites in the hippocampal CA1 region. / The inhibitory and excitatory effects of adenosine on CA1 pyramidal cells and inhibitory effects of adenosine on interneurones suggest that adenosine may be a homeostatic neuromodulator regulating neuronal excitability and synaptic transmission in the hippocampus in mammalian brain.

Adhesive interactions between neurons and astroglia are likely to play an important role during central nervous system development. Using a monoclonal antibody (designated MAb 1A1) raised against purified neonatal rat astrocytes, I have characterized some of the in vitro functional and biochemical properties of a potentially novel cell surface molecule. By indirect immunofluorescence, the antibody labels subpopulations of astrocytes (flat type-1 astrocytes and Bergmann glia) and cells derived from the mesenchyme (leptomeninges and fibroblasts). The latter showed 1A1$ sp{+}$ immunoreactivity only when grown to confluency. Cell-cell contact and extracellular matrix molecules were found to play a role in the regulation of 1A1 antigen expression on leptomeninges. 1A1 Fab fragments inhibited the binding of neuron to astrocyte and astrocyte to astrocyte, but not of neuron to neuron in an in vitro adhesion assay, thus indicating that this surface antigen may function as a cell adhesion molecule on astrocytes. Addition of Fab fragments of MAb 1A1 also reduced leptomeningeal cell adhesion. Further functional antibody perturbation experiments indicated that this surface molecule mediates neurite outgrowth on astrocytes and neuronal migration on Bergmann glia via a heterophilic-binding mechanism. Both immunoprecipitation and immunocytochemical analysis showed the timing of 1A1 antigen expression in postnatal rat cerebellum to coincide with the developmental period of granule cell migration along Bergmann glia. On SDS-PAGE, the immunopurified 1A1 surface molecule migrated as a single molecular weight band of $ approx$135 kd and appeared to be poorly glycosylated. Based on its unique cell-type distribution, functional properties and biochemical analysis, this 135 kd glycoprotein is likely to be distinct from other known cell adhesion molecules expressed on astrocytes.