Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs
- 642 Downloads
As best characterized for rats, it is clear that pulmonary neuroepithelial bodies (NEBs) are contacted by a plethora of nerve fiber populations, suggesting that they represent an extensive group of multifunctional intraepithelial airway receptors. Because of the importance of genetically modified mice for functional studies, and the current lack of data, the main aim of the present study was to achieve a detailed analysis of the origin and neurochemical properties of nerve terminals associated with NEBs in mouse lungs. Antibodies against known selective markers for sensory and motor nerve terminals in rat lungs were used on lungs from control and vagotomized mice of two different strains, i.e., Swiss and C57-Bl6. NEB cells were visualized by antibodies against either the general neuroendocrine marker protein gene-product 9.5 (PGP9.5) or calcitonin gene-related peptide (CGRP). Thorough immunohistochemical examination of NEB cells showed that some of these NEB cells also exhibit calbindin D-28 k (CB) and vesicular acetylcholine transporter (VAChT) immunoreactivity (IR). Mouse pulmonary NEBs were found to receive intraepithelial nerve terminals of at least two different populations of myelinated vagal afferents: (1) Immunoreactive (ir) for vesicular glutamate transporters (VGLUTs) and CB; (2) expressing P2X2 and P2X3 ATP receptors. CGRP IR was seen in varicose vagal nerve fibers and in delicate non-vagal fibers, both in close proximity to NEBs. VAChT immunostaining showed very weak IR in the NEB-related intraepithelial vagal sensory nerve terminals. nNOS- or VIP-ir nerve terminals could be observed at the base of pulmonary NEBs. While a single NEB can be contacted by multiple nerve fiber populations, it was clear that none of the so far characterized nerve fiber populations contacts all pulmonary NEBs. The present study revealed that mouse lungs harbor several populations of nerve terminals that may selectively contact NEBs. Although at present the physiological significance of the innervation pattern of NEBs remains enigmatic, it is likely that NEBs are receptor–effector end-organs that may host complex and/or multiple functional properties in normal airways. The neurochemical information on the innervation of NEBs in mouse lungs gathered in the present study will be essential for the interpretation of upcoming functional data and for the study of transgenic mice.
KeywordsSensory airway receptors Myelinated vagal afferents C-fibers ATP receptors Glutamate NEBs Airways
This work was supported by the following research grants: Fund for Scientific Research-Flanders (G.0085.04 and G.0081.08 to D.A.), NOI-BOF 2003 and GOA-BOF 2007 (to D.A.), and KP-BOF 2006 (to I·B.) from the University of Antwerp. We are grateful to Prof. G. Burnstock (Royal Free & University College Medical School) for his invaluable input in the ATP receptor studies, and acknowledge J. Van Genechten for his help with the study of the nitrergic innervation of mouse NEBs. We thank R. Spillemaeckers, G. Svensson, F. Terloo, S. De Geyter and G. Vermeiren for technical assistance, J. Van Daele and D. De Rijck for help with microscopy, imaging and illustrations, D. Vindevogel for aid with the manuscript, and H. De Pauw and S. Kockelberg for secretarial help.
- Adriaensen D, Timmermans J-P, Brouns I, Berthoud HR, Neuhuber WL, Scheuermann DW (1998) Pulmonary intraepithelial vagal nodose afferent nerve terminals are confined to neuroepithelial bodies: an anterograde tracing and confocal microscopy study in adult rats. Cell Tissue Res 293:395–405PubMedCrossRefGoogle Scholar
- Brouns I, Van Nassauw L, Van Genechten J, Majewski M, Scheuermann DW, Timmermans J-P, Adriaensen D (2002b) Triple immunofluorescence staining method with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors. J Histochem Cytochem 50:575–582PubMedGoogle Scholar
- Cadieux A, Springall DR, Mulderry PK, Rodrigo J, Ghatei MA, Terenghi G, Bloom SR, Polak JM (1986) Occurrence, distribution and ontogeny of CGRP immunoreactivity in the rat lower respiratory tract: effect of capsaicin treatment and surgical denervations. Neuroscience 19:605–627PubMedCrossRefGoogle Scholar
- De Proost I, Pintelon I, Wilkinson WJ, Goethals S, Brouns I, Van Nassauw L, Riccardi D, Timmermans J-P, Kemp PJ, Adriaensen D (2008b) ATP released from pulmonary neuroepithelial bodies activates Clara-like cells in the NEB microenvironment via P2Y2 receptors. FASEB J 22: 929.4 (Abstract)Google Scholar
- Gever JR, Cockayne DA, Dillon MP, Burnstock G, Ford APDW (2006) Pharmacology of P2X channels. Pflugers Arch Eur J Physiol 453:513–537Google Scholar
- Negoescu A, Labat-Moleur F, Lorimier P, Lamarq L, Guillermet C, Chambaz E, Brambilla E (1994) F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence. J Histochem Cytochem 42:433–437PubMedGoogle Scholar
- Pintelon I, Brouns I, Van Genechten J, Scheuermann DW, Timmermans J-P, Adriaensen D (2003) Pulmonary expression of the vesicular acetylcholine transporter with special reference to neuroepithelial bodies. Auton Neurosci 106:47 (Abstract)Google Scholar
- Sorokin SP, Hoyt RF (1989) Neuroepithelial bodies and solitary small-granule cells. In: Massaro D (ed) Lung cell biology. Marcel Dekker, New York, pp 191–344Google Scholar
- Sorokin SP, Hoyt RF (1990) On the supposed function of neuroepithelial bodies in adult mammalian lungs. News Physiol Sci 5:89–95Google Scholar
- Timmermans J-P, Adriaensen D (2008) Gastrointestinal mechanosensors: analysis of multiple stimuli may require complex sensors. Neurogastroenterol Mot 20:4–7Google Scholar