Figure 6. IGF1 is a Chemoattractant for Cerebellar Granule Neuron Growth Cones In Vitro.

Growth cone turning assays were performed on cultured rat cerebellar granule neurons. Actively migrating growth cones were exposed to a gradient of IGF1 from a point source set at an initial 45 degree angle. (A) Scatter plots showing the relative angle (X-axis) and total neurite extension (Y-axis) of individual growth cones following exposure to PBS, 20µg/ml IGF1, or 200 µg/ml IGF1, as indicated. For each plot, the origin corresponds to the center of the growth cone at the beginning of the experiment, and the direction of the gradient is indicated with an arrow. (B) Cumulative distribution of turning angles showing that growth cones are attracted to a gradient of IGF1 in a dose dependent manner compared to PBS control, and that this attraction is abolished in the presence of the PI3 kinase inhibitor, LY294002. PBS control: n = 31 growth cones assayed; 20µg/ml IGF1: n = 42; 200 µg/ml IGF1: n = 35; 200 µg/ml IGF1 + 10µM LY294002: n = 26. (C) Quantitation of the data shown in panel A demonstrates that the attraction of growth cones to IGF1 is statistically significantly different as compared to control (PBS; see text for details). Inhibition of PI3 kinase with LY294002 results in a significant reduction in IGF1-mediated attraction. (D) Measurement of net extension of the same growth cones over a 30 minute period shows no effect of IGF1 on neurite outgrowth under these assay conditions. (E) Western blotting for phospho-Akt, a downstream target of IGF1R/PI3 kinase, reveals a dose-dependent accumulation of phosphorylated Akt in response to IGF1 in cerebellar granule cells (top panel). An anti-actin blot from the same gel serves as a loading control (bottom panel). Lane 1, no IGF1; lane 2, 2 ng/ml IGF1; lane 3, 20 ng/ml IGF1; lane 4, 200 ng/ml IGF1.