Figure 3.

Neurite elongation and branching from peptidergic sensory neurons stimulated by BT13 in vitro is blocked by PI3K, MAPK, and Src kinase inhibitors. Neurons were isolated from lumbar (L4–L5) dorsal root ganglia taken from adult rats and cultured for 24 h. Treatment groups were vehicle (CTR), nerve growth factor (NGF: 10 ng/ml), artemin (ARTN, 30 ng/ml), BT13 (100 ng/ml), and either ARTN or BT13 with an inhibitor of PI3K (LY294002, 10 μM), MAPK (PD98059, 50 μM), or Src kinase (SU6656, 2 μM). (A) Representative images of βIII-tubulin immunofluorescence in neurons after 24 h in cultures. Image analysis of neurites was used to compare the effects of drug treatment on neurite initiation (proportion of neurons with primary neurites) and elongation (parameters: maximum length, total length, and branch points). Scale bar = 100 μm in all panels. (B) Quantification of the percentage of calcitonin gene-related peptide-positive (CGRP+ve) neurons did not detect any change in the proportions of peptidergic (CGRP+ve) and non-peptidergic (CGRP-ve) sensory neurons in cultures treated with NGF, ARTN, or BT13. In the same cultures, quantification of neurite initiation showed significant increases after treatment with NGF or ARTN, but not BT13. ^***P < 0.05 vs. CTR; ^###P < 0.001 vs. BT13, one-way ANOVA with Tukey post-hoc test, n = 4. (C) Quantification of neurite elongation showed all parameters (maximum length, total length, branch points) were significantly increased in peptidergic (CGRP+ve) sensory neurons after treatment with NGF and BT13, but not ARTN. No effect was detected in non-peptidergic (CGRP–ve) sensory neurons. BT13 had no significant effect on maximum length and total length when cultures were also treated with inhibitors of PI3K (LY294002, 10 μM, n = 3), MAPK (PD98059, 50 μM, n = 4), or Src kinase (SU6656, 2 μM, n = 6). ^**P < 0.01, ^***P < 0.001 vs. CTR; ^#P < 0.05, ^###P < 0.001 vs. inhibitor, one-way ANOVA with Tukey post-hoc test.