Age-related remodeling of neuromuscular junctions on type-identified diaphragm fibers

Previous studies have reported fiber-type differences in the morphological adaptations of neuromuscular junctions (NMJs) to aging by comparing limb muscles consisting of predominantly type I or II fibers. A confounding factor in these studies is age-related change in activity, which may differ between muscles. In the present study, we assessed age-related changes of the NMJ in type-identified fibers of the rat diaphragm muscle, which maintains consistent inspiratory-related activation throughout life. In 6- and 24-month-old rats, a fluorescent triple-labeling technique was used to visualize phrenic axons, presynaptic nerve terminals, and postsynaptic acetylcholine receptors (end-plates) on type-identified fibers. The NMJs were then imaged using three-dimensional (3D) confocal microscopy. On type IIx and IIb fibers, nerve terminal and end-plate 2D planar and 3D surface areas expanded, and the number of nerve terminal and end-plate branches increased, indicating fragmentation of the NMJ with aging. On the other hand, NMJs on type I and IIa fibers displayed little adaptation. These morphological adaptations may be geared toward maintaining the efficacy of inspiratory-related activity of the diaphragm muscle, but may affect the functional reserve of the aging diaphragm.