Synapses must continuously maintain stable function in order for neuronal circuits and higher-order systems to properly function. By necessity, tight regulation of molecules necessary for appropriate neurotransmission coupled with homeostatic forms of plasticity function to stabilize synaptic output. The Drosophila melanogaster larval neuromuscular junction (NMJ) is an excellent model synapse for investigating both homeostatic synaptic plasticity (HSP) and neurotransmission machinery. At the NMJ, post-synaptic impairments to neurotransmitter sensitivity (decreased quantal size) initiate HSP. A retrograde, muscle-to-nerve signal instructs the presynaptic neuron to increase neurotransmitter release (quantal content) to compensate for the post-synaptic impairment and maintain synaptic output. HSP can be separated into temporally distinct induction and maintenance phases, depending on the nature of the impairment. Acute blockade of glutamate receptors initiates rapid forms of HSP that restore synaptic output within minutes. Loss-of-function mutations in a gene encoding a glutamate receptor result in reduced quantal size, and as a result, expression of HSP over the lifespan of that animal. However, it is unclear whether these temporal phases are distinct processes with overlapping machinery, or whether both phases are part of a common process with temporal distinct signaling requirements. Here we show that, in addition to being molecularly distinct, the temporal phases are functionally distinct. We provide evidence that the long-term maintenance of HSP requires continuous inositol trisphosphate receptor (IP3R) and Ryanodine receptor (RyR) activities, but neither are necessary for the rapid induction phase of HSP. In addition, we investigated how mutations associated with Familial Hemiplegic Migraine Type 1 (FHM1) impact synapse function and seizure behavior. We show that flies expressing this mutant channel are susceptible to seizures. Further, neurons expressing a transgene for cacophony containing the FHM1 mutations R192Q and S218L in the analogous locations showed significant hyper-excitability. Concurrent knockdown of the gene Multiple inositol polyphosphate phosphatase 2 (Mipp2) attenuated hyper-excitable phenotypes. Additionally, Mipp2 knockdown or LiCl treatment, both of which should attenuate downstream IP3R signaling, mitigated susceptibility to seizures in adults. Together these results contribute to our understanding both of both the pathophysiology of migraine and seizures.

Le nématode Caenorhabditis elegans est un organisme polyvalent et unique pour l'étude de la biologie du développement, de la neurologie et des mécanismes complexes de signalisation des GTPases. Ce modèle animal offre une opportunité unique pour l'étude du rôle des protéines dans le développement neurologique et les maladies. Les recherches portaient sur trois gènes : pix-1, git-1 et pak-1. Chez les mammifères, GIT / PIX / PAK agissent comme une plateforme d'intégration de la signalisation des GTPases Rho et Arf dans les processus biologiques tels que : la polarité cellulaire, la migration, le trafic vésiculaire, la formation des synapses et la morphologie des épines dendritiques. Ces recherches ont amené l'utilisation des approches génétiques et microscopiques pour établir que pix-1, git-1 et pak-1 contrôlent les phases précoces et tardives de l'élongation de l'embryon par la régulation de l'activité des chaînes légères de myosine (CLM). Les résultats de recherche suggèrent un positionnement de pix-1 et pak-1 dans l'une des voies de signalisation contrôlant la phosphorylation de ces CLMs en parallèle de la voie mel-11 / let-502. MEL-11 est une phosphatase des CLMs agissant de façon antagoniste à LET-502 (une kinase effectrice des Rhos) dans l'une des deux voies de signalisation redondantes qui assurent l'étape précoce d'élongation embryonnaire. Les résultats suggèrent, de plus, l'implication de mel-11 et let-502 au cours de la phase tardive de l'allongement. Un certain nombre de résultats suggèrent aussi une implication des intégrines ina-1 dans ces processus. La caractérisation fonctionnelle de pix-1, git-1 et pak-1 chez les nématodes adultes démontre leur implication dans le contrôle du comportement de recherche de nourriture (comportement de forage). Ce comportement dépend de la neutotransmission glutamatergique et dopaminergique et implique des mécanismes cellulaires similaires à la plasticité synaptique chez les mammifères. Un lien a été établi entre l'expression des récepteurs au glutamate AMPA homologue de GLR-l et PIX-l en utilisant la microscopie quantitative, la cytométrie de flux et des tests de comportement dans des labyrinthes micro-fluidiques secs. Les résultats suggèrent que les animaux mutants pour pix-1 contrôleraient la neurotransmission glutamatergique de façon indirecte. La conservation fonctionnelle du complexe GIT / PIX / PAK chez les invertébrés permettrait d'utiliser notre modèle dans l'identification de cibles thérapeutiques et de composés actifs contre les pathologies associées à des mutations dans aPIX et PAK3. Il aidera en outre à fournir des éclaircissements sur la fonction et les mécanismes de régulation des GTPases. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : GTPases monomériques, GIT/PIX/PAK., C. elegans, signalisation cellulaire, développement embryonnaire, neuro-transmission glutamatergique.

No description available.

Cette recherche a pour objectif principal l'étude de l’impact de l'intégration d'outils informatiques et de la pédagogie par résolution de problèmes à l'enseignement de la neurophysiologie. La première partie de la thèse porte sur l'enseignement de la notion de la neurotransmission en classe de terminales Scientifiques : analyse épistémologique et historique du concept, étude des programmes et des manuels scolaires. Dans la deuxième partie, une analyse des conceptions des élèves de terminale concernant la transmission du message nerveux au niveau de la synapse a été effectuée. Dans une troisième partie des situations didactiques ont été conçues et testées : par résolution de problème et par des simulations informatisées. Les résultats de la recherche tendent à prouver que l’intégration des simulations informatisées ou la pédagogie par résolution des problèmes dans l'enseignement de la neurophysiologie augmente l’efficacité de l’enseignement du concept de la neurotransmission, et que par contre, la pédagogie par résolution de problèmes semble être une alternative plus efficace pour le changement conceptuel / This search aims to study the impact of the integration of computing tools and the pedagogy by resolution of problems in the teaching of the neurophysiology. The first part of the thesis concerns neurotransmission’s teaching in final year of secondary school: epistemological analysis, history of neurotransmission concept and analysis of the programs and the textbooks. The second part interests the pupils’ conceptions concerning the transmission of the nervous message in the synapse. In the third part didactic situations were conceived and tested: situation using PBL and simulations. The results of the search tend to prove that the integration of the simulations or the pedagogy by PBL increases the efficiency of the teaching of the concept of the neurotransmission, and that on the other hand, the pedagogy by PBL seems to be a more effective alternative

In vorliegender Arbeit sollte untersucht werden, in wieweit die Abwesenheit von P2Y1-Rezeptoren die cholinerge, GABAerge und glutamaterge Transmission beeinflusst. Hierfür wurden P2Y1-Rezeptor-defiziente Mäuse im Alter von 3-4 Monaten einem Screening nach repräsentativen Markern der genannten Transmissionssysteme unterzogen. So konnten in Hirnen P2Y1-Rezeptor-defizienter Mäuse reduzierte Aktivitäten der Acetylcholinesterase im Nc. arcuatus und Substantia nigra sowie Veränderungen in den Bindungsspiegeln der M1-muskarinischen Acetylcholinrezeptoren im ventromedialen Hypothalamus beobachtet werden, während die nikotinischen Acetylcholinrezeptoren kaum beeinflusst waren. Weiter wurden im cingulären Kortex, Nc. accumbens, Hippocampus, ventromedialer und lateraler Hypothalamus sowie im auditorischen und visuellen Kortex von P2Y1-Rezeptor-defizienten Mäusen erhöhte GABAA-Rezeptorbindungen im Vergleich zu Wildtyp-Mäusen gemessen, während kein Einfluss der P2Y1-Rezeptor-Defizienz auf NMDA-Rezeptoren gefunden wurde. In P2Y1-Rezeptor-defizienten Mäusen konnten keine Anzeichen von oxidativem Stress oder veränderter Expression des Amyloidvorläuferproteins beobachtet werden. Die Erhöhung der GABAA-Rezeptorbindung in den mesolimbischen Hirnregionen der P2Y1-Rezeptor-defizienten Mäuse steht im Einklang mit Verhaltensbefunden, die zeigen, dass diese Rezeptoren bei der Vermittlung appetitiver und aversiver Reaktionen eine wichtige Rolle spielen.

Over thirty years ago electrical coupling was observed in embryonic cells prior to chemical communication. This temporal relationship of electrical coupling preceding functional chemical neurotransmission occurs throughout neurogenesis, prompting the idea that gap junctional coupling synchronizes the synaptogenic establishment of functional neural networks. Helisoma neuronal pairs treated with trophic factors exhibit increased electrical coupling and subsequently delay the formation of inhibitory chemical connections. Studies in this thesis addressed the mechanism regulating this inverse relationship between electrotonic and chemical communication. Synaptogenesis between two neurons from the Helisoma buccal ganglia, B110 and B19, were examined using alternative culturing conditions that were either exposed to or deprived of trophic factors. Incubating neuronal pairs in trophic factors induced transient electrical synapses and postponed the formation of chemical connections. In electrically coupled neuronal pairs, presynaptic secretory vesicles were recruited to the sites of presynaptic contact, but did not respond to calcium elevation (i.e., photolytic release of calcium from NP-EGTA) with neurotransmitter release. These and other studies demonstrated that transient electrical coupling does not disrupt calcium handling or postsynaptic responsiveness. Rather, electrotonic coupling delays chemical synaptic transmission by imposing a functional block between the accumulation of presynaptic calcium and the synchronized vesicular release of neurotransmitter.

Autosomal-dominant mutations in the amyloid precursor protein (APP) gene increase the production and aggregation of toxic amyloid-β (Aβ) peptides and cause early-onset Alzheimer’s disease (AD). Noradrenergic cell loss is well documented in AD and has been posited to play a role in cognitive symptoms as well as disease progression. We investigated memory and affect, tissue levels of catecholamines, brain-derived neurotrophic factor (BDNF) mRNA and bioenergetic homeostasis in TgCRND8 mice that express a double mutant (K670N/M671L + V717F) human APP695 transgene. We found that TgCRND8 mice develop object memory impairment and behavioural despair, as well as reductions in noradrenaline and BDNF expression in the hippocampus and cortex, before the appearance of Aβ plaques. Animals with more advanced Aβ pathology exhibit disruptions in energetic status, along with diminished complex I+III activity in the electron transport chain. To test whether the AD-like phenotypes of TgCRND8 mice might be due to altered noradrenergic tone, pre-plaque mice were treated with dexefaroxan, an antagonist of presynaptic inhibitory α2-adrenoceptors that are highly expressed on both noradrenergic and cholinergic terminals. Effects of dexefaroxan were compared to those of rivastigmine, a cholinesterase inhibitor. Both dexefaroxan and rivastigmine improved behavioural phenotypes and BDNF expression without affecting tissue Aβ load. Drug treatments also restored complex I+III mitochondrial activity and increased ATP levels. Reductions in noradrenergic tone appear to underlie Aβ-induced functional impairment in TgCRND8 mice, in addition to BDNF deficits and bioenergetic stress. These studies suggest that α2-adrenoceptor targeting may warrant consideration as a therapeutic strategy in AD.

Autosomal-dominant mutations in the amyloid precursor protein (APP) gene increase the production and aggregation of toxic amyloid-β (Aβ) peptides and cause early-onset Alzheimer’s disease (AD). Noradrenergic cell loss is well documented in AD and has been posited to play a role in cognitive symptoms as well as disease progression. We investigated memory and affect, tissue levels of catecholamines, brain-derived neurotrophic factor (BDNF) mRNA and bioenergetic homeostasis in TgCRND8 mice that express a double mutant (K670N/M671L + V717F) human APP695 transgene. We found that TgCRND8 mice develop object memory impairment and behavioural despair, as well as reductions in noradrenaline and BDNF expression in the hippocampus and cortex, before the appearance of Aβ plaques. Animals with more advanced Aβ pathology exhibit disruptions in energetic status, along with diminished complex I+III activity in the electron transport chain. To test whether the AD-like phenotypes of TgCRND8 mice might be due to altered noradrenergic tone, pre-plaque mice were treated with dexefaroxan, an antagonist of presynaptic inhibitory α2-adrenoceptors that are highly expressed on both noradrenergic and cholinergic terminals. Effects of dexefaroxan were compared to those of rivastigmine, a cholinesterase inhibitor. Both dexefaroxan and rivastigmine improved behavioural phenotypes and BDNF expression without affecting tissue Aβ load. Drug treatments also restored complex I+III mitochondrial activity and increased ATP levels. Reductions in noradrenergic tone appear to underlie Aβ-induced functional impairment in TgCRND8 mice, in addition to BDNF deficits and bioenergetic stress. These studies suggest that α2-adrenoceptor targeting may warrant consideration as a therapeutic strategy in AD.

The lower esophageal sphincter (LES) is characterized by basal tone and appropriately timed neurogenic relaxation. The physiological mechanisms underlying these crucial LES functions remain poorly understood. The current studies were designed to characterize the electrophysiological properties and neural regulation of LES circular smooth muscle (CSM), and to determine whether interstitial cells of Cajal (ICC) play a role in neurotransmission. Conventional intracellular recordings were performed in CD1, nNOS knock-out, eNOS knock-out and W/Wv mutant mice. Mouse LES consists of “sling” and “clasp” smooth muscle, which were studied separately in CD1 mice. In subsequent studies of mutant mice and respective controls, only the clasp muscle was examined, Immunohistochemical c-Kit staining of ICC was performed in wild-type and W/Wv mutant mice that were first characterized electrophysiologically. The smooth muscle of the LES clasp and sling displayed unitary membrane potentials with a resting membrane potential (RMP) of ~ -43 mV. Spontaneous nifedipine-sensitive action potentials superimposed on the unitary potentials were usually recorded in the LES clasp, but not sling muscle. A monophasic inhibitory junction potential (IJP) was recorded in sling CSM, whereas a biphasic IJP consisting of an initial IJP, followed by long-lasting slow IJP (LSIJP) was recorded in clasp. Further pharmacological studies using control and various knockout mice suggest that: 1. the CSM of the mouse LES is innervated by cholinergic, nitrergic and purinergic nerves; 2. the LSIJP is mediated entirely by nitrergic nerves, whereas purinergic and nitrergic nerves produce the monophasic IJP in the LES sling and initial phase of biphasic IJP in the LES clasp; 3. Ca2+/CaM-kinase II is involved in the regulation of the nitrergic IJPs; 4. TREK-1 K+ channels are not involved in the nitrergic IJP; 5. purinergic and cholinergic neurotransmission is intact in LES CSM of W/Wv mutant mice, whereas nitrergic neurotransmission is impaired in about half of the animals. In animals in which nitrergic neurotransmission was intact, ICC-IM were markedly deficient immunohistologically, suggesting that ICC are not required for nitrergic neurotransmission; 6. impaired nitrergic neurotransmission in W/Wv mutant mice is associated with dysfunction of a Ca2+-dependent signaling cascade primed by spontaneous Ca2+ release from the sarcoplasmic reticulum. / Thesis (Ph.D, Physiology) -- Queen's University, 2008-01-24 15:54:52.175

This project involved the study of mechanisms which modulate autonomic transmission within the sympathetic nervous system using the mouse vas deferens as a model tissue. Data was collected using contraction studies, electrophysiological techniques with sharp microelectrodes, and fluorescent calcium imaging of both smooth muscle cells and nerve terminal varicosities. An additional series of experiments was conducted using the PC12 cell line, derived from a phaeochromocytoma of the rat adrenal medulla, for flow cytometry experiments using fluorescence-activated cell sorting. During the course of this project a novel technique for studying the activity of the norepinephrine transporter within a whole organ preparation was developed using the neurotransmitter uptake assay. The uptake of this assay within the nerve terminals of the vas deferens was abolished by desipramine whilst its rate of washout was increased by amphetamine. However, some non-neuronal, peri-nuclear staining which could not be prevented by a range of pharmacological means was also observed. This new technique was then used in other work exploring putative NET regulation by cannabinoids. The modulatory effects of two pharmacological groups were assessed: testosterone and cannabinoids. Testosterone was found to have a rapid, non-genomic effect inhibiting neurotransmission within the vas deferens. This was a postjunctional effect which appeared to involve modulation of the opening of L-type calcium channels on the smooth muscle cells. For the studies of cannabinoids, two broad areas of research were conducted. First the effects of Δ⁹-tetrahydrocannabinol were investigated with regard to the pre-junctional release of neurotransmitters and the effect of THC on calcium dynamics within individual nerve terminal varicosities. Secondly, a surprising novel effect upon the norepinephrine transporter was identified and examined. This inhibitory effect was revealed initially by contraction experiments demonstrating a decrease in the rate of uptake of noradrenaline from the junction. This work demonstrates that there are still novel modes of regulation of sympathetic transmission to be uncovered. The ongoing challenge is to establish their role within physiology and pathophysiology.