The dysfunction and degeneration of the nicotinic cholinergic system in the brain are integral physiopathological indicators of one of the most socially impacting neurological disorders, Alzheimer's disease (AD). In AD, the permanent loss of cholinergic neurons and nicotinic receptors (nAChRs) in brain areas that process cognitive functions, particularly the hippocampus and the frontal cortex, correlates well with the decline in cognition and memory. To date, treatment of patients with AD relies heavily on the use of acetylcholinesterases. These drugs, by increasing function of the cholinergic system, partially reverse the symptoms of AD patients. Recently, clinical trials have shown that nicotinic agonists (including nicotine) and drugs that allosterically potentiate the activity of nAChRs are more effective for treatment of patients with AD. The mechanisms underlying the effectiveness of these drugs remain unknown, because there is very little information on how function and expression of neuronal nAChRs in the brain are regulated by cholinergic afferents. In addition, detailed analysis of regulation of nAChR expression and function in the hippocampus by septal cholinergic afferents has been limited by the lack of a viable biological preparation that closely resembles the nicotinic cholinoceptive hippocampal system in vivo. Our initial characterization of the nicotinic properties of hippocampal neurons in organotypic, hippocampal and septal-hippocampal cultures constitutes the mainstay of the present proposal, as it establishes the septal-hippocampal co-cultures as an excellent model for in vitro study of the influences of septal innervation on nAChR expression in the hippocampus. Thus, this proposal is designed to use convergent, multidisciplinary approaches to address the central hypothesis that septal innervation and nicotine dynamically modify the hippocampal cholinergic system. The first goal of this study is to use electrophysiology, confocal microscopy, ligand binding and immunocytochemistry to determine whether septal innervation alters the nicotinic properties of different types of hippocampal neurons during development in organotypic cultures. The second goal is to use electrophysiological assays, recombinant DNA technology and """"""""knock-out"""""""" mice, which have a null mutation in the gene encoding alpha7 nicotinic receptors, to study nAChR targeting and to investigate the motifs in the nAChR subunits that account for final receptor targeting in hippocampal neurons. The final goal is to use electrophysiological, biochemical and molecular biological techniques to evaluate how nicotine affects alpha7 and alpha4beta2 nAChR expression in the hippocampus. The results of these studies will have far reaching implications in identifying cellular and molecular mechanisms regulating nAChR expression and function in the hippocampus and provide the basis for developing therapeutic agents targeting regions of the brain affected in AD.
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