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，神经元通过将带有活动生长锥的轴突发送到细胞环境中与目标细胞建立联系。这些生长锥通过响应分子信号引导轴突到达适当的目标。在哺乳动物CMS中，轴突分支的发育是轴突引导的一个重要方面。生长锥中的钙信号对于确定轴突生长速率和对引导信号的响应是重要的。树枝顶端的生长锥暴露在不同于原始轴突感知的环境引导信号中。重要的是，在体内和培养中，轴突及其分支表现出不同的生长速率。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与其侧支的生长速率中的作用。我将以发育中的啮齿动物皮层为模型系统，对培养的游离皮质神经元进行活细胞荧光钙成像。这种方法允许对活神经元中的钙动态进行精确成像。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信号分子，可以精确地检查它们对初级轴突及其分支钙动力学的影响。在Specific Aim 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多的体内模型，以进一步研究钙信号在哺乳动物皮层发育中调节轴突生长和分支的作用。这项研究的最终目的是研究轴突是如何在发育中的大脑中生长和分支到合适的目标。这些发育过程对于理解哺乳动物大脑如何对损伤作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.