Our goal is to combine in situ nucleic acid hybridization techniques and immunocytochemical methods to study cellular mechanisms of myelin formation, breakdown and regeneration. Current studies and major findings are: (1) Localization of mRNA encoding for Po glycoprotein in sections of developing peripheral nervous tissue by in situ hybridization. Aldehyde-fixed light microscopic vibratome and semithin sections of trigeminal nerves and ganglia from 15 day old rats were treated with biotinylated Po cDNA according to procedures described by Manuelidis (1985) and Lemke and Axel (1985). Reaction product that localized Pom RNA was detected light microscopically in perikarya of myelin-forming Schwann cells. It also was found along the inner and outer margins of developing myelin sheaths. Our results show that use of a biotinylated Po probe provides more precise localization of Po mRNA than has been obtained with the use of paraffin sections and radio-labelled probes. Experiments in progress include electron microscopic studies of Po mRNA localization. (2) Intracellular Distribution of Po glycoprotein in developing Schwann cells determined by electron microscopic immunogold staining. A method described by Graeber and Kreutzberg (1985) has been modified to preserve the fine structure of myelin-forming Schwann cells in LR White-embedded developing trigeminal nerves. Thin sections have been immunostained with a polyclonal antiserum. In electron micrographs, anti-Po immunoreactivity is present on developing myelin lamellae. Further observations, including those produced with monoclonal anti-Po are in progress. (3) Quantitative morphometric study of PNS nerve fibers in the mouse mutant, shiverer. Measurements in mutants and controls have shown that myelinated nerve fibers in shiverer PNS are significantly smaller in size, more densely packed and possess thin myelin sheaths relative to axon caliber. These results suggest mild PNS immaturity, growth retardation and hypomyelination in a mutant thought previously to have essentially normal myelinated fibers in the PNS.
