Fig 2: Reciprocal aggravation of serotonin and neurotrophin pathways via 5-HT7 activation in neurons.Human SH-SY5Y cells were stimulated with NGF or BDNF (100 ng/ml) for 48 h and the neurite length and protein levels were measured. A Representative immunostaining images of PGP9.5, TPH2, and 5-HT7 in cells with or without neurotrophin stimulation. Bar: 50 µm. B Longer nerve fibre length was observed after neurotrophin stimulation. a Average nerve fibre length and b percentage of neurons with neurite longer than 50 µm. *P < 0.05 vs. w/o. C Increased immunofluorescent intensity of (a) TPH2 and (b) 5-HT7 in neurons after neurotrophin stimulation. *P < 0.05 vs. w/o. A total of 250–300 neurons from each group were used for quantification. The bar graph represented the mean ± SEM of each group. D Transcript levels of tryptophan hydroxylases (TPH1 and TPH2) and 5-HT receptor subtypes (HTR3, HTR4, and HTR7) in neurons after neurotrophin stimulation. The neuroblastoma cells which are untreated (UT) or those differentiated with retinoic acid (RA) served as controls. *P < 0.05 vs. UT; #P < 0.05 vs. RA. N = 6/group. E Knockdown of NTRK1 gene in SH-SY5Y cells by lentiviral shRNA targeting TrkA (shTrkA) suppressed the NGF-induced TPH2 and HTR7 overexpression. The mock plasmid control is labeled as pLKO. a Gel images of semi-quantitative PCR band products showing gene silencing of NTRK1. b, c Quantitative PCR results showing reduction of TPH2 and HTR7 mRNA levels in the NTRK1-knockdown cells upon NGF stimulation. F Knockdown of NTRK2 gene by lentiviral shRNA targeting TrkB (shTrkB) decreased the BDNF-induced TPH2 and HTR7 overexpression. a Gel images of semi-quantitative PCR band products showing gene silencing of NTRK2. b, c Quantitative PCR results showing reduction of TPH2 and HTR7 mRNA levels in the NTRK2-knockdown cells upon BDNF stimulation. *P < 0.05 vs. pLKO. N = 6/group. G Neurotrophin-mediated nerve fibre elongation was inhibited by pretreatment with 5-HT7 antagonists CYY and SB7. *P < 0.05 vs. UT; #P < 0.05 vs. vehicle. A total of 250–300 neurons from each group were used for quantification. The bar graph represented the mean ± SEM of each group. H Proposed schema of a positive-feedback loop between serotonin and neurotrophin pathways via 5-HT7 activation for induction of neurite outgrowth and visceral hypersensitivity.

Fig 3: Increased mucosal nerve fibre densities were correlated with higher 5-HT7 expression in the colonic tissues of GW and PT mice.A,B Immunostaining for PGP9.5 in the colonic tissues of GW and PT mice and mice administered multiple doses (m.d.) of CYY. C, D Immunostaining for 5-HT7 in the colonic tissues of GW and PT mice and mice administered CYY (m.d.). The insets showed magnified images of the staining. Bar: 50 µm. E, F Quantification of immunoreactivity in the colonic mucosal tissues of GW and PT mice. Fluorescence intensity of (a) PGP9.5 and (b) 5-HT7 per area was quantified using an imaging software in a total of 25 images from each mouse group. *P < 0.05 vs. Ctrl or Sham; #P < 0.05 vs. GW or PT. N = 6–8/group.

Fig 4: Innervation of ameloblastomas. (A–H) Immunohistochemistry against (A–D) ßIII-Tubulin (brown color) and (E,F) PGP9.5 on ameloblastomas. Counterstaining with Toluidine Blue (blue color). Red arrowheads: neurons associated with vessels. (I–L) Double immunofluorescent staining against ßIII-Tubulin (green color) and Vimentin (red color). Blue color: DAPI. White arrowheads: ßIII-Tubulin+ Vimentin- nerve fibers; yellow arrowheads: double ßIII-Tubulin+ Vimentin+ mesenchymal cells. White boxes in (I) and (K) indicate the areas shown in (J) and (L), respectively. (M–P) Double immunofluorescent staining against neurofilament (red color) and keratins (green color). Blue color: DAPI. White arrowheads: nerve fibers in close contact with the ameloblastoma epithelium. Abbreviations: ep, epithelium; NF, neurofilament; pKrt, pan-Keratins; st, stroma; v, vessels. Scale bars: (A,E,I,K,M,N): 500 µm; (B,D,G,H,J,O,P): 100 µm; (C,L): 200 µm; (F): 400 µm.

Fig 5: TrkB signaling disruption prevents airway neuronal plasticity but has no effect on inflammation and structural remodeling. A, TrkBF616A mice were submitted to the same protocol described in Figure 2A. A solution of 20 µM of 1NMPP1 was added to the drinking water 1 week before the first challenge with saline (1NMPP1/saline) or OVA (1NMPP1/OVA). Control animals received vehicle (0.2% of DMSO) and were challenged with saline (vehicle/saline) or OVA (vehicle/OVA). B, Eosinophil number surrounding the airways normalized by the basement membrane area. C, a-SMA positive area surrounding the airways normalized by basement membrane area. D, Quantification of PGP 9.5+ and VAChT+ area surrounding the airways normalized by the basement membrane area. Representative images showing fluorescently labeled PGP 9.5 stained with Alexa Fluor 488 in green and VAChT labeled with Cy3 in red as a merged image. Scale bar 50µm. Data shown as mean ± SEM. The differences were tested for statistical significance using two-way analysis of variance (ANOVA) followed by Tukey post hoc test. *P < .05; **P < .01; ***P < .001. AHR, airway hyperresponsiveness; OVA, ovalbumin; VAChT, vesicular acetylcholine transporter; a-SMA, alpha-smooth muscle actin