Figure 3.
Blocking glycosphingolipid biosynthesis reverses MAG-mediated inhibition of neurite outgrowth. Rat CGNs were treated without (control, A) or with (B) the glycosphingolipid biosynthetic inhibitor P4 (2.5 μM) for 48 h. Cell surface complex gangliosides were detected by neuraminidase treatment followed by labeling with FITC-cholera toxin B-subunit as described in Materials and Methods. Fluorescent images of the cholera B-subunit-labeled cultures (A and B) demonstrate nearly complete depletion of complex cell surface gangliosides after P4 treatment. In separate experiments, cells were cultured on MAG-containing inhibitory substrata in the absence (control, C) or presence of 2.5 μM P4 (D). After 48 h, cells were immunostained for GAP-43 and visualized by Cy3-conjugated secondary Ab to detect neurites [images are presented as the reverse grayscale (black on white) for clarity], revealing enhanced neurite outgrowth by P4-treated neurons. Neurite outgrowth inhibition, quantified as described in the legend to Fig. 2, is presented as the mean ± SD of at least 3 independent experiments (E). *, P < 0.005. (Bars = 50 μm.) Equivalent reversal of neurite outgrowth inhibition by P4 treatment was quantified with semiautomated image analysis (see Fig. 7).