Fig. 7. Intra-axonal protein synthesis maintains the growth cone. To address the functional significance of intra-axonal protein synthesis, we used video microscopy to monitor changes in axons during inhibition of protein synthesis. Axons were anucleated using a glass capillary tube that had been pulled to a closed tip. Elapsed time is indicated in the top left corner of each image of a time-lapse sequence (A–E).A and B show the results of one experiment. A shows anucleated axons that were incubated for 20 min in complete medium. Scale bar, 20 μm. Thearrowhead indicates where the axon was severed with the axon coursing from left to right. Note that although the proximal portion of the axon retracts slightly (arrowheads), the distal axon remains intact. Theboxed region is shown at higher magnification in the time-lapse sequence in B. Scale bar, 10 μm. In the first two images the distal axon remains stable over 20 min. After addition of 10 μg/ml cycloheximide, the distal tip of the axon begins to retract over the 30–40 min of the time-lapse sequence (10–20 min after treatment). C shows high-magnification time-lapse sequence of a second experiment in which an axon was treated with cycloheximide immediately after severing or anucleation. Scale bar, 10 μm. Note that this anucleated axon is stable over the first 10 min but then begins to retract, similar to that seen in B. In a third series of experiments, the relevance of local protein synthesis in intact neurons was addressed by treating cultures with colchicine to decrease the influence of cell body-synthesized proteins by impeding axonal transport (D). A low-magnification time-lapse sequence of a culture preparation that was treated with 10 μg/ml colchicine for 25 min followed by 10 μg/ml cycloheximide for 25 min as indicated is shown in D. Most axonal branches were stable over the course of colchicine treatment (D, top two panels). However, during treatment with cycloheximide, many of the axonal branches retracted (D, bottom five panels). Scale bar, 60 μm. This axonal retraction required pretreatment with colchicine or axotomy because treatment of intact neurons with 10 μg/ml cycloheximide alone was without effect (E). Scale bar, 20 μm. The above series of time-lapse experiments have been repeated in three different DRG preparations in at least five neurons per preparation, using the protein synthesis inhibitor anisomycin, and yielded similar results (data not shown). To quantitate the axonal retraction in colchicine plus cycloheximide-treated neurons, 16 hr DRG cultures were treated with colchicine for 60 min (COL), cycloheximide for 30 min (CHX), or colchicine for 60 min plus cycloheximide for 30 min (COL + CHX). Control (Cntl) for these studies consisted of cultures that were allowed to grow in normal medium for 17 hr. The average length of most terminal axon branches was measured for each treatment paradigm. Axonal branch lengths in the CHX + COL were approximately half that of control, COL, or CHX paradigms (F) (average ± 2 × SEM). The differences between the COL + CHX, COL, and Cntl samples was statistically significant (p ≤ 0.0001 for COL + CHX vs Cntl, COL, and CHX samples).