Precision of Reinnervation and Synaptic Remodeling Observed in Neuromuscular Junctions of Living Frogs

Abstract

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.

Reciprocal interactions between perisynaptic Schwann cells and regenerating nerve terminals at the frog neuromuscular junction

“…Unlike mammalian NMJs, where reinnervation appears to be highly faithful to the original pattern (Rich and Lichtman, 1989), frog NMJs show a high degree of extension and retraction of NT branches during regeneration. On average, only 57% of the final NT length at an NMJ coincides with the original synaptic site (Astrow et al, 1996). Our in vivo study points strongly to PSC sprouting as the cause of this imprecision of reinnervation, which we also observed (only 65% of NT length per NMJ at 10 -12 weeks is at the original site, as calculated from Fig.…”

“…Finally, the overall geometry of regenerated NT branches at an NMJ typically mimics the geometry established earlier by PSCs and PSC sprouts. These results provide an explanation for the finding that reinnervation at frog NMJs is imprecise, in that the pattern of innervation after nerve injury is significantly altered with respect to the original pattern (Astrow et al, 1996). Unlike mammalian NMJs, where reinnervation appears to be highly faithful to the original pattern (Rich and Lichtman, 1989), frog NMJs show a high degree of extension and retraction of NT branches during regeneration.…”

“…For example, at 10 -12 weeks, 164 Ϯ 18 m of the total 468 Ϯ 29 m NT length per NMJ, or 35%, is comprised of NT sprouts. This lack of precise restoration of the original innervation pattern (Astrow et al, 1996) appears to be a consequence of PSC sprouting.…”

Qualitative and quantitative changes in acetylcholine receptor distribution at the neuromuscular junction following free muscle transfer

“…The new junction did not occupy precisely the site of the original neuromuscular junction in our study, in contrast to the observations of Rich and Lichtman,25 who held that the axons reoccupied the original endplate sites during the process of reinnervation following nerve crush. Other studies support our observation 1…”

Neuregulin Inhibits Acetylcholine Receptor Aggregation in Myotubes

“…The relative stability of the postsynaptic complex in denervated muscle is evidence for the existence of a nerve-mediated destabilizing factor (36,37). However, if a denervated myofiber is re-innervated and if the original NMJ is only partially reoccupied by the axon terminal, then the unoccupied portions of the postsynaptic apparatus become disassembled (38,39). The factor is able to destabilize AChRs in neighboring inactive regions of muscle, whereas if it is generated in active regions of muscle, it does not destabilize the AChRs there.…”