Release of protein from frog sciatic nerve during rapid axonal transport

An in vitro system was used to study the release of protein from frog sciatic nerve during rapid axonal transport. This preparation included the dorsal root ganglia, sciatic n erve , and gastrocnemius. It was placed in a chamber with three comparments ; each compartment was separated by a silicon grease barrier . The ganglia were incubated in 14c leucine for five hours at 20° c. in compartment A. Protein, synthesized in the ganglial, was transported down the axon in compartment B toward the muscle in compartment C. The axons in compartment B were superfused throughout the experiment with aerated Ringer solution. This solution was collected in hourly samples, dialyzed to remove any unincorporated leucine, and analyzed for the presence of released protein. A plot of the time course of the release of radiolabeled protein revealed that, at 20°, four hours after the superfusion (of compartment B) was begun, the maximum protein efflux was obtained.The peak of maximum efflux corresponds to the time required for the pulse to reach the superfused compartment. Research with cycloheximide, a known protein synthesis inhibitor, placed, during separate experiments , in compartment A and compartment B, indicated that (1) the released protein is axonal (as opposed to glial) in origin ; and ( 2) the released protein is synthesized in the cell bodies in the ganglia and transported to the axon where it is released. Experiments using temperature modifications (5°, 15°, and 20° C.) indicated that at 20°, the peak of maximum protein efflux occurred at two hours, at 15° the peak occurred at four hours, and at 5° the peak occurred at eight hours after the onset of efflux collection had begun. This further demonstrated that the protein being released from the axon depends on axonal transport for its movement from the cell body to the axon where it is released.