ULTRASTRUCTURAL EVIDENCE FOR THE LACK OF COTRANSPORT OF B-50 GAP-43 AND CALMODULIN IN MYELINATED AXONS OF THE REGENERATING RAT SCIATIC-NERVE/

Following peripheral nerve injury, neurons respond with synthesis of p roteins required for axonal regeneration. Newly synthesized membrane p roteins, Like B-50/GAP-43, are transported with the fast component of anterograde axonal transport. Structural proteins and calmodulin are t ransported by the slow component. Since B-50/GAP-43 can bind calmoduli n, it has been hypothesised that B-50/GAP-43 may act as a carrier for fast anterograde transport of calmodulin, so that both proteins are de livered rapidly to the distally outgrowing axons ('the fast carrier hy pothesis'). We have investigated whether this hypothesis is valid in m yelinated axons of the regenerating rat sciatic nerve. Seven days afte r crush, the nerve was ligated to accumulate fast transported proteins . Nerve pieces were dissected proximal to the ligation and processed f or immunofluorescence and quantitative electron microscopy by postembe dding single and double immunogold labelling. By Light microscopy, we observed a qualitative increase in B-50/GAP-43 immunofluorescence in t he axonal element immediately proximal to the nerve ligation (termed ' accumulated') compared to an upstream site (termed 'regenerating') clo ser to the cell. body. The immunofluorescence for calmodulin appeared to be the same at both sites. Using electron microscopy, we observed t hat organelles had collected at the 'accumulated' site, moreover the d ensity of B-50/GAP-43 immunolabelling was significantly increased comp ared to the 'regenerating' site, where the axoplasmic structure was un disturbed. The increase in B-50/GAP-43 immunolabelling was largely ass ociated with vesicles. The density of calmodulin immunolabelling was s imilar at both sites. Approximately 25% of the total B-50/GAP-43 was a ssociated with vesicles of which only 15% also contained labelling for calmodulin, Thus, ligation of the nerve resulted in accumulation of v esicles, including those carrying B-50/GAP-43, largely without calmodu lin. Therefore, contrary to 'the fast carrier hypothesis', the bulk of calmodulin is not co-transported with B-50/GAP-43 in myelinated axons of the sciatic nerve.