University, Diss--Heidelberg, 2004.

Stuttgart, Univ., Diss., 2003.

Rationale: Chronic cough (cough >8 weeks) is common, leads to an impaired quality of life, and is difficult to treat. Despite intensive investigation, ~40% of patients referred to a specialist cough clinic will remain resistant to treatment targeted at peripheral triggers such as reflux disease, rhino-sinusitis or airways inflammation. An improved understanding of underlying mechanisms in such patients would facilitate drug development. I propose that there are several important similarities between pain and cough that can be exploited better to understand underlying mechanisms. In chronic pain, a long-lasting up-regulation of afferent pain processing may be generated by changes within the central nervous system, mediated by the NMDA receptor and/or by impaired inhibitory mechanisms. A similar central neuronal up-regulation of cough may also be responsible for the pathogenesis of chronic cough (CC). Methods: A series of experimental studies were performed to address this hypothesis. Firstly, the anti-tussive and analgesic effect of ketamine, an NMDA receptor antagonist, was investigated in CC patients and healthy controls (HC). Pain thresholds were measured using electrical stimulation in the oesophagus, pharynx and chest wall. Cough sensitivity was measured using standard capsaicin cough challenges. Secondly, I designed and tested novel capsaicin cough challenges in CC patients, asthmatics (A) and HC. ED50 (dose inducing and least 50% maximal cough frequency) and Cmax (maximal cough frequency) was compared by group and gender. Finally, I investigated 2 independent mechanisms of cough inhibition. Results:(i) CC patients, but not HC, had cough induced by oesophageal electrical stimulation, whilst pain thresholds were similar. Ketamine had a significant analgesic effect but no antitussive effect in CC or HC.(ii) CC patients had both cough hypersensitivity (lower ED50) and cough hyper-responsiveness (higher Cmax) on full capsaicin dose-response curves. (iii) Both a painful cold stimulus applied to the hand and conscious cough suppression significantly inhibited capsaicin-induced cough responses in CC and HC.Conclusions:CC patients exhibited increased oesophageal sensitivity to cough, but not pain, providing evidence for a process of central sensitisation in the brainstem. Higher capsaicin-induced cough frequencies in CC may also be mediated by an increased gain within the CNS, possibly because of failed tonic inhibitory mechanisms. Furthermore, CC patients may have poorer conscious control of coughing. In conclusion, an improved understanding of mechanisms in cough will provide a strong scientific rationale for the development of novel therapeutics.

High frequency stimulation of the perforant pathway triggers a prolonged field potential in the dentate gyrus that far outlasts that obtained with single pulses. The late rising component of this field potential has recently been shown to be mediated by N-methyl-D-aspartate (NMDA). In the present thesis, rats were implanted with stimulating electrodes in the perforant pathway and recording electrodes in the dentate gyrus of the hippocampus. Baseline input/output functions of field potentials (or population EPSPs) were established for each rat. Ketamine, an NMDA receptor antagonist, was then administered to confirm its effects on the late component of the EPSP. The late component was measured by subtracting the pulse-evoked from the train-evoked response. Ketamine was shown to significantly attenuate the late component. Diazepam, a GABA agonist, had no significant effect on the late component. Having established an NMDA component in the field potential allows for the monitoring of the levels of NMDA activation over prolonged periods. Hence, the effect of kindling, an animal model of temporal lobe epilepsy, was also determined. Fully kindled rats--defined as those who had experienced four stage 5 seizures--also had significantly attenuated late components. In contrast to decreased late components, kindled rats displayed increased population spike amplitudes and EPSP slopes. Such a decrease in the late component suggests that the NMDA receptor plays a role in kindling. Subjects were also given ketamine and diazepam following kindling, whereby the effects were proportionately the same as those observed prior to kindling. / Thesis / Master of Science (MS)

No description available.

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

This study investigated the role of NMDARs in the differentiation of MEG-01 cells and in the activation of human platelets. This investigation demonstrated that the NR1, NR2D and NR3 subunit proteins are expressed in human platelets, with the NR1 subunit also expressed in MEG-01 cells. The NR2A subunit protein was not detectable in either MEG-01 cells or human platelets. PMA-induced differentiation of MEG-01 cells did not appear to stimulate changes in expression of any of the subunit proteins tested. Using assays to measure the changes in [Ca2+]i and ATP secretion, it was determined that donors could be separated into those who responded to the agonists applied and those who did not; responses also decreased over time in both assays. Human platelets from responding donors demonstrate an increase in [Ca2+]i in response to extracellular glutamate, and that increases in ATP secretion are detected at a 10-fold lower concentration. The same is also true with extracellular glycine. Increases in [Ca2+]i were elicited on the addition of extracellular NMDA; extracellular D-serine had no effect. NMDAR inhibitors, MK-801 and D-AP5, inhibited ATP secretion evoked by either glutamate alone or in combination with glycine. D-serine inhibited responses elicited by extracellular glycine. NMDARs play a role in MK differentiation, with the adhesion of MEG-01 cells cultured on a fibrinogen-surface and differentiated with PMA reduced by both inhibitors. PMA-treated MEG-01 cells increased both in size and irregularity, with the addition of NMDAR-specific inhibitors having no effect. S-nitrosylation also inhibits activation of NMDAR, and a new molecule has been developed which can detect S-nitrosylated proteins through a single step process in live cells. Overall, this study has shown that both human platelets and MEG-01 cells express NMDAR subunits, which have been demonstrated to form functional receptors in human platelets.

Obesity is an accumulation of adipose tissue to the point of negative health outcomes; the association of obesity with type 2 diabetes and cardiovascular disease is particularly profound. Notably, obesity has begun to be associated with impairments in cognitive function; however, the underlying cellular mechanisms are poorly understood. Behavioural studies have demonstrated a link between a high fat diet and impaired hippocampal function, and our aim was to characterize protein level changes in the hippocampus of an obese female rat model. Female rats were fed either a control diet (CD; 10% kcal from fat), or a high-fat diet (HFD; 45% kcal from fat) for 16 weeks. Body weight, food consumption, fasting blood glucose levels, and glucose tolerance were monitored. Upon sacrifice brain, liver, adrenal glands, spleens and fat pads were harvested and analyzed. Plasma leptin and insulin levels were also measured. The distribution of NMDA receptor subunits was examined by using either cell-surface biotinylation, or differential filtration-centrifugation followed by immunoblotting. The feeding protocol induced an obese phenotype in female rats characterized by larger fat pads, spleens and adrenal glands, as well as greater problems handling a glucose load. However, cellular, surface and synaptic expression of NMDA receptor subunits (GluN1, GluN2A & GluN2B) were not significantly altered, which suggests that changes downstream of the receptor may be responsible for the effects of HFD on cognitive behaviour.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal