We will focus on membrane lipid-derived second messenger during seizures. We will study alterations in the metabolism of phospholipids, diacylgycerols and fatty acids, specifically arachidonic acid and arachidonoyl-glycerolipids in discrete brain regions, and in synaptosomes and synaptoneurosomes of the rat, during experimentally-induced seizures. These studies will define the role of synaptic membrane components in the development of epileptic activity and the molecular pathogenesis of epileptic brain damage. Three models will be investigated: 1) a single electroconvulsive shock with minimal neuronal damage; 2) bicuculline-induced status epilepticus, where, brain damage is severe; and 3) audiogenic seizures in genetically epileptic rats. We will test the hypothesis that seizures lead to receptor-mediated activation of phospholipase A2 and phospholipase C, resulting in the degradation of synaptic membrane polyphosphoinositides and ether-linked phosphatidylcholine, and in the altered release of lipid-derived messengers, e.g. arachidonic acid (20:4) and its oxygenated metabolites, diacylglycerols (DG), platelet activating factor (PAF) and inositol phosphates (IP). We will assess exitotoxic amino acid agonists and antagonists in relation to epileptic brain damage as well as polyunsaturated fatty acid peroxidation. Specifically, we will use radiotracer techniques and measurement of endogenous lipids to study the metabolism of alkyl- acylglycerophosphocholine (the PAF cycle precursor) and polyphosphoinositides as targets for seizure-activated phospholipase. We will use hippocampus, striatum and cerebral cortex isolated from microwave-fixed rat brain for in vivo studies, and synaptosomes and synaptoneurosomes prepared from these brain areas for in vitro studies. In some experiments 14C choline or (14C) arachidonic acid will be injected intraventricularly to follow precursor-product relationships. This proposal will employ in vivo models and subcellular fractions. Very rapid fixation of the tissues within 1 second will be achieved by high-powered, head- focused microwave irradiation. Powerful analytical techniques, such as high performance liquid chromatography, gas-liquid chromatography, and gas chromatography-mass spectrometry, will be used to examine biochemical changes in phospholipids and fatty acids of neuronal membranes. The results of this study will have application in the management of epileptic seizures and will provide a model to evaluate drugs potentially capable of halting or reversing membrane lipid breakdown, consequently preventing or limiting the brain damage caused by epilepsy.
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