Figure 5.
Perturbations in netrin signaling alter presynaptic site and axon branch dynamics. A–D, Netrin and anti-DCC influence presynaptic site dynamics. A, The number of GFP-synaptobrevin-labeled presynaptic sites added was significantly higher in RGCs axons in tadpoles treated with netrin-1 at all observation intervals (y-axis; absolute values). The number of newly added GFP-synaptobrevin-labeled presynaptic sites, however, was decreased after anti-DCC treatment, an effect that became significant in the 8–24 h observation interval. B, A small and gradual increase in the number of stabilized presynaptic clusters was observed after netrin treatment, with the number of stabilized presynaptic sites becoming significantly higher than controls in the 8–24 h interval. C, When expressed as percentage of initial value, the number of presynaptic clusters added after netrin treatment was significantly different from controls at the 0–4 h observation interval only. This suggests that the rate of presynaptic cluster addition was rapidly increased after netrin treatment, to then be maintained at a rate that matched controls. In contrast, when compared with its initial value (percentage of total), the number of presynaptic clusters added was significantly lower in RGC axons in anti-DCC-treated tadpoles both at 4–8 and 8–24 h when compared with controls. Anti-DCC had no effect, however, on the number of GFP-synaptobrevin clusters stabilized (B). D, The rates of increase in total presynaptic site (top), and branch number (bottom), in RGC axons treated with netrin (red) relative to controls (green) are also illustrated by the line graphs. E–H, Netrin and DCC influence branch addition but not stabilization. E, Netrin-1 increased the number of branches added throughout the imaging period, an effect that was significant from 8 to 24 h. The number of branches added in RGC axons in tadpoles treated with anti-DCC, in contrast, was significantly lower at the 8–24 h observation interval when compared with controls. F, Netrin and anti-DCC did not alter the number of branches stabilized. G, H, The rates of branch addition (G), and elimination (H), were significantly higher at all observation intervals after netrin treatment and, conversely, the rates of branch addition were significantly lower after anti-DCC treatment when compared with controls (Δ4 h: 0–4 h, 4–8 h data combined). Anti-DCC did not affect the rate of branch elimination (H). *Significance with p ≤ 0.05. #Trend toward significance with 0.05 > p < 0.10. Error bars indicate SEM.