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.
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