AXON-TARGET INTERACTIONS IN DEVELOPING CEREBELLUM

这项拟议的研究将研究轴突生长锥体之间的相互作用。 和它们的突触靶神经元，以解决细胞 指导和伴随突触发生的机制。最近的证据来自 我们的实验室表明，两者之间存在互惠关系。 传入轴突和靶细胞对传入神经生长的调节作用 靶细胞，以及靶细胞分化的调节 传入器。这种关系是这项提议的主题。我们将使用 发育中的小鼠小脑及完整脑组织的分析 培养模型，通过静态和动态显微镜观察。 为了说明神经传入细胞的生长如何受到靶细胞的影响，我们 将考察传入生长的时机与目标的协调 体内细胞利用度。在染料标记传入轴突后，细胞 苔藓纤维的接触将在两种情况下进行分析 纤维在颗粒细胞出现之前到达：前庭苔藓纤维 在胚胎15岁时向内生长，在神经学突变的曲折尾巴中， 小脑前部颗粒细胞缺失。第二，模式 在其旺盛时期攀升纤维生长和生长锥行为 生长到多个Purkinje细胞上，并在聚焦树枝期间 单个浦肯野细胞将在小脑切片中进行研究，使用 延时视频显微镜。第三，在体外模型系统中基于 纯化的浦肯野细胞--轴突靶标的神经特异性 将通过分析轴突的调节来研究相互作用 适宜攀缘平行纤维的生长和分枝规律 传入和不适当传入，静态和实时 显微镜。第四，使用相同的组织培养模型，细胞 与浦肯野和颗粒细胞突触发生相关的事件，包括 传入生长锥体行为，突触连接的形成，以及 突触相关蛋白的表达，将被检测。 对浦肯野细胞存活和分化的调节作用 将研究传入-靶和其他细胞-细胞相互作用，通过 纯化的浦肯野细胞与神经元和非神经元细胞共培养 小脑和其他脑细胞的固有和传入 类型。这些相互作用在多大程度上调节着 目标细胞中的突触机制也将被检查。 这些研究将描绘轴突-靶点的时间和空间特征 接触，以及调节传入轴突的关键细胞相互作用 生长、生存和分化的靶标。最基本的 从对完整大脑的分析中获得的信息，加上 从体外实验中获得的对细胞机制的洞察，威尔 对于解释神经元的新分子方法是必不可少的 发展。

This proposed research will study the interactions of axonal growth cones and their synaptic target neurons, in order to address the cellular mechanisms that direct and accompany synaptogenesis. Recent evidence from our laboratory suggests that a reciprocal relationship exists between afferent axon and target cell regulation of the growth of afferents by target cells, and the regulation of target cell differentiation by afferents. This relationship is the theme of this proposal. We will use the developing mouse cerebellum and analyze intact brain and tissue culture models, by static and dynamic microscopy. To address how the growth of afferents is influenced by target cells, we will examine the coordination of timing of afferent ingrowth with target cell availability in vivo. After dye labeling afferent axons, the cell contacts of mossy fibers will be analyzed in two situations where mossy fibers arrive before granule cells are present: vestibular mossy fiber ingrowth at E15, and in the neurological mutant meander tail, in which granule cells are missing in the anterior cerebellum. Second, the patterns of climbing fiber growth and growth cone behavior during their exuberant growth onto multiple Purkinje cells, and during focused arborization on individual Purkinje cells will be studied in slices of cerebellum, using time-lapse video microscopy. Third, in an in vitro model system based on purified Purkinje cells, the neural specificity of axon-target interactions will be investigated by analyzing the regulation of neurite growth and branching patterns of appropriate climbing and parallel fiber afferents and inappropriate afferents, with static and real time microscopy. Fourth, with the same tissue culture model, the cellular events associated with Purkinje and granule cell synaptogenesis, including afferent growth cone behavior, formation of synaptic junctions, and expression of synapse-associated proteins, will be examined. The regulation of survival and differentiation of Purkinje cells by afferent-target and other cell-cell interactions will be investigated, by co-culturing purified Purkinje cells with neurons and nonneuronal cells intrinsic and afferent to the cerebellum as well as other brain cell types. The extent to which these interactions regulate the construction of synaptic machinery in the target cell will also be examined. These studies will delineate temporal and spatial features of axon-target contacts, and the key cellular interactions that regulate afferent axonal growth and survival and differentiation of targets. The fundamental information obtained from the analysis of intact brain, coupled with the insight into cellular mechanisms obtained from in vitro experiments, will be essential to interpreting new molecular approaches to neuronal development.