Localization of neural cell adhesion molecule in denervated muscle to both the plasma membrane and extracellular...

The location of neural cell adhesion molecule in mouse skeletal muscle has been investigated using two immuno-electron microscopical techniques. In the first method, 6-micron frozen sections of a normal and a hemi-denervated gluteus muscle were stained by an indirect immunoperoxidase method using a rabbit-derived polyclonal antiserum to neural adhesion molecule as the primary antibody. The stain was visualized with the electron-dense chromogen, diaminobenzidine and the sections fixed, osmicated and processed for electron microscopy whilst still on the slide. Ultrathin sections were cut and viewed in the electron microscope, where the reaction product appeared to be localized in the plasma membrane and on the basal lamina of the muscle fibres. The second method, using a 5-nm immunogold-labelled secondary antibody, confirmed and extended these initial observations. In these experiments, the primary antibody to neural cell adhesion molecule was applied in vivo to hemi-denervated mouse gluteus muscles. The muscles were dissected out 24 h later, divided into denervated and innervated halves and then into junctional and extrajunctional regions. Together with the junctional and extrajunctional regions of normal, control pieces of muscle which had not been incubated with anti-neural cell adhesion molecule, they were lightly fixed, and incubated with the secondary gold-labelled antibody, before further fixation and processing for electron microscopy. Semi-thin, 0.5-micron sections of each were cut and viewed at 20,000 x magnification. Randomly sampled fibres from the extrajunctional regions of the denervated and innervated halves of the hemi-denervated gluteus and the normal, control gluteus were photographed for quantitative analysis. Five micrographs were randomly selected from each group and in these, the numbers, density and position of gold particles were measured using a digitizing tablet. By far the highest number of gold particles was found in the denervated half of the gluteus muscle, there being much less in the innervated half and practically none in the control. The density of gold particles in the denervated muscles was maximal in the plasma membrane-basal lamina complex, but most were located in the extracellular spaces outside this region, where they usually occurred in clusters apparently in association with collagen fibres. We conclude that neural cell adhesion molecule in denervated skeletal muscle is present not only in the plasma membrane but also in the basal lamina and extracellular space.