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 调节细胞发育、分化和功能的因素 神经系统。在过去的几年里，我们和其他人产生了 靶向突变导致神经营养因子或 神经营养素受体。我们的实验室还培育出了小鼠品系 当神经营养因子基因的编码区被LacZ取代时， 一种简便、灵敏的神经营养因子表达检测方法 在单个细胞内。此外，我们还制备了功能抑制药 其中一些分子的抗体对 它们分布的免疫细胞化学研究和它们的检测 功能。在一个项目中，将研究神经营养因子的作用。 利用靶向突变的小鼠和异位表达的小鼠 个别神经营养因子。单细胞水平上的分布 感觉神经节和海马区的神经营养因子将使用 小鼠的LacZ替代菌株和这些分布将是 与神经营养素受体TrkA、TrkB和TrkC相比。 神经营养素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，以及 分化表型的获得将在感觉神经节进行检查。 和海马体。避免神经营养素的营养不良影响 缺陷，旨在空间上针对缺陷的方法，以及 将暂时尝试。首先，注射Fab片段 将使用功能抑制抗TrkA、B或C抗体来评估疗效 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-负性trk突变体 一些神经元。在第三个阶段，将努力产生有条件的 通过在基本trk受体外显子两侧引入loxP位点而导致突变。 通过病毒或交配将cre重组酶导入转基因小鼠 菌株将被用来消除神经营养因子或神经营养因子受体 在受控时间定义神经元的子集。这应该会让它 可以在几个细胞中检查缺乏的后果 在其他正常环境中的分化和/或功能。 最后，我们将扩大初步观察表明，海马体 神经元表达几个整合素亚单位，其中一些表达 受谷氨酸受体激活的强烈调节。我们将努力 完整地描述了整合素亚基的组成 海马神经元。我们将研究mRNA和蛋白质的调节。 神经营养因子在体外的表达及对 体内转基因小鼠品系中的神经营养素缺乏。 谷氨酸受体激活剂对其表达的影响 否则将检查LTP。对这些分子的研究有可能 重要是因为他们是帮助调解更长期的有力候选者 对突触活动的反应，包括发芽、收缩 流程和其他结构性变化。

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.