Vagotomy-induced changes in mucin levels and clock gene expression. A, Mucin levels (AB/PAS) and Muc5b gene expression (in situ hybridization) at CT4 and CT16 in the trachea of C57BL/6 mice, one week after hemivagotomy. Middle, Low-magnification photographs of the AB/PAS-stained trachea, showing the ipsilateral vagotomized [VGT(+)] side and the contralateral intact side. Top, bottom, High-magnification photographs of the AB/PAS-stained ipsilateral vagotomized side and contralateral intact side, respectively. In addition, Muc5b mRNA levels (as determined by in situ hybridization) at the ipsilateral side (top panels) and the contralateral intact side (bottom panels) are shown at high magnification. B, Tracheal PER2 protein after vagotomy at CT4 and CT16. Br, Bronchiole; Ep, tracheal epithelium; Gl, submucosal gland. The number of PER2 protein-positive nuclei in epithelial cells and glandular cells in trachea was calculated in the ipsilateral vagotomized side at CT4 (Ipsi-VGT(+)-CT4), ipsilateral vagotomized side at CT16 [Ipsi-VGT(+)-CT16], contralateral intact side at CT4 (Contra-Intact-CT4), and contralateral intact side at CT16 (Contra-Intact-CT16). Note the absence of rhythmic mPER2 expression at the vagotomized side, in contrast to the clear rhythm in contralateral intact side in both epithelial and glandular cells. Statistics was performed by Student's t test (mean ± SEM; n = 3; *p < 0.05). ns, Not significant. C, Model for the circadian control of mucin secretion in respiratory organs. Circadian expression of the muscarinic cholinergic receptor genes Chrm2, Chrm3, and Chrm4 is under control of the local peripheral circadian clock. The peripheral oscillator receives input from the central circadian oscillator in the SCN via the autonomous nervous system, in which vagal innervation is highly dominant over sympathetic innervation. Mucin production is constant, but the release of this glycoconjugate from the submucosal gland is increased at night time via increased muscarinic receptor expression and increased vagal tone. Scale bars: 100 μm.