The major goal of this research proposal is to elucidate the mechanisms regulating the synthesis of the neurotransmitter acetylcholine (ACh) in brain, with an emphasis on determining how alterations in the availability of the precursor choline modify these processes. Evidence has supported the hypothesis that an increased supply of choline to the brain provides substrate for ACh synthesis that is of functional significance only when neurotransmitter synthesis is increased as a consequence of stimuli that increase ACh release. While this effect is manifest following the acute parenteral administration of choline, it has not been demonstrated following chronic dietary supplementation, despite evidence that both treatments increase choline availability in the brain. Furthermore, when fire choline is excluded from the diet, although steady-state levels of choline in brain are unaltered, the mobilization of free chorine from esterified sources decreases, with a concomitant reduction in the synthesis of ACh. Thus, the specific aim of this proposal is to elucidate the neurochemical mechanisms regulating the synthesis of ACh in brain, and determine, at the subcellular level, how alterations in choline availability modulate these processes. The studies outlined will use a combined in vivo/in vitro approach and investigate the effects of acute chorine administration, as well as the consequences of chronic dietary alterations; for the latter, rats will be maintained on chorine-deficient or chorine-supplemented diets for one month. Brain slices from these animals will be used for neurochemical investigations in vitro. The synthesis and release of ACh, the release and production of free chorine, and the esterified sources of chorine that provide precursor for ACh synthesis will be characterized in subcellular fractions from brain regions that contain a dense population of cholinergic nerve terminals, viz., striatum, hippocampus, and cerebral cortex. Specifically, the experiments will investigate: 1) the subcellular mechanisms responsible for the increased synthesis of ACh in brain slices from chorine-injected rats when these slices are exposed to stimuli that increase the demand for precursor by increasing neurotransmitter release; 2) whether chronic (dietary) supplementation with chorine has a direct effect on cholinergic neurons or whether observed in vivo effects are secondary to generalized membrane phospholipid perturbations; 3) the mechanism mediating the decreased synthesis of ACh in brain from rats fed a choline-deficient diet; and 4) the interactions among neuronal activity, the demand for choline, and phospholipid and ACh metabolism. Results from these studies will determine the nature and localization of the esterified choline pool that supplies free choline for ACh synthesis, and how this source is modulated by altering the availability of precursor. This knowledge is essential for a basic understanding of brain function and how such function can be impaired by the dietary restriction of an essential nutrient such as choline. Furthermore, results will provide a basis for the development of possible therapeutic strategies for the treatment of neuropsychiatric disorders postulated to involve central hypocholinergic activity such as Alzheimer's disease.
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