TROPIC FACTOR, INTEGRIN, AND CAM FUNCTIONS IN THE CNS

这项工作的总体目标是描绘神经营养的作用， 调节发育、分化和功能的因素 神经系统 在过去的几年里，我们和其他人已经产生了 导致神经营养因子缺乏的靶向突变， 神经营养因子受体 我们的实验室还培育出了 其中神经营养蛋白基因的编码区被lacZ取代， 为神经营养因子的表达提供了方便和灵敏的测定 在单个细胞内。 此外，我们还制备了功能抑制剂， 这些分子的抗体对于 免疫细胞化学研究其分布和测试其 功能协调发展的 在一个项目中，神经营养因子的作用将被检查 使用具有靶向突变的小鼠和异位表达 单个神经营养因子。 在单细胞水平上， 感觉神经节和海马中的神经营养因子将使用 小鼠的lacZ替代品系和这些分布将是 与神经营养因子受体trkA、trkB和trkC相比。 神经营养因子缺乏对神经前体细胞的影响 增殖，神经元命运决定，靶神经支配模式，和 将在感觉神经节中检查分化表型的获得 和海马体。 为了避免神经营养因子的多营养作用 缺陷，旨在针对空间缺陷的方法， 暂时将尝试。 在第一种方法中，注射人肝癌细胞的Fab片段， 将使用功能抑制性抗trkA、B或C IgG评估效应 在关键发育时期的受体抑制。 在第二种方法中， 腺病毒载体将用于表达显性阴性trk突变体， 一些神经元。 在第三种情况下，将努力产生有条件的 通过引入侧接必需trk受体外显子的loxP位点进行突变。 通过病毒或交配将cre重组酶导入转基因小鼠 菌株将用于消除神经营养因子或神经营养因子受体， 在控制时间内定义的神经元子集。 这应该能让 可以检查几个细胞缺乏的后果， 分化和/或在其他正常环境中发挥功能。 最后，我们将扩大初步观察表明，海马 神经元表达几种整合素亚基，其中一些亚基的表达是 受谷氨酸受体激活的强烈调节。 我们将努力 完整表征由以下表达的整合素亚基的库： 海马神经元 我们将研究mRNA和蛋白质的调节 表达的神经营养因子在体外和响应于 神经营养素缺乏症的基因改变的小鼠品系在体内。 对影响谷氨酸受体活化的药物表达的影响 或将检查LTP。 对这些分子的研究可能是 重要，因为他们是帮助进行长期调解的强有力的候选人 对突触活动的反应，包括发芽，收缩， 以及其他结构性变化。

The overall goal of this work is to delineate the roles of neurotrophic factors in regulating development, differention, and function of the nervous system. During the past few years, we and others have generated targeted mutations that result in deficiencies in neurotrophins or neurotrophin receptors. Our laboratory has also generated mouse strains where athe coding region of a neurotrophin gene is replaced by lacZ, providing a convenient and sensitive assay for neurotrophin expression within individual cells. In addition, we have prepared function-inhibitory antibodies to some of these molecules which have been invaluable for immunocytochemical studies of their distributions and tests of their functions. In one project, the roles of the neurotrophins will be examined using mice with targeted mutations in and mice expressing ectopically individual neurotrophins. The distributions on the single cell level of neurotrophins in sensory ganglia and the hippocampus will be examined using the lacZ replacement strains of mice and these distributions will be compared to those of the neurotrophin receptors trkA, trkB, and trkC. Consequences of neurotrophin deficiency on neuronal precursor cell proliferation, neuronal fate determination, target innervation pattern, and acquisition of differentiated phenotype will be examined in sensory ganglia and the hippocampus. To avoid pleitrophic effects of neurotrophin deficiency, methods designed to target deficiencies spatially and temporally will be attempted. In the first, injection of Fab fragments of function-inhibitory anti-trkA, B, or C IgG will be used to assess effects of receptor inhibition at key developmental times. In a second approach, Adenovirus vectors will be used to express dominant-negative trk mutants in a few neurons. In a third, efforts will be made to generate conditional mutations by introducing loxP sites flanking essential trk receptor exons. Introduction of cre recombinase by virus or mating to a transgenic mouse strain will be used to eliminate neurotrophin or neurotrophin receptor in defined subsets of neurons at controlled times. This should make it possible to examine consequences of deficiency in a few cells on differentiation and/or function in an otherwise normal environment. Finally, we will extend initial observations indicating that hippocampal neurons express several integrin subunits, expression of some of which are strongly regulated by glutamate receptor activation. We will try to completely characterize athe repertory of integrin subunits expressed by hippocampal neurons. We will examine regulation of mRNA and protein expression by the neurotrophic factors in vitro and in response to neurotrophin deficiency in genetically altered mouse strains in vivo. Effects on expression of agents that affect glutamate receptor activation or LTP will be examined. Investigation of these molecules is potentially important because they are strong candidates to help mediate longer term responses to synaptic activity, involving sprouting, retraction of processes, and other structural changes.