Release of protein from axons during rapid axonal transport: An in vitro preparation

Abstract

An in vitro system from the frog was used to study fast axonal transport and determine if transported protein is released from the axons. This preparation included the eighth and ninth dorsal root ganglia with their roots, sciatic nerve and gastrocnemius muscle. The preparation was placed in a three-compartment chamber with each compartment separated by a silicone grease barrier. The dorsal root ganglia were incubated in [ 14C]leucine for 5 h in compartment A. The labeled protein was transported down the axon from compartment A to compartment B. The sciatic nerve in compartment B was superfused with frog Ringer. This solution was collected in hourly samples and dialyzed to remove unincorporated leucine before counting. Incubating the ganglia in 100 μg/ml cycloheximide in frog Ringer blocked the release of labeled protein from the axon. Superfusing compartment B with solution containing 100 μg/ml cycloheximide inhibited axonal and Schwann cell protein synthesis, but did not block the release of labeled protein. It was concluded that the labeled protein released into the superfusing solution was synthesized in the ganglia and transported to the axon before release. SDS acrylamide gels were used to separate the labeled proteins. Sectioning the gels in 2 mm slices and determining the radioactivity showed that 80–85% of the counts were contained in two fast moving bands.