The long-term goal of this research is an understanding of the events within the axon of a neuron that causes it to grow and to steer the correct course. Such an understanding is basic to an understanding of how the complex circuitry of the brain is correctly connected during development and of how some neurons regain appropriate functioning after injury. Thus the work is directly relevant to research on spinal cord injury, where paralysis results from the inability of central neurons to regenerate. It is also relevant to certain neuromuscular diseases, such as amyotrophic lateral sclerosis, whose course is markedly affected by new axon growth, in the form of collateral sprouting of the axons of motor neurons. Experiments are designed to examine events occurring in the tip of the growing axon, the growth cone, that underlie the sequence of morphological transformations in which membrane protrudes forward and matures into the axon cylinder. All of the experiments will use single giant neurons from the sea here, Aplysia californica, growing in culture. The size of these neurons will allow microinjection of large amounts of proteins. The core of the experimental approach will be the observation of living growth cones by video-enhanced differential interference contrast and flu orescence microscopy. Changes in the organization of the cytoskeleton will be monitored by the microinjection of fluorescent actin, tubulin, and actin-associated proteins into the cells. Sites of membrane addition will be identified by microinjection of fluorescent lipid and surface labeling with fluorescent lectin. Possible roles of protein phosphorylation will be probed by microapplication of protein kinase activators and inhibitors. Lastly, video microscopy will be combined with electron microscopy to probe the possible role of a large organelle (LIRB) unique to the growth cone.
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