Regulation of Cortical Axon Outgrowth and Branching by Calcium Activity

描述(申请人提供)：在发育中的大脑中，神经元通过将带有可移动生长锥体的轴突发送到细胞环境中来与目标细胞建立联系。这些生长锥体通过对分子信号的反应将轴突引导到适当的靶点。在哺乳动物CMS中，轴突分支的发育是轴突引导的重要方面。生长锥体中的钙信号对于决定轴突生长的速率和对指导信号的反应是重要的。树枝顶端的生长锥体暴露在不同于初级轴突感受到的环境指导信号中。值得注意的是，在体内和在培养中，轴突及其分支显示出不同的生长速度。这项在《特定目标1》中提出的工作将调查局部钙信号在调节初级轴突与其侧枝的生长速率中的作用。以发育中的啮齿动物皮质为模型系统，对培养的分离的皮质神经元进行活体细胞荧光钙成像。这种方法可以对活神经元中的钙动力学进行精确成像。在同一轴突的不同分支中直接局部应用引导因子和光释放笼状信号分子将能够精确地检测它们对初级轴突及其分支中钙动力学的影响。在具体目标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活体的模型，以进一步研究钙信号在调节发育中的哺乳动物皮质轴突生长和分支中的作用。这项研究的最终目标是研究轴突如何生长，并在发育中的大脑中分支成适当的目标。这些发育过程对于理解哺乳动物的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.