SIGNALS GUIDING AXONAL GROWTH AND BRANCHING

这个项目的长期目标是确定细胞外信号 以及参与轴突生长和突触发生的细胞内信号。 在这里，我们建议在体内检查视神经的生长和分化， 非洲爪蟾的神经轴突。 最近开发的技术 允许直接显微镜检查单个视神经纤维， 它们在初生树体内长成分支形成顶芽乔木 目标视顶盖 在体内观察已识别轴突的能力 提供了一个实验性的机会， 在生物学相关环境中的轴突生长。 将跟踪单个视神经纤维的生长和分支 使用微光视频显微镜和激光扫描共聚焦 显微镜;在相同的轴突荧光成像技术将被使用 测定细胞内Ca ~（2+）和cAMP的含量。 任何 生长事件与第二信使水平变化之间的相关性 将通过实验干扰第二信使， 水平或新陈代谢。 在第二个系列的实验中，我们将检查定义的 生长因子对个体视神经生长和分支的影响 纤维 BDNF有望成为相关的生长因子;我们将证实 这是通过改变视顶盖中BDNF的水平（通过增加 外源性BDNF，通过过表达或通过抑制其合成）。 平行研究将改变受体的表达。 转染 与另一种生长因子受体将用于验证任何阳性 方面的影响.

The long term goal of this project is to determine the extracellular cues and the intracellular signals involved in axonal growth and synaptogenesis. Here we propose to examine in vivo the growth and differentiation of optic nerve axons in the frog Xenopus laevis. Recently developed techniques permit direct microscopic examination of individual optic nerve fibers as they grow into and branch to form terminal arbors within their primary target, the optic tectum. The ability to observe identified axons in vivo offers the opportunity to experimentally dissect the signals that guide axonal growth in a biologically relevant setting. The growth and branching of individual optic nerve fibers will be followed using low light level video microscopy and laser scanning confocal microscopy; in the same axons fluorescent imaging techniques will be used to assay the levels of the intracellular messengers Ca2+ and cAMP. Any correlations between growth events and changes in second messenger levels will be further explored by experimentally perturbing the second messenger levels or metabolism. In a second series of experiments, we will examine the effects of defined growth factors on the growth and arborization of individual optic nerve fibers. BDNF is expected to be the relevant growth factor; we will confirm this by altering the levels of BDNF in the optic tectum (by adding exogenous BDNF, by overexpressing it or by inhabiting its synthesis). Parallel studies will alter the expression of the receptor. Transfection with another growth factor receptor will be used to validate any positive effects.