Synapse withdrawal at the neuromuscular junction in a mouse with slow Wallerian degeneration (WLDs)

Motor nerve injury results in a sequence of events known as Wallerian degeneration, which takes 1 - 2 days in rodents. Wallerian degeneration is significantly delayed in the naturally occurring C57BL/Wldˢ mutant mouse. The ability to produce compound action potentials and the degradation of axonal material are delayed for up to 3 weeks following nerve injury.This thesis aims to examine the processes that occur after nerve section at neuromuscular junctions of mutant Wldˢ and normal mice. Functional morphological measurement of actively recycling synaptic vesicles at wild type nerve terminals using the vital dye FM1-43 indicated that the loss of the ability to recycle synaptic vesicles occurred within 24 hours, coinciding with disruption of neurofilament and SV2 proteins (stained using immunocytochemical methods). In contrast, the degeneration of nerve terminals in Wldˢ mice following nerve injury was significantly delayed, and once initiated was further slowed, degeneration occurring between 3 and 10 days after axotomy. The morphological appearance of Wldˢ nerve terminals after lesion differed greatly from that of degenerating wild type terminals: Wldˢ terminals appeared to be retracting from the endplate region rather than simply degenerating. Electrophysiological recordings from degenerating junctions demonstrate that the delay of degeneration/withdrawal from nerve terminals of Wldˢ mice is accompanied by a coincident loss of synaptic function. These data indicate that, as in normal mice, the first structural and functional changes to take place after nerve injury in mutant Wldˢ mice is the disruption of nerve terminals at the NMJ. This thesis proposes that nerve terminal retraction in the mutant mouse is caused by a withdrawal mechanism similar to that observed during synapse elimination, a naturally occurring activity dependant developmental phenomenon, rather than by classical Wallerian degeneration as previously assumed. To test this hypothesis, botulinum toxin (which abolishes neurotransmitter release) was administered to the nerve terminal region at the same time as the nerves were lesioned. The results indicate that nerve terminal withdrawal still occurs, but that it is reduced and further delayed in muscles treated with botulinum toxin by 1.14 days, in comparison with denervated alone Wldˢ muscles.Additional studies assessed the role of Schwann cells in nerve terminal withdrawal. Terminal Schwann cells that overly the NMJ sprout in response to denervation, and have been shown to guide axonal processes back to endplates during reinnervation. Experiments were conducted to evaluate the Schwann cell reaction to the absence of degeneration after nerve section, as observed during the first few days after axotomy in the Wldˢ mouse. Schwann cell sprouts were present at various endplates in the mutant mouse after nerve injury. However, when compared with denervated wild-type controls there were fewer Schwann cell sprouts in the Wldˢ and sprouting in general appeared less extensive. Thus, the presence of an intact nerve terminal and spontaneous activity are sufficient to delay, but not abolish, Schwann cell sprouting in the Wldˢ mouse after nerve lesion.In sum, these studies provide an ideal opportunity for the further investigation of the molecular and cellular mechanisms of synapse withdrawal