TARGETING OF THE BENZODIAZEPINE BINDING SITE

说明： 拟议研究的总体目标是确定 小鼠GABA/A受体上苯二氮(BZ)结合部位的研究 神经元发育、突触功能和某些行为。基因 通过Cre介导的重组在工程LOX/P站点上进行靶向将是 使用。BZ结合位点在体内将被破坏，无论是有条件的 GABA/A最近的γ/2亚单位基因敲除或工程a GABA/A受体α/1亚基的特异点突变。在这两个地方 小鼠品系的GABA/A受体总数预计为 与野生动物的数量相当，但亲和力 对于BZ配体，预计将大幅减少。具体目标 详情如下： 1.去除小鼠体内GABA/A受体高亲和力BZ结合部位 靶向缺失必需外显子对小鼠特定脑区的影响 在β/2亚基中。淘汰赛的时空控制 小鼠大脑中的这个亚单位将通过与另一个亚基杂交来执行 在特定脑内转基因表达Cre重组酶的小鼠 地区。 2.阻断GABA/A受体高亲和力BZ结合部位I 通过阿尔法/1亚基中特定氨基酸的靶向突变。这个 靶向氨基酸是当突变为Arg时的残基100 产生对BZ激动剂基本上不敏感的受体，尽管 GABA正常门控。 3.研究BZ调制缺失对两个品系小鼠的影响 对于中枢神经系统功能，特别是关于神经元发育 突触传递。分析将涉及电生理， 放射配基和免疫细胞化学技术。 4.研究BZ缺失对两个品系小鼠的影响 对几种BZ配体的行为效应的调节(每个都有一个 不同的本征活性)以及诸如乙醇的其他物质， 挥发性麻醉剂和巴比妥酸盐。

DESCRIPTION: The overall goal of the proposed research is to define the role of the benzodiazepine (BZ) binding site in the GABA/A receptors of mice during neuronal development, synaptic function and certain behaviors. Gene targeting by Cre-mediated recombination on engineered lox/P sites will be used. The BZ binding site will be disrupted in vivo either by conditional gene knockout of the GABA/A recently gamma/2 subunit or engineering a specific point mutation in the GABA/A receptor alpha/1 subunit. In both mouse lines the total number of GABA/A receptors is expected to be comparable to the number found in the wild type animal, but the affinity for BZ ligands is expected to be drastically reduced. The specific aims are as follows: 1. To remove the high affinity BZ binding site of GABA/A receptors in specific brain regions in mice by targeted deletion of an essential exon in the beta/2 subunit. The spatial and temporal control of the knockout of this subunit in mice brain will be performed by intercrossing with another mice that is transgene expressing Cre recombinase in specific brain regions. 2. To disrupt the high affinity BZ binding site type I in GABA/A receptors by targeted mutations of a specific amino acid in the alpha/1 subunit. The targeted amino acid is the resides His 100 which when mutated to Arg yields receptors which are essentially insensitive to BZ agonists, despite the normal gating by GABA. 3. To investigate the impact of lack of BZ modulation in both mouse lines for CNS function, in particular, regarding neuronal development with synaptic transmission. The analyses will involved electrophysiological, radio-ligand and immunocytochemical techniques. 4. To investigate, in both mouse lines, the impact of lack of a BZ modulation on the behavioral effects of several BZ ligands (each with a different intrinsic activity) and of other substances such as ethanol, volatile anesthetics and barbiturates.