Regulation of Cortical Axon Outgrowth and Branching by Calcium Activity

描述（由申请人提供）：在发育中的大脑中，神经元通过将顶端带有能动生长锥的轴突发送到细胞环境中来与其靶细胞建立连接。这些生长锥通过对分子信号的反应来引导轴突到达合适的目标。轴突分支的发育是哺乳动物CMS中轴突导向的一个重要方面。生长锥中的钙信号对于决定轴突生长的速率和对引导线索的响应是重要的。树枝顶端的生长锥暴露在与初级轴突感觉不同的环境引导线索中。重要的是，在体内和在文化轴突和它们的分支表现出不同的增长率。具体目标1中提出的工作将研究局部钙信号在调节初级轴突相对于其侧支的生长速率中的作用。以发育中的啮齿类动物皮层为模型系统，对培养的皮层神经元进行活细胞荧光钙成像。这种方法可以精确成像活神经元中的钙动力学。在同一轴突的不同分支中直接局部应用引导因子和笼状信号分子的光释放将能够精确地检查它们对初级轴突及其分支中的钙动力学的影响。在特定目标2中，使用皮质切片制备和双光子荧光显微镜将这项工作扩展到更体内的模型，以进一步研究钙信号在发育中的哺乳动物皮质中调节轴突生长和分支的作用。这项研究的最终目标是研究轴突如何在发育中的大脑中生长和分支到它们合适的目标。这些发育过程对于理解哺乳动物大脑如何对损伤做出反应以及如何诱导其再生非常重要。

DESCRIPTION (provided by applicant): In the developing brain, neurons make connections with their target cells by sending axons tipped with motile growth cones into the cellular environment. These growth cones guide the axon to appropriate targets by responding to molecular cues. Development of axon branches is an important aspect of axon guidance in the mammalian CMS. Calcium signaling in the growth cone is important for determining rates of axon outgrowth and in responding to guidance cues. Growth cones at the tips of branches are exposed to environmental guidance cues different from those that the primary axon senses. Importently, in vivo and in culture axons and their branches exhibit different rates of outgrowth. The work proposed in Specific Aim 1 will investigate the role of localized calcium signaling in regulating the rates of outgrowth of primary axons versus their collateral branches. Using the developing rodent cortex as a model system, I will carry out live cell fluorescence calcium imaging of dissociated cortical neurons in culture. This approach permits precise imaging of calcium dynamics in living neurons. Direct local application of guidance factors and photorelease of caged signaling molecules in different branches of the same axon will enable precise examination of their effects on calcium dynamics in the primary axon ans its branches. In Specific Aim 2, the use of a cortical slice preparation and two-photon fluorescence microscopy will extend this work to a more in vivo model to further investigate the role of calcium signaling in regulating axon growth and branching in the developing mammalian cortex. The ultimate goal of this research is to study how axons grow and branch into their appropriate targets in the developing brain. These developmental processes are important in understanding how the mammalian brain responds to injury, and how it may be induced to regenerate.