The goals of the proposed research are to determine the role of GABAA/benzodiazepine receptors in the development of seizure activity in animal models of inherited epilepsy and to determine whether changes in specific amino acid residues of the receptors or in regulation of expression of unchanged receptors are responsible for any such role. The major approach will involve cloning the receptors from brains of inbred rodent strains chosen on the basis of their susceptibility (SP, seizure prone) or resistance (SR, seizure resistant) to various forms of seizure activity. Gammaaminobutyric acid (GABA) is the major inhibitory neurotransmitter of the brain. Its receptor, which includes a chloride channel as part of its structure, is thought to be the site of action of a number of important anticonvulsant drugs, including benzodiazepines and barbiturates, and certain convulsants. Blockade of GABA receptor function causes seizures and its enhancement prevents them. Recent evidence for changes in GABA-related synaptic markers, including receptor binding, in several forms of inherited epilepsy in rodents suggests that seizure susceptibility may result, at least in part, from changes in GABA receptor structure or expression. To test this hypothesis, we will prepare cDNA libraries in lambda gt10 vector from brains of SP and SR rodents (initially audiogenic seizure prone mice, DBA/2J, vs. 2 controls, one C57BL/6J and one congenic), screen them with probes representing pieces of the cloned bovine GABA receptor, sequence positive clones, look for linkage of receptor differences, as detected with specific oligonucleotide probes, with seizure susceptibility and other markers in recombinant inbred strains, and examine levels of GABA receptor mRNA in different brain regions by in situ hybridization. Crude mRNA from SP and SR brains, prepared as the first step in making cDNA libraries, will also be injected into Xenopus oocytes for electrophysiological studies by patch clamp and intracellular microelectrode techniques. This should reveal any differences in GABA receptor function between SP and SR mice and also give data for later comparison with that obtained using artificial mRNAs prepared from cloned cDNAs, verifying the cloning. These studies may eventually lead to gene therapy of inherited epilepsy.
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