Morphologic and electrophysiologic identification of multiply innervated fibers in rat extraocular muscles

Mammalian extraocular muscles comprise singly and multiply innervated fibers (SIFs, MIFs) that are distributed in both the global and orbital layers of the muscles. Previously it has been concluded solely on the basis of electrophysiologic analysis that in rat, MIFs of the global layer show graded responses or slow peak potentials instead of the action potentials exhibited by SIFs. To confirm directly this conclusion, a protocol that allows the combined electrophysiologic-morphologic characterization of muscle fibers has been developed. It is based on electrophysiological analysis of the muscle fibers followed by intracellular labeling with Lucifer yellow and staining of nerve endings with a modified Koelle stain for cholinesterase activity. Labeled fibers have been recognized and followed in sequential 20-micron cross-sections of the whole muscle.