Netrin-1 does not chemoattract GnRH neurites.

<p>(A) Schematic diagram showing the fixed source with explant set-up and definition of toward and away sides. (B) Bar graph showing the toward:away (T/A) ratio of the lengths of GnRH neurites (y-axis) as a function of treatment (x-axis). Numbers at the base of each bar indicate the number of explants used, and are from fetal brains derived from 3 independent pregnancies. Error bars indicate SEM. *, <i>P</i><0.05 with Mann-Whitney test of significance. (C) Example of explants cultured for 2 DIV in the presence of a fixed source of Netrin-1, either with (lower panel) and without (upper, control) nifedipine, and immunostained to reveal GnRH neurons and their processes. The arrow indicates the toward side (ie., the direction of the Netrin-1 source), and arrowheads indicate representative GnRH⁺ neurites. Scale bar, 40 µm. (D) Schematic drawing of turning angle analysis. Upper indicates two possible orientations in which neurites may grow as they exit the explant, either growing relatively straight (left) or turning toward the Netrin-1 source (right). i and ii indicate the two quadrants from which neurites were counted. Lower indicates how turning angle was measured, where emergence refers to the point at which the neurite exits the explant (set to 0), and termination indicates the position of the terminal swelling that demarcates the end of the neurite. (E) Bar graph showing the mean turning angle (y-axis) of GnRH neurites extending from the explant across the conditions indicated (x-axis). Error bars represent SEM. Numbers at the base of each bar indicate the number of explants used, and are from fetal brains derived from 3 independent pregnancies. Abbreviations, Netrin-1 (sym) indicates explants that were exposed to a symmetrical Netrin-1, Netrin-1 (asym) indicates explants that were exposed to asymmetrical Netrin-1 from a fixed source.