ALTERED KCa CHANNEL EXPRESSION IN DEVELOPING CEREBELLUM

小脑发育过程中 KCa 通道表达的改变

• 批准号: 6392468
• 负责人: ANDREA J YOOL
• 金额: $ 18.02万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6392468
• 关键词: antisense nucleic acid; biological models; brain electrical activity; calcium channel; calcium flux; cell membrane; cerebellar Purkinje cell; cerebellar cortex; developmental neurobiology; gene expression; green fluorescent proteins; heart pacemaker tissue; immature animal; immunocytochemistry; laboratory rat; nucleic acid sequence; polymerase chain reaction; potassium channel; tissue /cell culture; transfection /expression vector; voltage /patch clamp

DESCRIPTION (Verbatim from the Applicant's Abstract): The proposed research uses a disarmed Herpes viral vector carrying the marker gene for Green Fluorescent Protein (GFP) to mediate antisense knockdown of the K-Ca channel, rslo, of rat cerebellar Purkinje neurons. Preliminary data show that antisense knockdown of rslo causes a measurable decrease in the abundance of K-Ca channels in Purkinje neurons, and that viral-driven antisense against another K+ channel beta subunit (Kvbeta1.1) dramatically affects whole cell firing patterns, thus demonstrating the feasibility of the viral vector antisense approach. Expression of the alpha subunit of the K-Ca channel rslo is regulated in cerebellum by depolarization and Ca2+ entry in a temporal pattern that is comparable in vivo and in vitro. Electrophysiology, molecular biology, immunocytochemistry and fluorescence imaging are used to analyze the consequences of viral infection, molecular antisense manipulations, and to evaluate the role of the K-Ca channel in the generation of the classic pacemaker firing pattern of Purkinje neurons. Aim 1 uses viral-driven antisense against the alpha sequence to analyze the function of K-Ca conductance in generating pacemaker firing. Aim 2 analyzes the expression of the rslo beta subunit in cerebellum, and uses viral-mediated overexpression and underexpression to dissect the contribution of the beta subunit to the maturation of the pacemaker firing pattern. The disarmed viral vector is a powerful new approach for manipulating gene expression in neurons in vivo and in vitro. A number of human neurological developmental disorders affect the cerebellum; its extended postnatal developmental period constitutes a window of vulnerability to clinical agents with neurological side effects. The rat provides a useful model for studying postnatal cerebellar development and neuropathological damage, and may provide insights into methods to manipulate developmental abnormalities in the cerebellum.

描述（逐字摘自申请人摘要）：拟议的研究用途 携带绿色荧光标记基因的解除武装的疱疹病毒载体 介导大鼠 K-Ca 通道 rslo 反义敲低的蛋白 (GFP) 小脑浦肯野神经元。初步数据表明，反义敲低 rslo 导致浦肯野 K-Ca 通道丰度显着减少 神经元，以及病毒驱动的针对另一个 K+ 通道 β 的反义 亚基 (Kvbeta1.1) 显着影响全细胞放电模式，因此 证明病毒载体反义方法的可行性。 K-Ca 通道 rslo 的 α 亚基的表达受调节 小脑通过去极化和 Ca2+ 进入的时间模式是 体内和体外具有可比性。 电生理学、分子生物学、免疫细胞化学和荧光 成像用于分析病毒感染的后果，分子 反义操作，并评估 K-Ca 通道在 浦肯野神经元经典起搏器放电模式的生成。目标1 使用病毒驱动的针对 alpha 序列的反义来分析功能 K-Ca 电导在产生起搏器放电中的作用。目标 2 分析 rslo beta 亚基在小脑中的表达，并使用病毒介导的 过度表达和表达不足来剖析 beta 的贡献 亚单位促进起搏器放电模式的成熟。 解除武装的病毒载体是操纵基因的强大新方法 在体内和体外的神经元中表达。人类神经系统的一些 发育障碍影响小脑；其延长的产后 发育期构成了对临床药物的脆弱性窗口 具有神经系统副作用。老鼠为研究提供了一个有用的模型 出生后小脑发育和神经病理损伤，并可能提供 深入了解操纵发育异常的方法 小脑。