No description available.

No description available.

Glutamate is the principal excitatory neurotransmitter throughout the CNS, including the spinal cord. It acts on ionotropic (iGluR) and metabotropic glutamate receptors. Three iGluR families have been identified by the development of more-or-less selective agonists: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. Both AMPA (GluR1-4) and NMDA (NR1, NR2A-D) receptors have been detected in the spinal cord and these play a major role in physiological processes such as fast excitatory transmission, synaptic plasticity and neuronal development. In addition, they have also been implicated in pathological conditions including neuropathic pain and neurodegenerative disorders. However, very little is known about the synaptic distribution of these receptors in the spinal gray matter. This is because conventional immunocytochemical techniques, generally used to investigate the location of proteins in the CNS, fail to detect these subunits at synapses due to the presence of an elaborate protein meshwork associated with the postsynaptic membrane, which masks synaptic receptors. Postembedding immunocytochemistry on freeze-substituted, Lowicryl-embedded material is a technique which has been used exclusively for the detection of synaptic AMPA and NMDA receptors. This project initially set out to use this method to examine these receptors on neurons of the adult rat spinal cord, with emphasis on their involvement in the sensory processing of the dorsal horn. With antibodies against the GluR1, GluR2/3, NR1, NR2A and NR2B subunits, heavy labelling was observed at many asymmetrical synapses and where the plane of section was perpendicular to the cleft, most of the immunogold particles were associated with the postsynaptic density. To examine the receptor expression pattern of selected cell populations a new method was developed which involved the combination of postembedding electron microscopy with immunofluorescence and confocal microscopy. However, during the course of this study heavy immunogold labelling of dense-cored vesicles (dcvs) inside axonal boutons was observed with all NMDA antibodies. Several studies have found iGluRs in primary afferent terminals in the spinal gray matter and these are thought to function as presynaptic receptor, in order to determine whether gold particles found over dcvs corresponded to presynaptic receptors in transit, immunogold reactions were carried out on transgenic mice which lacked the NR2A subunit. Surprisingly, not only did the dev labelling remain in these knock-out animals, but there was also a significant synaptic labelling. This suggested that the postembedding immunogold labelling observed with the NR2A antibody was non-specific. Since the labelling patterns were similar with other NMDA antibodies this cast doubts on the validity of the postembedding method for detecting NMDA receptors.

La maladie d'Alzheimer (MA) et l'angiopathie amyloïde cérébrale (AAC) sont respectivement caractérisées par des dépôts de peptides amyloïdes β (Aβ) dans le cerveau et la vascularisation cérébrale. Étant donné qu’un régime riche en DHA est associé à une réduction du risque de la MA, l'objectif principal de ma thèse a été d'évaluer l'impact d'un régime enrichi en DHA sur l'inflammation systémique et ses conséquences sur les dépôts Aβ parenchymateux et vasculaires au cours du vieillissement dans un modèle de souris transgéniques de MA/AAC, les Tg2576. Les dépôts amyloïdes ont été détectés en utilisant un anticorps anti-Aβ et les hémorragies avec du bleu prussien sur des coupes cérébrales. A 10, 14 et 18 mois, nous avons démontré une réduction des dépôts vasculaires amyloïdes et des hémorragies en régime DHA par rapport au régime placebo, alors que les dépôts parenchymateux ne sont pas affectés. De plus, nous avons démontré une forte corrélation entre les dépôts amyloïdes vasculaires, les hémorragies et un médiateur pro-inflammatoire lipidique, le 12-HETE. Nous avons ensuite évalué in vitro les effets du peptide Aβ1-40 sur la production de 12-HETE par les cellules musculaires lisses vasculaires. Nous avons démontré que les niveaux d'ARNm de l'enzyme 12-LOX, impliquée dans la synthèse de 12-HETE, était augmenté lorsque les cellules ont été incubées avec du peptide Aβ1-40, suggérant une relation de cause à effet entre les dépôts Aβ et les lipides pro-inflammatoires. Mon travail a de nouveau souligné l'importance de l'inflammation dans la pathogenèse de l'AAC tout en ouvrant une nouvelle voie pour des cibles potentielles dans l'intervention préventive de cette pathologie. / Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) are characterized by Amyloid β-peptides depositions in the brain and cerebral vasculature respectively. Because DHA-diet is associated with a reduced risk of AD, the main objective of my thesis was to evaluate the impact of a DHA-diet on cerebrovascular and peripheral inflammation and its consequences on parenchymal and vascular Aβ-deposits during aging in a transgenic mouse model of AD/CAA (Tg2576). The Aβ-peptide deposits were detected using an anti-Aβ peptides and hemorrhages were detected with Prussian Blue on brain sections. At 10, 14 and 18 month-old, we demonstrated a reduction of amyloid vascular deposits and hemorrhages under DHA-diet compared to placebo, while parenchymal Aβ-peptides deposits remain unaffected. Moreover, we demonstrated a strong negative correlation between amyloid vascular deposition, hemorrhage and a lipid-derived pro-inflammatory mediator, 12-HETE. We next evaluated the in vitro effects of Aβ1-40-peptide on 12-HETE production by the Vascular Smooth Muscle Cells. We demonstrated that the mRNA level of the 12-LOX enzyme, involved in 12-HETE synthesis, was increased when cells where incubated with Aβ1-40-peptide, suggesting a cause-to-effect relationship between Aβ deposits and pro-inflammatory biolipids. The work carried out during my thesis made it possible to demonstrate DHA protective effect on evolution and consequences of Aβ peptide cerebrovascular accumulation and link it to plasma 12-HETE level. My work emphasized once again the importance of inflammation in AAC pathogenesis while opening up a new pathway for potential targets in the preventive intervention of this pathology.