Dissociated sensory neurons from E11 chick embryos were cultured for 2 d in vitro and treated with either B2-ECD (control) or B4-ECD for 1 or 24 h

(A) Quantification of surface or total pools of α7* nAChR by I-αBgTx radiolabeling in sensory neurons treated with either B2-ECD (control) or B4-ECD for 24 h. In response to a 24-h B4-ECD treatment, we detected an ∼2.7-fold increase in surface I-αBgTx binding compared with control conditions (B2-ECD [control], 1,339.15 ± 329.77 cpm; and B4-ECD, 3,562.81 ± 1,111.19 cpm). B4-ECD treatment did not induce a change in total I-αBgTx binding as compared with the control (B2-ECD [control], 11,159.74 ± 1,059.79 cpm; and B4-ECD, 12,258.85 ± 580.11 cpm). The graph shows means ± SEM. Data were pooled from three independent experiments with greater than or equal to three wells per condition per experiment. Statistical significance was determined by ANOVA. *, P < 0.05 (Statview). (B) Immunoblot analysis of total α7 subunit protein in sensory neurons treated with B2-ECD (control) or B4-ECD treatment for 24 h. In response to B4-ECD treatment, we did not detect a difference in total α7 subunit protein. NF probing in bottom panel shows equivalent lysate loading. (C) Sensory neurons were treated with B2-ECD (control) or B4-ECD for 1 h. In parallel, neurons pretreated with CHX for 45 min were treated with B2-ECD or B4-ECD for 1 h. Neurons were labeled with αBgTx-488 (green), fixed, permeabilized, and colabeled for NF protein (blue). CHX treatment (c and d) did not affect either the basal number of αBgTx-488 clusters on control neurons (c) or the response to B4-ECD (d). Linescans of fluorescence intensity profiles of αBgTx-488 along representative axons (see Materials and methods) are shown. Bar, 5 μm. (D) Quantification of surface αBgTx-488 clusters along NF-labeled axons. B4-ECD treatment induced an ∼1.9-fold increase in surface αBgTx-488 clusters along axons, and B4-ECD treatment in the presence of CHX induced an ∼2.1-fold increase. 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 = 0.01; **, P < 0.0001 (Statview). (E) Quantification of surface αBgTx-488 cluster area. B4-ECD treatment in the presence or absence of CHX induced an increase in αBgTx-488 cluster area. Data pooled from three independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance was determined by the Kolmogorov-Smirnov Test. *, P ≤ 0.0001 (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

Dissociated sensory neurons from E11 chick embryos were treated with B2-ECD (control) or B4-ECD for 1 h

In parallel, neurons were pretreated with WM or an Akt inh. for 45 min before treatment with B2-ECD or B4-ECD for an additional hour. Neurons were labeled for surface α7* nAChRs with αBgTx-488 (green), fixed, permeabilized, and costained for NF protein (blue). (A) Representative micrographs of αBgTx-488 staining along NF-positive axons. B4-ECD treatment increased surface αBgTx-488 clusters (b), which did not occur in the presence of WM (d). Linescans of fluorescence intensity profiles of αBgTx-488 along representative axons (see Materials and methods) are shown. Bar, 5 μm. (B) Quantification of surface αBgTx-488 clusters along sensory neuron axons represented in A. B4-ECD treatment induced an ∼1.9-fold increase of surface αBgTx-488 clusters but not in the presence of WM or Akt inh. 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 < 0.0001 (Statview). (C) Quantification of surface αBgTx-488 cluster area. B4-ECD treatment induced an increase in αBgTx-488 cluster area but not in the presence of WM or Akt inh. Data pooled from three independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance determined by the Kolmogorov-Smirnov Test. *, P = 0.0001 (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

Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons-1

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

(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

Type III Nrg1^+/− sensory axons have reduced numbers of Nrg1⁺ punctae that contain a signaling-competent intracellular domain (Nrg1-ICD) relative to WT sensory axons.

<p>(A) Conventional microscopic images of Nrg1-ICD⁺ punctae (green) found along peripherin⁺ axons (blue) from WT and Type III Nrg1^+/− sensory cultures. Scale bars equal 10 µm. (B) Quantification of the average number of punctae per 100 µm axon length from 14 WT and 15 Type III Nrg1^+/− images. Comparison by genotype illustrates that Type III Nrg1^+/− sensory axons have significantly fewer Nrg1-ICD⁺ punctae than WT sensory axons (Mann-Whitney Rank Sum test, ***p<0.001).</p

Acute stimulation of Type III Nrg1 signaling in Type III Nrg1^+/− sensory axons does not rescue functional TRPV1 deficits.

<p>(A) Representative traces of intracellular calcium along Type III Nrg1^+/− sensory axons in response to repeated applications of 1 µM capsaicin followed by application of 56 mM KCl. The maximum percent change in intracellular calcium ([(F−F₀)/F₀]*100) in response to capsaicin decreased between the 4^th and 5^th capsaicin application in Type III Nrg1^+/− axons under control conditions (left) and did not increase when Type III Nrg1 signaling was stimulated by sErbB4-ECD application during that interval (right). (B) Quantification of percent change in maximum response to capsaicin between the 4^th and the 5^th capsaicin application ([(F₅−F₄)/F₄]*100) by treatment. Results from WT sensory axons are included for comparison (WT CON, n = 10; WT B4, n = 7; ***p<0.001). Type III Nrg1^+/− axons did not show a statistically significantly enhanced response to capsaicin when Type III Nrg1 signaling was stimulated (Type III Nrg1^+/− CON, n = 6 animals; Type III Nrg1^+/− B4, n = 8). All comparisons between genotypes and treatments were made using an ANOVA with a Holm-Sidak post-hoc test for multiple comparisons. Graph shows mean±SEM.</p

Type III Nrg1^+/− sensory axons have reduced numbers of Type III Nrg1⁺ punctae relative to WT sensory axons.

<p>(A) Conventional microscopic images of Type III Nrg1⁺ punctae (green) found along peripherin⁺ axons (blue) from WT and Type III Nrg1^+/− sensory cultures. Scale bars equal 10 µm. (B) Quantification of the average number of punctae per 100 µm axon length from 16 WT and 15 Type III Nrg1^+/− images. Comparison by genotype illustrates that Type III Nrg1^+/− sensory axons have significantly fewer Type III Nrg1⁺ punctae than WT sensory axons (Mann-Whitney Rank Sum test, *p<0.05).</p

Sensory axons, but not soma, from Type III Nrg1^+/− mice show reduced capsaicin responsiveness compared to axons from WT mice.

<p>(A) Representative traces of intracellular calcium along sensory axons in response to 1 µM capsaicin or 56 mM KCl. The change in intracellular calcium from baseline over time ([(F−F₀)/F₀]*100) is shown for WT (left) and Type III Nrg1^+/− (right) axons. Hatched diagonal lines indicate where the time course was non-continuous. (B) Quantification of the maximum change in intracellular calcium in response to application of 1 µM capsaicin or 56 mM KCl by genotype. Averages of 5 animals per genotype were compared using a Student's t-test. Type III Nrg1^+/− axons showed a significantly decreased response to capsaicin (p<0.05), but not to KCl, relative to WTs. Graph shows mean±SEM. (C) Type III Nrg1^+/− sensory soma show normal response to capsaicin. Quantification of maximal change in fluorescence from baseline ([(F−F₀)/F₀]*100) in WT or Type III Nrg1^+/− sensory neuron <i>soma</i> in response to 1 µM capsaicin or 56 mM KCl. Average responses from 4 WT and 4 Type III Nrg1^+/− animals to application of capsaicin or KCl were compared by genotype using a Student's t-test. There was no statistically significant difference between genotypes. Graphs show mean±SEM. (D) Type III Nrg1 (green) and TRPV1 (red) are co-expressed along P21 WT cultured sensory neuron axons identified with a pan-axonal (PA) marker (blue). White arrows indicate examples where Type III Nrg1 and TRPV1 are in close proximity. Scale bar equals 10 µm. (E) P21 WT and Type III Nrg1^+/− sensory neuron cultures have equivalent levels of total TRPV1 protein. Total TRPV1 protein measurement by immunoblot. The 95 kD TRPV1 band and the 35 kD GAPDH band are shown from a representative experiment comparing protein from P21 WT and Type III Nrg1^+/− cultures. Quantification of fold change in intensity of TRPV1∶GAPDH normalized to WT average. There was no statistically significant change in the ratio of TRPV1 to GAPDH between genotypes (WT, Type III Nrg1^+/−, n = 3 animals). Genotype comparisons were made using a Student's t-test. Graph shows mean±SEM.</p

Stimulation of Type III Nrg1 signaling enhances response to capsaicin in WT sensory axons.

<p>(A) Representative traces of intracellular calcium along WT sensory axons in response to repeated applications of 1 µM capsaicin followed by application of 56 mM KCl. The maximum percent change in intracellular calcium ([(F−F₀)/F₀]*100) in response to capsaicin decreased between the 4^th and 5^th capsaicin applications under control conditions (left), but increased when Type III Nrg1 signaling was stimulated by sErbB4-ECD application during that interval (right). (B) Quantification of percent change in maximum response to capsaicin between the 4^th and the 5^th capsaicin application ([(F₅−F₄)/F₄]*100) by treatment. WT sensory axons showed a significantly enhanced response to capsaicin when Type III Nrg1 signaling was stimulated with sErbB4-ECD (WT CON, n = 10 animals; WT B4, n = 7 animals; Student's t-test, ***p<0.001). Graph shows mean±SEM.</p

Adult WT and Type III Nrg1^+/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections.

<p>(A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1^+/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1^+/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1^+/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1^+/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.</p