Abstract
Abstract The purpose of my study was to develop a convenient method for quantifying the growth rate the axons. This method would also be applied to study how extrinsic molecules, such as extracellular matrix molecules (ECM) and cell adhesion molecules (CAM), affected the growth of axons and to further investigate the cellular signaling paths that might involve in axonal growth. Here, we made the CAM-coated fine lines on the surface of glass coverslips for guiding the growth of axons by micro-contact printing technique. The cell bodies of dissociated rat hippocampal neurons were confined to a designated area of the same coverslip by using a stencil made from Polydimethylsinloxane (PDMS). By using this setup, I could make continual observations of the growth of the same sets of axons over periods of many days. By using this method, I found that neuroligin-1, a postsynaptically residing CAM, could enhance the growth of neuronal axons. By using a conventional method, which involved the quantifying the lengths of axons as processes positively fluorescence immunostained by an axon marker, neuroligin-1 and laminin, but not collagen was found enhance the axonal growth. The results indicate that the methodology developed herein will be a useful tool for neuroscientists while studying the issues related to the growth of axons in the future. Furthermore, I started to investigate the phosphotyrosine levels of the proteins of cultured rat hippocampal neuron grown on glass coverslip with its surface coated with laminin, poly-L-lysine or collagen. The results indicated that neurons growing on the surface coated with these different molecules exhibited a similar set of proteins, as indicated by silver-stained SDS-PAGE, and nearly identical phosphotyrosine protein patterns, as indicated by Western blotting analysis. It will be of interest in the future if such studies could be conducted on isolated axons and at different times after axons are exposed to these extracellular molecules.