In vivo visualization of the growth of pre- and postsynaptic elements of neuromuscular junctions in the mouse

In order to study how neuromuscular junctions grow, we have repeatedly viewed the same junctions in mouse sternomastoid muscles at monthly intervals from 2 weeks to 18 months of age. Motor nerve terminals were stained with the nontoxic fluorescent dye 4-Di-2-ASP (Magrassi et al., 1987), and postsynaptic ACh receptors were labeled with fluorescently tagged alpha-bungarotoxin. Neuromuscular junctions grew primarily by expansion of existing motor nerve terminal and postsynaptic receptor regions without the addition or loss of synaptic areas. The expansion of pre- and postsynaptic specializations was precisely matched, suggesting that as neuromuscular junctions grow, the opposing specializations enlarge simultaneously. Each neuromuscular junction grew in length and width at the same rate that muscle fibers enlarged in those 2 dimensions, suggesting that junctional growth might be a mechanical consequence of muscle fiber growth. Repeated visualization of ACh receptors over time showed that previously labeled receptors spread apart in the membrane occupying a progressively larger area as muscle fibers grew. At the same time, new receptors were intercalated throughout the enlarged postsynaptic area. Thus, the growth of postsynaptic regions appears to be directly related to the expansion of the muscle fiber membrane as muscle fibers grow. The maintained alignment between growing motor nerve terminals and postsynaptic regions suggests that nerve terminal growth may be a consequence of its adhesion to growing postsynaptic specializations. This conclusion is supported by the coextensive stretching of motor nerve terminals and postsynaptic regions when muscle fibers are stretched. Thus, the growth of motor nerve terminals is coupled to the growth of postsynaptic regions, and the growth of the postsynaptic regions is in turn coupled to the growth of muscle fibers. In this way, the branching pattern of neuromuscular junctions may be stably maintained despite ongoing enlargement of synaptic area.