NEURAL PLASTICITY IN COLLAGEN LATTICE CULTURE

提出的实验的总体目的是评估 细胞外基质（ECM）底物，其可能影响生长， 在培养物中的中枢神经系统（CNS）神经元的分化。 数据 从这个实验室显示，当生长在一个三维（3-D） 水合胶原晶格（HCL），单个鸡胚脑神经元 不与任何其他类型的细胞接触， 分化神经元的最显著特征：形态学上 可识别的轴突和树突，其快速生长并分化 随着时间的推移。 可以对指标进行定量分析 表征生长和分化。 实验将测试和 扩展这些观察的有效性。 具体目标是：（1） 建立关于生长和分化的定量基线数据， 随着时间的推移，在3D HCL中生长的单个verbetrate神经元。 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）进行分析。 轴突的长度和 每个神经元单独和组合的树突;初级突起的数量， 每个神经元的分支点、节段和终末; 轴突;将测量每个轴突是否存在静脉曲张 使用耦合到相位对比器的图像分析系统的时间周期 显微镜和微型计算机。 轴突和树突的生长速率 单独和组合，因为每个神经元的总新过程生长将是 计算了 将计算并比较每个时间段的平均值 采用方差分析或学生t检验进行统计分析。 （二） 通过实验确定重要的ECM部件是否包括a） 层粘连蛋白，B）纤连蛋白和c）神经胶质细胞，或是否CNS组织老化 改变这些特征。 3)a的定量测定 与生长和分化相关的神经元特异性产物， 使用生化ELISA方法检测神经丝（NF）蛋白，和 将其与生长和分化的定量分析相关联， 随着NF在单个神经元中的免疫细胞化学定位， 平行三维HCL培养。 4)评估分化的表达 使用形态学技术的形态特征。 本研究 应导致更好地了解调节中枢神经系统生长的因素， 在体外分化，可能是重要的中枢神经系统再生， vivo.

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.