The role of target muscle fibers in the maintenance of the frog motor nerve terminals.

摘要

The neuromuscular junction is the site where signals are transmitted from a nerve to a target muscle fiber. The mechanisms responsible for the maintenance of motor nerve terminals at synaptic sites are not understood. Here, I investigated the role of target-muscle fibers in the maintenance of frog motor nerve terminals. Cutaneous pectoris muscle fibers were selectively removed and prevented from regenerating while leaving the motor innervation intact. The role of target muscle fibers in nerve terminal structure and function was examined.;First, the maintenance of presynaptic activity in the absence of target was assayed with the activity-dependent dye FM1-43. I found that target-deprived nerve terminals maintain their presynaptic function of synaptic vesicle recycling for up to 5 months of target deprivation. These results indicated that the molecular machinery required for vesicular release is maintained in a functional state for long periods of target deprivation.;Second, I quantified the stability of target-deprived nerve terminals using in vivo repeated imaging. I found that most target-deprived nerve terminals were remarkably well maintained for several months after muscle fiber removal. These data indicate that the cues that confer stability to frog motor nerve terminals reside outside the muscle fibers such as in the synaptic basal lamina or terminal Schwann cells. Destabilization observed at some nerve terminals after extended target-deprivation, could result from the turning over of the stabilizing cues.;Finally, the molecular organization of target-deprived nerve terminals was analyzed. I found that the levels of two synaptic vesicle proteins, SV-2 and synaptotagmin were reduced in target-deprived nerve terminals when compared to intact neuromuscular junctions. Analysis of cytoskeletal proteins revealed that F-actin was located at discrete bands along synaptic sites that do not colocalize with synaptic vesicle clusters. F-actin is suggested to be located at either the Schwann cell processes and/or the nerve terminal immediately above them. A possible adhesion between nerve terminals and Schwann cell processes, could contribute to the maintenance of the frog nerve terminal at the synaptic site. Finally, all target-deprived synaptic sites were found to be associated with variable levels of agrin immunoreactivity, implicating agrin as a possible maintenance molecule.