The phenotype of developing neurons is influenced not only by selective gene expression but also by the local environment which neurons encounter during development. Given the complex interactions known to occur between glia, neurons and their target cells during development, it is possible that neurological disease results from genetic interference with the intercellular signalling. It is, therefore, of considerable importance to discover all the factors which normally influence neuronal development, and to find out how these signals work. Both in vivo and in vitro studies have revealed the nature of one of the environmental signals which influence differentiation decisions. It is a diffusable factor which is produced by certain types of non-neuronal cells, and mediates the transmitter decision of developing sympathetic neurons in culture without affecting their survival or growth. In order to understand how this factor works at the molecular level, it is essential to obtain a pure factor in quantities large enough to carry out biochemical and physiological studies necessary for understanding its mechanism of action. Progress has been made that the factor has been purified to homogeneity.
The specific aims of this study are: (1) to develop methods of obtaining the pure factor in milligram quantities by improving the current protocol of collecting conditioned medium from which the factor is purified and by introducing a more efficient method to use antibodies as immunoaffinity reagents for the purification of the factor, and (2) as a first step toward understanding its mechanism of action, to study the nature and function of its binding sites and its short-term effects on neurons. Fundamental research such as the one proposed here is essential for understanding the nature of the developmental diseases, particularly those involving interactions between the neurons and their targets.
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