Detection of Neuromuscular Junctions on Isolated Branched Muscle Fibers: Application of Nitric Acid Fiber Digestion Method for Scanning Electron Microscopy

Three-dimensional morphology and neuromuscular junctions of the branched muscle fibers of the rat skeletal muscles isolated from the rat were examined by application of the nitric acid digestion method for scanning electron microscopy. Treatment with fifteen percent nitric acid and a supersonic wave bath were effective in isolating the branched muscle fibers and for detecting their innervations by scanning electron microscopy. Mean numbers of total and branched fibers in the rat plantaris muscles were 11,130 +/- 314 and 32.4 +/- 4.8 (n = 12), respectively. Bifurcation of these fibers resembled either the letter X (X type) or letter Y (Y type). The Y type fibers were most frequently seen (84% of total branched fibers), and X types accounted for 14%. The remaining 2% of the fibers were subtypes of X and Y with overlapping bifurcation. These branched fibers were differentiated by their length from the bifurcated point, i.e., short, medium and long branches. Neuromuscular junctions on these branched fibers were located almost at the center of their length. The number of innervations was decided by the number of bifurcating fibers across the central part of the length.