ALTERED KCa CHANNEL EXPRESSION IN DEVELOPING CEREBELLUM

描述（来自申请人摘要的逐字记录）：拟议的研究使用 携带绿色荧光标记基因的解除武装的疱疹病毒载体 介导反义敲除大鼠K-Ca通道的蛋白质（GFP） 小脑浦肯野神经元。初步数据显示，反义敲除 rabbits引起浦肯野细胞K-Ca通道丰度的可测量的减少 神经元，以及针对另一种K+通道β的病毒驱动的反义 Kv β 1.1亚基显著影响整个细胞的放电模式， 证明了病毒载体反义方法的可行性。 K-Ca通道蛋白α亚基的表达在细胞内受到调节， 小脑通过去极化和Ca 2+进入的时间模式， 在体内和体外相当。 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通道的作用， 产生浦肯野神经元的经典起搏器放电模式。要求1 使用病毒驱动的针对α序列的反义来分析功能， K-Ca电导的变化。Aim 2分析了 在小脑中表达，并使用病毒介导的 过度表达和低表达，以分析β 亚单位的成熟的起搏器放电模式。 解除武装的病毒载体是一种强有力的基因操纵新方法 在体内和体外的神经元中表达。许多人类神经系统 发育障碍影响小脑;其延长产后 发育期是临床药物的易感窗口期 有神经副作用大鼠为研究 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

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.