Abstract
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.
Similar content being viewed by others
References
Adriaensen D, Scheuermann DW (1993) Neuroendocrine cells and nerves of the lung. Anat Rec 236:70–85
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 293:395–405
Adriaensen D, Brouns I, Van Genechten J, Timmermans J-P (2003) Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors. Anat Rec 270A:25–40
Adriaensen D, Brouns I, Pintelon I, De Proost I, Timmermans J-P (2006) Evidence for a role of neuroepithelial bodies as complex airway sensors: comparison with smooth muscle-associated airway receptors. J Appl Physiol 101:960–970
Brouns I, Adriaensen D, Burnstock G, Timmermans J-P (2000) Intraepithelial vagal sensory nerve terminals in rat pulmonary neuroepithelial bodies express P2X3 receptors. Am J Respir Cell Mol Biol 23:52–61
Brouns I, Van Genechten J, Scheuermann DW, Timmermans J-P, Adriaensen D (2002a) Neuroepithelial bodies: a morphologic substrate for the link between neuronal nitric oxide and sensitivity to airway hypoxia? J Comp Neurol 449:343–354
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 50:575–582
Brouns I, Van Genechten J, Hayashi H, Gajda M, Gomi T, Burnstock G, Timmermans J-P, Adriaensen D (2003) Dual sensory innervation of pulmonary neuroepithelial bodies. Am J Respir Cell Mol Biol 28:275–285
Brouns I, Pintelon I, Van Genechten J, De Proost I, Timmermans J-P, Adriaensen D (2004) Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies. Histochem Cell Biol 121:1–12
Brouns I, De Proost I, Pintelon I, Timmermans J-P, Adriaensen D (2006a) Sensory receptors in the airways: neurochemical coding of smooth muscle-associated airway receptors and pulmonary neuroepithelial body innervation. Auton Neurosci 126–127:307–319
Brouns I, Pintelon I, De Proost I, Alewaters R, Timmermans J-P, Adriaensen D (2006b) Neurochemical characterisation of sensory receptors in airway smooth muscle: comparison with pulmonary neuroepithelial bodies. Histochem Cell Biol 125:351–367
Burnstock G (2007) Physiology and pathophysiology of purinergic transmission. Physiol Rev 87:659–797
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 19:605–627
Castelucci P, Robbins HL, Furness JB (2003) P2X2 purine receptor immunoreactivity of intraganglionic laminar endings in the mouse gastrointestinal tract. Cell Tissue Res 312:167–174
Cutz E, Jackson A (1999) Neuroepithelial bodies as airway oxygen sensors. Respir Physiol 115:201–214
Cutz E, Yeger H, Pan J (2007) Pulmonary neuroendocrine cell system in pediatric lung disease—recent advances. Pediatr Dev Pathol 10:419–435
Dakhama A, Kanehiro A, Mäkelä MJ, Loader JE, Larsen GL, Gelfand EW (2002) Regulation of airway hyperresponsiveness by calcitonin gene-related peptide in allergen sensitised and challenged mice. Am J Respir Crit Care Med 165:1137–1144
De Proost I, Pintelon I, Brouns I, Timmermans J-P, Adriaensen D (2007) Selective visualisation of sensory receptors in the smooth muscle layer of ex vivo airway whole mounts by styryl pyridinium dyes. Cell Tissue Res 329:421–431
De Proost I, Pintelon I, Brouns I, Kroese ABA, Riccardi D, Kemp PJ, Timmermans J-P, Adriaensen D (2008a) Functional live cell imaging of the pulmonary neuroepithelial body microenvironment. Am J Respir Cell Mol Biol 39:180–189
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)
Dinh QT, Groneberg DA, Peiser C, Joachim RA, Frossard N, Arck PC, Klapp BF, Fisher A (2005) Expression of substance P and nitric oxide synthase in vagal sensory neurons innervating the mouse airways. Regul Pept 126:189–194
Gever JR, Cockayne DA, Dillon MP, Burnstock G, Ford APDW (2006) Pharmacology of P2X channels. Pflugers Arch Eur J Physiol 453:513–537
Guembe L, Villaro AC (1999) Histochemical demonstration of neuronal nitric oxide synthase during development of mouse respiratory tract. Am.J.Respir.Cell Mol.Biol 20:342–351
Haxhiu MA, Kc P, Moore CT, Acquah SS, Wilson CG, Zaida SI, Massari VJ, Ferguson DG (2005) Brain stem excitatory and inhibitory signaling pathways regulating bronchoconstrictive responses. J Appl Physiol 98:1961–1982
Hung K-S, Loosli CG (1974) Bronchiolar neuro-epithelial bodies in the neonatal mouse lungs. Am J Anat 140:191–200
Hung K-S, Hertweck MS, Hardy JD, Loosli CG (1973) Ultrastructure of nerves and associated cells in bronchiolar epithelium of the mouse lung. J.Ultrastr.Res 43:426–437
Ichikawa H (2002) Innervation of the carotid body: immunohistochemical, denervation, and retrogade tracing studies. Microsc Res Tech 59:188–195
Kraus T, Neuhuber WL, Raab M (2007) Distribution of vesicular glutamate transporter 1 (VGLUT1) in the mouse esophagus. Cell Tissue Res 329:205–219
Lachamp P, Crest M, Kessler JP (2006) Vesicular glutamate transporters type 1 and 2 expression in axon terminals of the rat nucleus of the solitary tract. Neuroscience 137:73–81
Lauweryns JM, Van Ranst L (1987) Calcitonin gene related peptide immunoreactivity in rat lung: light and electron microscopic study. Thorax 42:183–189
Lauweryns JM, Van Ranst L (1988) Protein gene product 9.5 expression in the lungs of humans and other mammals. Immunocytochemical detection in neuroepithelial bodies, neuroendocrine cells and nerves. Neurosci Lett 85:311–316
Lauweryns JM, Cokelaere M, Theunynck P (1972) Neuroepithelial bodies in the respiratory mucosa of various mammals. A light optical, histochemical and ultrastuctural investigation. Z Zellforsch Mikrosk Anat 135:569–592
Lawrence AJ (1995) Neurotransmitter mechanisms of rat vagal afferent neurons. Clin Exp Pharmacol Physiol 22:869–873
Linnoila RI (2006) Functional facets of the pulmonary neuroendocrine system. Lab Invest 86:425–444
Llewellyn-Smith IJ, Costa M, Furness JB (1985) Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations. J Histochem Cytochem 33:857–866
Luts A, Uddman R, Absood A, Håkanson R, Sundler F (1991) Chemical coding of endocrine cells of the airways: presence of helodermin-like peptides. Cell Tissue Res 265:425–433
Ogura T, Margolskee RF, Tallini YN, Shui B, Kotlikoff MI, Lin W (2007) Immuno-localization of vesicular acetylcholine transporter in mouse taste cells and adjacent nerve fibers: indication of acetylcholine release. Cell Tissue Res 330:17–28
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 42:433–437
Pan J, Luk C, Kent G, Cutz E, Yeger H (2006) Pulmonary neuroendocrine cells, airway innervation, and smooth muscle are altered in Cftr null mice. Am J Respir Cell Mol.Biol 35:320–326
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)
Pintelon I, De Proost I, Brouns I, Van Herck H, Van Genechten J, Van Meir F, Timmermans J-P, Adriaensen D (2005) Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di–2-ASP in various animal species. Cell Tissue Res 321:21–33
Raab M, Neuhuber WL (2003) Vesicular glutamate transporter 2 immunoreactivity in putative vagal mechanosensor terminals of mouse and rat esophagus: indication of a local effector function. Cell Tissue Res 312:141–148
Raab M, Neuhuber WL (2005) Number and distribution of intraganglionic laminar endings in the mouse esophagus as demonstrated with two different immunohistochemical markers. J Histochem Cytochem 53:1023–1031
Raab M, Neuhuber WL (2007) Glutamatergic functions of primary afferent neurons with special emphasis on vagal afferents. Int Rev Cytol 256:223–275
Scheuermann DW, Adriaensen D, Timmermans J-P, De Groodt-Lasseel MH (1992) Comparative histological overview of the chemical coding of the pulmonary neuroepithelial endocrine system in health and disease. Eur J Morphol 30:101–112
Shan J, Carbonara P, Karp N, Tulic M, Hamid Q, Eidelman DH (2007) Localization and distribution of NOS1 in murine airways. Nitric Oxide 17:25–32
Shimosegawa T, Said SI (1991) Pulmonary calcitonin gene-related peptide immunoreactivity: nerve–endocrine cell interrelationships. Am J Respir Cell Mol Biol 4:126–134
Shirahata M, Balbir A, Otsubo T, Fitzgerald RS (2007) Role of acetylcholine in neurotransmission in the carotid body. Respir Physiol Neurobiol 157:93–105
Song P, Sekhon HS, Jia Y, Keller JA, Blusztajn JK, Mark GP, Spindel ER (2003) Acetylcholine is synthesized by and act as an autocrine growth factor for small cell lung carcinoma. Cancer Res 63:214–221
Sorokin SP, Hoyt RF (1989) Neuroepithelial bodies and solitary small-granule cells. In: Massaro D (ed) Lung cell biology. Marcel Dekker, New York, pp 191–344
Sorokin SP, Hoyt RF (1990) On the supposed function of neuroepithelial bodies in adult mammalian lungs. News Physiol Sci 5:89–95
Springall DR, Cadieux A, Oliveira H, Su H, Rayston D, Polak JM (1987) Retrograde tracing shows that CGRP-immunoreactive nerves of rat trachea and lung originate from vagal and dorsal root ganglia. J Auton Nerv Syst 20:155–166
Stahlman MT, Gray ME (1997) Immunogold EM localization of neurochemicals in human pulmonary neuroendocrine cells. Micros Res Tech 37:77–91
Takamori S (2006) VGLUTs: ‘Exciting’ times for glutamatergic research? Neurosci Res 55:343–351
Thompson RJ, Doran JF, Jackson P, Dhillon AP, Rode J (1983) PGP 9.5—a new marker for vertebrate neurons and neuroendocrine cells. Brain Res 278:224–228
Timmermans J-P, Adriaensen D (2008) Gastrointestinal mechanosensors: analysis of multiple stimuli may require complex sensors. Neurogastroenterol Mot 20:4–7
Van Genechten J, Brouns I, Burnstock G, Timmermans J-P, Adriaensen D (2004) Quantification of neuroepithelial bodies and their innervation in Fawn-Hooded and Wistar rat lungs. Am J Respir Cell Mol Biol 30:20–30
Van Lommel A, Lauweryns JM (1993) Neuroepithelial bodies in the Fawn Hooded rat lung: morphological and neuroanatomical evidence for a sensory innervation. J Anat 183:553–566
Van Lommel A, Lauweryns JM, Berthoud H-R (1998) Pulmonary neuroepithelial bodies are innervated by vagal afferent nerves: an investigation with in vivo anterograde Dil tracing and confocal microscopy. Anat Embryol 197:325–330
Verástegui C, Oliveira AP, Fernández-Vivero J, Romero A, de Castro JM (1997) Calcitonin gene-related peptide immunoreactivity in adult mouse lung. Eur.J.Histochem 41:119–126
Wasano K (1977) Neuroepithelial bodies in the lung of rat and mouse. Arch Histol Jpn 40:207–219
Widdicombe JG (2001) Airway receptors. Respir Physiol 125:3–15
Youngson C, Nurse C, Yeger H, Cutz E (1993) Oxygen sensing in airway chemoreceptors. Nature 365:153–155
Zhong Y, Dunn PM, Bardini M, Ford APDW, Cockayne DA, Burnstock G (2001) Changes in P2X receptor responses of sensory neurons from P2X3-deficient mice. Eur J Neurosci 14:1784–1792
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Brouns, I., Oztay, F., Pintelon, I. et al. Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs. Histochem Cell Biol 131, 55–74 (2009). https://doi.org/10.1007/s00418-008-0495-7
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-008-0495-7