NEURAL PLASTICITY IN COLLAGEN LATTICE CULTURE

所提出的实验的总体目的是评估 细胞外基质(ECM)底物可能影响生长和 培养的中枢神经系统(CNS)神经元的分化。数据 来自这个实验室的研究表明，当生长在三维(3-D) 水合胶原网(HCL)，鸡胚脑单个神经元 不与任何其他类型的细胞进行物理接触的情况下，快速表达 分化神经元最显著的形态特征 可识别的轴突和树突，生长迅速并分化 随着时间的推移。可以对指标进行量化分析 以生长和分化为特征。这些实验将测试和 扩大这些观察的有效性。具体目标是；1) 建立有关儿童生长和分化的量化基线数据 随着时间的推移，在3-D HCL中生长的单锥体神经元。在功能上 鸟类和哺乳动物中枢神经系统的重要区域(大脑、脊髓、 下丘脑、小脑)将被分析。轴突的长度和 每个神经元单独和合并的树突；初级突起的数量， 每个神经元的分支点、节段和终末； 轴突；静脉曲张的存在或不存在 使用耦合到相位对比度的图像分析系统的时间段 显微镜和微型计算机。轴突和树突的生长速度 单独和组合作为每个神经元的总新过程增长将是 计算出来的。将计算和比较每个时间段的平均值 统计分析采用单因素方差分析或t检验。2) 通过实验确定重要的ECM组件是否包括) 层粘连蛋白，b)纤维连接蛋白和c)神经胶质细胞，还是中枢神经系统组织的年龄 改变了这些特征。3)定量测定 与生长和分化相关的神经元特异性产物， 用生化ELISA法测定神经丝(NF)蛋白； 将其与生长和分化的定量分析相关联， 用免疫细胞化学方法研究神经营养因子在生长中单个神经元的定位 平行三维HCL培养。4)评估分化基因的表达 使用形态技术的形态特征。这项研究 应有助于更好地了解调控中枢神经系统生长的因素 在体外分化可能对中枢神经系统的再生很重要 活着。

The overall purpose of experiments proposed is to evaluate the effects of extracellular matrix (ECM) substrates which may influence growth and differentiation of central nervous system (CNS) neurons in culture. Data from this laboratory show that when grown in a three-dimensional (3-D) hydrated collagen lattice (HCL), single embryonic chick cerebral neurons not in physical contact with cells of any other kind quickly express the most distinctive feature of differentiated neurons: morphologically identifiable axons and dendrites, that grow rapidly and differentiate further over time. Quantitative analysis can be performed for indices characterizing growth and differentiation. The experiments will test and extend the validity of these observations. Specific aims are to; 1) Establish quantitative base-line data on growth and differentiation of single verbetrate neurons growing in 3-D HCL over time. Functionally important regions of the avian and mammalian CNS (cerebrum, spinal cord, hypothalamus, cerebellum) will be analyzed. The length of axons and dendrites alone and combined per neuron; the number of primary processes, branch points, segments and terminals per neuron; the maximum length of axons; the presence or absence of varicosities will be measured for each time period using an image analysis system coupled to a phase contrast microscope and microcomputer. Rates of growth for axons and dendrites alone and combined as total new process growth per neuron will be calculated. The means will be calculated and compared for each time period using ANOVA or Student's t test for statistical analysis. 2) Experimentally determine whether important ECM components including a) laminin, b) fibronectin and c) glial cells, or whether age of CNS tissue alters these characteristics. 3) Quantitatively assay for a neuron-specific product related to growth and differentiation, neurofilament (NF) protein using the biochemical ELISA method, and correlate this with quantitative analysis of growth and differentiation and with the immunocytochemical localization of NF in single neurons growing in parallel 3-D HCL culture. 4) Evaluate the expression of differentiated morphological features using morphological techniques. This research should lead to a better understanding of factors regulating CNS growth and differentiation in vitro that could be important for CNS regeneration in vivo.