EXCEED THE SPACE PROVIDED. Neuroanatomical changes in Alzheimer's disease Although genetic studies causally link A6 to Alzheimer's disease, the mechanism that links A6 deposits to neuronal dysfunction, loss, and dementia remains unknown. This application for continued support builds on the results of quantitative anatomical and neuropathological studies in Alzheimer's disease and transgenic mouse models carried out in the last 5 years, which have led to the hypothesis that ABdeposition is a dynamic process, leading to the formation of a toxic 6 pleated sheet containing subset of plaques. Rather than testing these hypotheses solely on static tissue sections, we developed a novel in vivo imaging approach - using multiphoton microscopy - and applied it to imaging the cortex of living, (anesthetized) APP transgenic mice. Multiphoton microscopy has a resolution equivalent to confocal microscopy, and can image several hundred microns deep to the surface (into layer HI of the mouse cortex). After imaging, the mouse recovers uneventfully and the exact same site can be re-imaged days to months later. We have successfully imaged the exact same AB deposits using thioS as a fluorophore up to 5 months after initial imaging. We have also used this imaging technology to show that direct application of anti-AB antibodies to the cortical surface clears existing plaques in a remarkably short period of time -just 3 days. These advances in in vivo microscopy set up the possibility of studying functional changes in the livingbrain due to ABdeposits, and of asking the critical question of whether clearance of ABleads to recovery or resolution of those changes. We have developed several in vivo histology and functional imaging tools: fluorescein angiograms to define vascular changes and provide an internal 'road map' for subsequent imaging sessions, Hoescht stain for nuclei, tomato lectin for microglia, thioS for plaques, in vivo immunofluorescence using directly labelled antibodies to visualize diffuse AB deposits, green fluorescent protein (EGFP) imaging via amplicon gene transfer delivery techniques in the adult mouse, and calcium imaging using indo-1-dextran. We believe that the further development of in vivo multiphoton microscopy will have broad applicabilityfor neuroscience. These techniques allow us to test specific hypotheses about ABtoxicity in vivo, and, combined with analyses of Alzheimer tissue, will provide insight into the pathophysiology of ABusing dynamic methods of extraordinary power. KfcHf-ORMANCt blTE(S) (organization, city, state) Massachusetts General Hospital 42 Fruit Street Boston, MA 02114 KEY PERSONNEL ========================================Section End===========================================
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