CLONED GABA RECEPTORS IN INHERITED EPILEPSY

拟议研究的目标是确定 GABAA/苯二氮卓受体在癫痫发作中的作用 在遗传性癫痫动物模型中的活性，并确定 受体的特定氨基酸残基的变化 或调节未改变的受体的表达， 对任何这样的角色负责。 主要方法将涉及 从选择的近交系啮齿动物的大脑中克隆受体， 根据其易感性（SP，癫痫发作倾向）或 对各种癫痫发作的抵抗力（SR，抗癫痫发作） 活动 γ-氨基丁酸（GABA）是主要的 大脑的抑制性神经递质。 它的受体， 包括氯离子通道作为其结构的一部分，被认为 是许多重要抗惊厥药的作用部位 药物，包括苯二氮卓类和巴比妥类，以及某些 惊厥药 GABA受体功能的阻断导致癫痫发作 而它的增强可以阻止它们。 最近的变化证据 GABA相关的突触标记物，包括受体结合， 啮齿类动物的几种遗传性癫痫表明， 癫痫易感性可能至少部分是由于 GABA受体结构或表达。 为了验证这个假设， 我们将在λ gt 10载体中制备来自脑的cDNA文库， SP和SR啮齿类动物（最初是听源性癫痫倾向小鼠， DBA/2 J，对比2个对照，一个C57 BL/6 J和一个同类基因），筛选 用代表克隆牛GABA片段的探针 受体，测序阳性克隆，寻找受体的连锁 用特异性寡核苷酸探针检测到的差异， 重组近交系中癫痫易感性和其它标记 菌株，并检查GABA受体mRNA水平在不同的 大脑区域的原位杂交。 来自SP的粗mRNA和 SR大脑是制作cDNA文库的第一步， 也将被注射到非洲爪蟾卵母细胞中进行电生理检查。 膜片钳和细胞内微电极研究 技术. 这应该能揭示GABA受体 功能之间的SP和SR小鼠，并提供数据，为以后 与使用制备的人工mRNA获得的结果进行比较 从克隆的cDNA，验证克隆。 这些研究可能 最终导致遗传性癫痫的基因治疗。

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.