(A) Dissociated sensory neurons from E11 chick embryos were treated for 5 min with B2-ECD (control), B4-ECD, 50 ng/ml NGF, or 10 ng/ml of soluble Nrg1β peptide (Nrg1-ECD)
In parallel, neurons were treated with WM for 45 min before B4-ECD stimulation (WM + B4-ECD). Neurons were fixed, permeabilized, and costained for PIP (red) and tau protein (blue) to label axons. Both B4-ECD (g) and NGF (i) treatment induced puncta of PIP along tau-positive axons. Neither B4-ECD stimulation in the presence of WM (c and h) nor that of Nrg1-ECD (e and j) induced an increase in PIP. Confocal images were obtained with a 40× oil objective. Bar, 10 μm. (B) Immunoblot analysis of phospho-Akt (Ser 473) in WT or Type III Nrg1 sensory neurons treated with either B2-ECD (control) or B4-ECD for 10 min. In WT neurons, B4-ECD treatment induced an approximately threefold increase in phospho-Akt, whereas no response was detected in mutant neurons. Total Akt in the bottom panel shows equal lysate loading. The bar graph represents phospho-Akt normalized to total Akt immunoreactive bands. Data are representative of three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.002 (Statview). (C and D) E14.5 WT (a and b) or Type III Nrg1 (c and d) DRG explants were treated with B2-ECD (control) or B4-ECD for 10 min. Surface-bound B4-ECD or B2-ECD were labeled with an antibody against the human Fc domain (anti-Fc; green) before fixation. Neurons were fixed, permeabilized, and stained for phospho-Akt (red) and NF protein (blue). B4-ECD treatment increased phospho-Akt along Fc-positive axons of WT neurons (b and D) but did not do so along axons of mutant neurons (d). Note the close proximity of anti-FC and phospho-Akt puncta in the high-power micrograph shown in e. The asterisk denotes an axon negative for both anti-Fc and phospho-Akt immunolabeling (c). A 63× oil objective was used (a–d). Confocal imaging was obtained with a 100× oil objective (D). Bar: (a–d)10 μm; (D) 5 μm. (E) Quantification of the average fluorescence intensity (AFI) of phospho-Akt along axons of WT or Type III Nrg1 sensory neurons treated with B2-ECD (control) or B4-ECD for 10 min or 1, 2, or 6 h (see Materials and methods). Along WT axons, B4-ECD treatment induced increases in phospho-Akt. Along axons of mutant neurons, we did not detect an increase in phospho-Akt in response to B4-ECD treatment. The graph shows means ± SEM. Data are from three independent experiments. Statistical significance was determined by ANOVA. *, P < 0.02.<p><b>Copyright information:</b></p><p>Taken from "Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons"</p><p></p><p>The Journal of Cell Biology 2008;181(3):511-521.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364689.</p><p></p
