AXON TARGET INTERACTIONS IN THE DEVELOPING CEREBELLUM

这项研究将研究轴突生长锥的相互作用 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。的最新证据 我们的实验表明， 传入轴突和靶细胞调节传入神经的生长 靶细胞，以及通过调节靶细胞分化 传入神经这种关系是本提案的主题。我们将使用 发育中的小鼠小脑并分析完整的大脑和组织 培养模型，通过静态和动态显微镜。 为了说明传入神经的生长是如何受到靶细胞的影响，我们 将检查传入神经向内生长的时间与靶点的协调 体内细胞利用率。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， 纤维先于颗粒细胞到达：前庭苔藓纤维 在E15的向内生长，以及在神经学突变体曲折尾中，其中 小脑前部的颗粒细胞缺失。第二，模式 攀援纤维生长和生长锥行为在其旺盛的 生长到多个浦肯野细胞上，并在集中分支期间， 将在小脑切片中研究单个浦肯野细胞，使用 延时视频显微镜第三，在体外模型系统中， 纯化的浦肯野细胞，轴突靶向的神经特异性， 将通过分析神经突的调节来研究相互作用 适当的攀援和平行纤维的生长和分枝模式 传入和不适当的传入，静态和真实的时间 显微镜第四，在相同的组织培养模式下， 与浦肯野和颗粒细胞突触发生相关的事件，包括 传入生长锥行为，突触连接的形成，以及 将检测突触相关蛋白的表达。 细胞因子对浦肯野细胞存活和分化的调节 传入-靶和其他细胞-细胞相互作用将被研究，通过 将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内在和传入 类型这些相互作用在多大程度上调节了 还将检查靶细胞中的突触机制。 这些研究将揭示轴突靶向的时空特征 接触，以及调节传入轴突的关键细胞相互作用 目标的生长、存活和分化。根本 从完整大脑的分析中获得的信息，加上 从体外实验中获得的细胞机制的洞察力，将 对于解释神经元的新分子方法至关重要 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.