Figure 5.

Quantitative analysis of neurofilament protein accumulation proximal and distal to the constriction site after constriction for various durations and under various experimental conditions. Each column represents the mean accumulation ratio for the number of cells indicated and the error bars represent the standard deviation about the mean. An accumulation ratio >1 indicates an accumulation of neurofilament protein in the constricted axon relative to the control sister axon and an accumulation ratio <1 indicates a depletion. (A) Time course of accumulation. For the sham experiment, no constriction was performed so as to allow comparison of the normal variability between sister axons. For the other experiments, the axons were constricted for either 5 s, 30 min, or 2 h. The mean proximal accumulation ratio after 2 h was significantly greater than the corresponding distal accumulation ratio (P = 0.01, t test), and significantly greater than the proximal accumulation ratios after 5 s (P = 0.01, t test) and 30 min (P = 0.04, t test). All other accumulation ratios were not significantly different from each other. (B) Constriction in the presence of an inhibitor of glycolysis. Cells were preincubated in medium containing 2 mM sodium iodoacetate for 1 h and then constricted for 2 h in the same medium. The mean proximal accumulation ratio was significantly less than for axons constricted for 2 h in the absence of inhibitor (P = 0.02, t test). (C) Permeabilization of constricted axons with detergent to extract soluble neurofilament protein. Cells were constricted for 2 h and then extracted with 0.02% saponin before fixation as described in Materials and Methods. The mean proximal accumulation ratio was significantly greater than the corresponding distal accumulation ratio (P = 0.01, t test) and significantly greater than the proximal accumulation ratio in unextracted axons after constriction for 5 s (P = 0.01, t test).