Effects Of Denervation And Neuronal Contact On The Distribution Of Cell Surface Molecules On Single Rat Skeletal Muscle Fibers In Vitro.

Abstract

In order to investigate the molecular basis for reinnervation of adult mammalian skeletal muscle a tissue culture system was developed. Single young adult (3-5 month old) rat skeletal muscle fibers were cultured in the absence or presence of embryonic rat ventral spinal cord explants. Since basal lamina components and molecules located in the synaptic region may be important for reinnervation, initial experiments were directed towards following the effects of denervation and neural influence on the presence or absence of two well-characterized synaptic molecules: acetylcholinesterase (AChE) which is partially located in the basal lamina and acetylcholine receptors (AChRs) which are plasmalemmal proteins. These molecules persist at neuromuscular junctions for some time after denervation in vivo. Neurite-muscle fiber interactions were also examined. Observations of the frequency of neurites in junctional versus extrajunctional regions of young adult muscle fibers suggested that neurites may become stable in the junctional region over time. Functional innervation was demonstrated electrophysiologically. The extent to which AChE and AChRs were maintained at junctional regions over time depended on the muscle source. Early in culture (24 hrs) both molecules were observed at the majority of neuromuscular junctions on young adult, aged (17-25 month old) muscle fibers, and on fibers from muscles that had been denervated for 2-5 months. However, over time fewer muscle fibers maintained both synaptic molecules. Young adult and aged muscle fibers responded differently in culture. Extrajunctional AChE was observed on young adult, but not on aged, muscle fibers. Late in culture (120 hrs) more young adult muscle fibers near neurites had extrajunctional AChE compared to muscle fibers cultured alone or muscle fibers not near neurites. Disappearance of AChE and AChRs increased after 6 months of denervation, but as denervation time became longer (up to 24 months), more muscle fibers lacked AChRs than AChE. The tissue culture system is amenable to a variety of experimental techniques and should prove useful in further studies of reinnervation of adult mammalian skeletal muscle at the cellular and subcellular levels.