Re labeled for surface α7* nAChRs with αBgTx-488 (green), fixed, permeabilized, and labeled for NF protein (blue). (A) Representative micrographs of axons from WT (a and b) or Type III Nrg1 (c and d) sensory neurons under control (a and c) versus B4-ECD (b and d) conditions. B4-ECD treatment increased the number of surface αBgTx-488 clusters along NF-positive processes of WT neurons (b). Linescans of fluorescence intensity profile for αBgTx-488 staining along representative axons (see Materials and methods). Bar, 5 μm. (B) Quantification of surface αBgTx-488 clusters along NF-positive axons from WT versus Type III Nrg1 DRG explants treated with either B2-ECD (control) or B4-ECD for 24 h. In WT cultures, B4-ECD treatment induced an ∼1.6-fold increase in surface αBgTx clusters along NF-positive axons compared with the control. There was no detectable change in αBgTx clusters along axons of Type III Nrg1 neurons. The graph shows means ± SEM. Data were pooled from three independent experiments. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.03; **, P < 0.001 (Statview). (C) After 2 d in vitro, dissociated sensory neurons from E11 chick embryos were treated with B2-ECD (control) or B4-ECD for 1, 2, 6, or 12 h and labeled as described in A. Axonal surface αBgTx-488 clusters were quantified. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Statview). (D) Axonal-bound B4-ECD and αBgTx-488 were detected in puncta along axons treated with B4-ECD (b, d, and e). Sensory neurons from E14.5 WT mouse embryos were cultured for 2 d in vitro and treated with B2-ECD (control) or B4-ECD for 1 h. Before fixation, surface α7* nAChRs and axonal-bound B2-ECD (control) or B4-ECD were labeled with αBgTx-488 (green) and an antibody against the human Fc domain (anti-Fc; red), respectively. c and d and e are magnifications of the areas shown in dotted squares in a and b, respectively. Bar: (a and b) 5 μm; (c–e) 1 μm. (E) Sensory neurons from E11 chick embryos were treated with B2-ECD (control), B4-ECD, or soluble Nrg1β peptide (Nrg1-ECD) for 1 h. In parallel, neurons pretreated with an ErbB tyrosine kinase inhibitor (ErbB inh.) for 45 min were treated with B2-ECD, B4-ECD, or Nrg1-ECD for 1 h. Neurons were labeled as described in A, and surface αBgTx-488 clusters along axons were quantified. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.005; **, P < 0.01 (Statview).<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
