Ultrastructural evidence for the lack of co-transport of B-50/GAP-43 and calmodulin in myelinated axons of the regenerating rat sciatic nerve

Summary Following peripheral nerve injury, neurons respond with synthesis of proteins required for axonal regeneration. Newly synthesized membrane proteins, like B-50/GAP-43, are transported with the fast component of anterograde axonal transport. Structural proteins and calmodulin are transported by the slow component. Since B-50/GAP-43 can bind calmodulin, 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 delivered rapidly to the distally outgrowing axons (the fast carrier hypothesis). We have investigated whether this hypothesis is valid in myelinated axons of the regenerating rat sciatic nerve. Seven days after crush, the nerve was ligated to accumulate fast transported proteins. Nerve pieces were dissected proximal to the ligation and processed for immunofluorescence and quantitative electron microscopy by postembedding single and double immunogold labelling. By light microscopy, we observed a qualitative increase in B-50/GAP-43 immunofluorescence in the axonal element immediately proximal to the nerve ligation (termed accumulated) compared to an upstream site (termed regenerating) closer to the cell body. The immunofluorescence for calmodulin appeared to be the same at both sites. Using electron microscopy, we observed that organelles had collected at the accumulated site, moreover the density of B-50/GAP-43 immunolabelling was significantly increased compared to the regenerating site, where the axoplasmic structure was undisturbed. The increase in B-50/GAP-43 immunolabelling was largely associated with vesicles. The density of calmodulin immunolabelling was similar at both sites. Approximately 25% of the total B-50/GAP-43 was associated with vesicles of which only 15% also contained labelling for calmodulin. Thus, ligation of the nerve resulted in accumulation of vesicles, including those carrying B-50/GAP-43, largely without calmodulin. 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.