Figure 4

Fig. 4. Quantitative assessment of the movement of bleached zones. The rate of the movement of the center of the bleached zone relative to the substrate is plotted as a function of neurite elongation rate. (A) Neurites growing on laminin-coated substrate. The average rate of neurite extension was 59 ± 7 µm/hour (mean ± sem, n = 18). In 11 experiments the bleached zone was located at the distal axonal segment (open circles) and in 7 experiments at the proximal segment (filled squares). The average rate of bleached zone movement was 32 ± 5 µm/hour (mean ± sem). In each experiment the accuracy of the measurements of the positions of growth cone and bleached zone was about 1 µm. Detection of the MT translocation rate was more accurate for neurites with relatively stable MTs (slow fluorescence recovery). Generally the bleached zone could be reliably traced for a period of 10-20 min and therefore the accuracy of the measurements was about 3-6 µm/hour. In 7 experiments we were not able to measure reliably the rate of bleached zone movement. In 2 out of these 7 cases the bleached zone was visible only for a few minutes (t1/2 about 3 min) which precluded accurate measurements of the MT movement. In the remaining 5 cases the lateral movement of the whole axonal structure was very fast and the measurements of the bleached zone position were meaningless. Results of these 7 experiments have been excluded from the analysis of MT movement. (B) Neurites growing on Con A-coated substrate. Summary of 25 different experiments. In 5 experiments (filled triangles) neurotrophic factors NT-3, BDNF and CNTF (50 ng/ml each) were added to the culture medium during cell culture preparation and were present throughout the experiment. No neurotrophic factors were added to the culture medium in the remaining 20 experiments (open circles). The bleached zone was at the distal axonal segment in 18 experiments and at the proximal segment in 7 experiments. In the absence of NT-3 in the culture medium the average rate of axonal growth and the rate of bleached zone movement were 25.6 ± 3.7 µm/hour and -0.4 ± 0.5 µm/hour, respectively (mean ± sem, n = 20). In the presence of NT-3 the rates of axonal growth and the bleached zone movement were 55 ± 8 µm/hour and 1.4 ± 1.2 µm/hour, respectively (mean ± sem, n = 5). The position of the bleached zone could be measured with an accuracy of about 1 µm. Typically the movement of the bleached zone was followed for 30 - 60 min. Therefore we estimate the accuracy of the measurements of the MT movement rate to be about 1 - 2 µm/hour.