ALTERED KCa CHANNEL EXPRESSION IN DEVELOPING CEREBELLUM

描述(逐字摘自申请人的摘要)：拟议的研究使用 一种携带绿色荧光标志基因的解除武装疱疹病毒载体 介导大鼠钾-钙通道rslo反义敲除的蛋白质(GFP) 小脑浦肯野神经元。初步数据显示，反义基因敲除 RSLO导致浦肯野细胞钾-钙通道丰度显著降低 神经元，以及病毒驱动的针对另一个K+通道Beta的反义 亚基(Kvbeta1.1)显著影响整个细胞的放电模式，因此 论证了病毒载体反义方法的可行性。 钾-钙通道α亚基rslo的表达受 小脑通过去极化和钙离子以一种时间模式进入 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 成像被用来分析病毒感染的后果，分子 反义操作，并评估钾-钙通道在 浦肯野神经元经典起搏器放电模式的产生。目标1 使用病毒驱动的针对阿尔法序列的反义来分析功能 钾-钙电导在起搏器放电中的作用。目标2分析了 Rsloβ亚基在小脑中的表达，并使用病毒介导法 高表达和低表达来剖析贝塔的贡献 亚单位与起搏器放电模式的成熟有关。 解除武装的病毒载体是一种强有力的基因操作新方法 在体内和体外神经元中的表达。一些人类神经学 发育障碍影响小脑；其延长的出生后 发育期是临床药物易感性的窗口 有神经副作用。大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害，并可能提供 对操纵儿童发育异常的方法的见解 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.