Figure 6. Collapse of axonal terminals of cortical neurons induced by recombinant α_Vβ₃-Fc.

(A, B) Differentiated cortical neurons, 12–15 days in culture in vitro, were treated with supernatants containing α_Vβ₃-Fc fusion protein (α_Vβ₃-Fc) or with α_Vβ₃-Fc-depleted supernatants (DS), supplemented or not with Protein A. Then, neurons were fixed and immunostained for Thy-1 (A–D) and Tau protein (D) and photographed using conventional fluorescence microscopy. (B) Digital zoom was applied to marked areas (red squares in A) and indicated areas were inverted by gray-tone scaling using ImageJ. (C) Quantification of Thy-1-positive retracted axonal terminals in neurons for each condition is plotted as a percentage of total cells. NT, non-treated neurons; DS, α_Vβ₃-Fc-depleted supernatants; α_Vβ₃-Fc, α_Vβ₃-Fc-containing supernatant; PA, Protein A; α_Vβ₃-Fc+PA, α_Vβ₃-Fc-containing supernatant supplemented with Protein A (see Methods). Graphs show means+s.e.m. from at least 25 neurons per condition, n = 4. *, &, P<0.05 compared to their respective control. (D) Representative axonal terminals are shown from neurons treated with α_Vβ₃-Fc-depleted supernatants (DS); α_Vβ₃-Fc-containing supernatant (α_Vβ₃-Fc); or 1 U/ml of PI-PLC added prior addition of α_Vβ₃-Fc-containing supernatants (PI-PLC/α_Vβ₃-Fc). Arrows point to Thy-1 clusters in axon and growth cone. Asterisk indicates a retracted axonal terminal.