.) The long-term goal of this proposal is to determine how axotomy leads to regeneration in the CNS. Specifically, these studies will: 1) Use immunohistochemistry with phosphorylation state-dependent mAbs to determine whether there is a correlation between the level of phosphorylation of neurofilaments (NF) in an axon and its cross-sectional area. 2) Determine with EM whether NFs are spaced farther apart in large axons than in small axons. 3) Determine post-axotomy changes in CNS NF contents and in mAb labeling of neuronal perikarya and axons of various sizes. 4) Inject Lucifer Yellow into regenerating giant reticulospinal axons (GRA) and determine whether their fluorescently labeled fine sprouts express the same highly phosphorylated isoform of NF180 as the parent axon or are labeled with mAbs specific for lightly phosphorylated NFs, as are normal small diameter axons. 5) Determine whether axotomy-induced changes in NF are associated with changes in NF180 gene expression. Probes for mammalian NF will be used to screen a lamprey genomic library. The gene for NF180 will be cloned and DNA probes prepared. These will be used to determine the timecourse of changes in NF180 mRNA in brain and spinal cord following spinal transection. In Situ hybridization will then be used to identify the neurons in which expression is altered. 6) Determine whether other cytoskeletal proteins show a large change in expression in CNS following axotomy. This will be accomplished by use of existing mAbs raised to lamprey cytoskeletal proteins in combination with methods similar to that described for NF180.
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