MECHANISM OF NERVE GROWTH AND NAVIGATION

了解轴突寻路的机制是 发育神经生物学。这项提案中概述的工作重点是 对生长和生长的分子和细胞机制的研究 神经突起的导航。L将使用的实验系统 非洲爪哇神经元的体外研究与最先进的成像技术相结合 蒲慕明博士实验室中的分子操作技术。 这项提案中涉及的主要问题是，第二信使和 细胞骨架蛋白参与指导生长锥体的运动。 这项提议有两个具体目标。 在目标1中，我将确定钙离子和cAMP作为第二信使的作用 在不同条件下调控神经突起伸展的速度和方向 环境条件。这将通过测量[Ca2+]i在 使用标准Fura-2比率成像方法的规则时间间隔和 记录明视场图像测量轴突延长率 使用延时录像。无论是胞浆内钙离子还是cAMP 坡度足以诱导生长锥体转动将通过以下测试 利用紫外光激活的聚焦释放笼状化合物中的钙离子和cAMP 生长锥体。钙离子和cAMP依赖的通路是否相互作用 彼此也将接受检查。在AIM 2中，我将确定早期的 与生长锥体转动相关的细胞事件 不同的趋化梯度(谷氨酸、Forsklin和NT-3)。这 将通过分析丝状伪足活动来完成， 微管/微丝聚合/解聚和 联合DIC法检测肌动蛋白结合蛋白明胶蛋白的分布 以及荧光成像方法。完成这两个目标将 洞察生长锥体转动之前的分子事件。 这些结果都将促进我们对神经发育的理解 并为设计引导增长的方法提供概念框架 神经损伤后轴突的再生。

Understanding the mechanism of axonal pathfinding is a central issue in developmental neurobiology. The work outlined in this proposal is focused on the molecular and cellular mechanisms underlying the growth and navigation of nerve processes. The experimental system l will use combines in vitro study of Xenopus neurons with state-of-the-art imaging molecular manipulation techniques in the laboratory of Dr. Mu-ming Poo. The main question addressed in this proposal is how second messengers and cytoskeletaI proteins are involved in directing growth cone motility. There are two specific aims of this proposal. In AIM 1, I will determine the role of Ca2+ and cAMP as second messengers in regulating the rate and direction of neurite extension under different environmental conditions. This will be assayed by measuring [Ca2+]i at regular time intervals using the standard fura-2 ratio imaging method and measuring the rate of neurite extension by recording bright-field images using time-lapse video recording. Whether cytoplasmic Ca2+ or cAMP gradient is sufficient in inducing growth cone turning will be tested by using focal UV-activated release of Ca2+ and cAMP from caged compounds at the growth cone. Whether Ca2+ and cAMP-dependent pathways interact with each other will also be examined. In AIM 2, I will determine the early cellular events associated with the growth cone turning induced by three different chemotropic gradients (glutamate, forskolin, and NT-3). This will be accomplished by analyzing the filopodia activity, microtubule/microfilament polymerization/depolymerization and distribution of gelsolin, an actin binding protein, using combined DIC and fluorescence imaging methods. Completion of these two aims will provide insight into molecular events that precede growth cone turning. These results will both advance our understanding of neurodevelopment as well as provide conceptual framework for designing means to direct growth of regenerating axons after nerve injury.