Transgenic Electrical Silencing of a Neural Circuit

描述（由申请人提供）：这是一个NIDA尖端基础研究奖（CEBRA）在R21格式的建议。 我们已经取得了进展，开发了一套修饰的K+通道亚基作为分子工具，操纵在果蝇体内特定的神经回路中的神经元电兴奋性。从我们的初步结果，我们可以有效地改变特定的行为和细胞特异性的信号转导事件通过表达修饰的K+通道基因。这种转基因操作似乎对体内神经元存活没有不良影响（Nitabach等人，2002 a; Nitabach等人，手稿正在准备中）。在果蝇中的成功实施为将这项新技术转化为转基因小鼠的特定神经回路提供了强大的动力。 我的具体目标是： (1)在分离的哺乳动物神经元中体外表达修饰的钾通道基因，并测试通道基因表达的稳健性、神经元电沉默的有效性和神经元活力。符合这些标准的候选基因将用于随后的目标。 (2)构建和表征表达由高度特异性神经元启动子L7靶向的修饰的钾通道基因的转基因小鼠。表征将包括电生理和成像分析。 将这项技术成功转化为转基因小鼠将提供一种强大的基于基因靶向的方法，以补充广泛使用的现有药理学和损伤策略，用于研究与成瘾和药物滥用有关的神经回路。

DESCRIPTION (provided by applicant): This is a NIDA Cutting-Edge Basic Research Awards (CEBRA) proposal in the R21 format. We have made progress towards developing a set of modified K+ channel subunits used as molecular tools for manipulating neuronal electrical excitability in vivo in specific neural circuits in Drosophila. From our preliminary results, we can effectively alter specific behaviors and cellspecific signal transduction events by expressing modified K+ channel genes. This transgenic manipulation appears to exert no ill effects on neuronal survival in vivo (Nitabach et al., 2002a; Nitabach et al., manuscript in preparation). This successful implementation in Drosophila provides strong motivation to translate this new technology to specific neural circuits in transgenic mice. My Specific Aims are: (1) Express modified potassium channel genes in vitro in dissociated mammalian neurons and test for the robustness of channel gene expression, the effectiveness of neuronal electrical silencing, and neuronal viability. Candidate genes that meet these criteria will be used for the subsequent Aim. (2) Construct and characterize transgenic mice that express modified potassium channel genes targeted by the highly specific neuronal promoter L7. Characterization will consist of electrophysiological and imaging analysis. Successful translation of this technology to transgenic mice will provide a powerful gene-targeted based method to complement widely-used existing pharmacologic and lesion strategies for studying neural circuits that are linked to addiction and drug abuse.