Regeneration of neuromuscular connections in crayfish allotransplanted neurons

Abstract

Transplantation of whole ganglia was used to study the regeneration of four of the neurons that innervate the superficial flexor muscles of the crayfish Procambarus clarkii. The isolated ganglia containing the somas of these neurons were successfully transplanted from one crayfish to another. Reinnervation proceeded across the muscle surface and by 8 to 10 weeks connections were detected across the entire target field. At different time periods after the transplant, junction potentials (JPs) produced in phase with spontaneous neuronal spikes were recorded. The distribution of JP sizes and their decay times were examined. JPs from transplanted preparations were smaller than JPs from control or normal regeneration animals. These JPs also failed to facilitate when stimulated at 1 and 10 Hz. These are normal characteristics of immature terminals, but in the transplant preparations, once established, they remained stable for the duration of the study. Thus, synaptogenesis appears to be arrested at a stage before synaptic efficacy is established in the allotransplants. In addition, connectivity maps were plotted for each axon over the muscle surface. Some muscle fibers did not receive any contacts, and overall innervation leveled off at around 60% of the muscle fibers, remaining stable for the duration of this study. Despite the incomplete physiological innervation, however, three of the four neurons showed the same medial/lateral preferences observed in control animals, regenerating their original patterns of connectivity across the muscle surface.

Structure of allotransplanted ganglia and regenerated neuromuscular connections in crayfish

“…Reinnervation by the transplanted motoneurons was complete by 6 weeks and persisted for up to 50 weeks (Krause and Velez, 1995). As confirmation of this physiological evidence, we found in both short-and long-term transplants, well-defined nerve terminals populated with mitochondria and synaptic vesicles and making synaptic contacts with the muscle membrane [ Fig.…”

“…Six to eight weeks and 24-30 weeks after the transplant, the implanted ganglia and the superficial flexor muscles were examined with the electron microscope. The 6-8 week transplants were chosen to represent the time when regeneration of neuromuscular connections is fully established ( Krause and Velez, 1995 ), and the 28-30 week transplants were chosen to represent longterm survival of the transplanted and regenerated tissue. The condition of the transplanted tissue varied among the preparations that were examined.…”

“…Our examination of the microstructure of the transplanted neural tissue provides some clues to questions raised by an earlier physiological study (Krause and Velez, 1995) regarding how these allotransplanted ganglia remain alive and how their motoneurons are able to find and make appropriate connections with their host muscle fibers.…”

“…The technique for transplanting ganglia from donor to host crayfish was previously described in detail (Krause and Velez, 1995). Briefly, the third and fourth abdominal ganglia together with the connecting ventral nerve cord are removed from a donor crayfish.…”

“…Even more remarkable was the recent finding by Krause and Velez ( 1995) that the transplanted nerve and ganglia from a donor crayfish, when allowed to regenerate in a host crayfish, formed synapses on about 60% of the muscle fibers with a regional pattern that resembled the intact condition. The regeneration of neuromuscular contacts implies that the motoneurons survive in the transplanted ganglia, even though these ganglia are from another crayfish and are placed in a foreign location without any visible connection to the rest of the nervous system or to the circulatory system.…”