Precision of reinnervation and synaptic remodeling observed in neuromuscular junctions of living frogs

Repeated in vivo observations were used to study regenerated nerve terminals in neuromuscular junctions of the adult frog Rana pipiens. Sartorius junctions in living animals were stained with the fluorescent vital dye RH414 and viewed with video fluorescence microscopy. Each junction was observed in the intact muscle and then again 7, 10, and 13 weeks after nerve crush. At 13 weeks, junctions were determined to be mono- or polyneuronally innervated using intracellular recording. Between 7 and 13 weeks, most identified junctions were reinnervated less precisely and completely than described previously. Although some of the original synaptic gutters were reoccupied by regenerated terminal branches, other gutters were only partially occupied, and many appeared abandoned. Junctions showing precise recapitulation of original terminal arborizations comprised a small number of the total examined, as did those where reinnervation was very imprecise. Striking differences in the precision of reinnervation were found within the muscle such that distal terminals regenerated more precisely and completely than did proximal terminals. Terminals in reinnervated muscles were more dynamic than terminals in unoperated muscles over equivalent times. In singly innervated junctions, terminal growth was favored over regression. In doubly innervated junctions, regressive events were more common. Imprecise reinnervation is explained in terms of multisite innervation of muscle fibers and the activity dependence of synaptic stability. We hypothesize that when axons reinnervate the second or third junctions on a fiber, they do so less precisely, because the activity restored by reinnervation of the first junction renders later sites less attractive or less stable.