Skip to main content
SpringerLink
Log in

Selective visualisation of sensory receptors in the smooth muscle layer of ex-vivo airway whole-mounts by styryl pyridinium dyes

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

Recently, we established the location, morphology and neurochemical coding of vagal smooth-muscle-associated airway receptors (SMARs) in rat lungs. These receptors were characterised as branching laminar terminals that originated from myelinated nerve fibres and were intercalated between airway smooth-muscle bundles. To allow the direct physiological examination of these receptors, the present investigation aimed at visualising SMARs in airway whole-mounts of rat and mouse lungs ex vivo. Short incubation with various styryl pyridinium dyes (AM1-43, FM2-10, FM4-64 or 4-Di-2-ASP) gave a highly selective fluorescent visualisation of both laminar nerve terminals and myelinated fibres from which they originated throughout the intrapulmonary airway tree in mouse and in rat. The reliable and specific labelling of SMARs ex vivo with these lipophilic membrane dyes was confirmed via immunostaining for protein gene-product 9.5 and vesicular glutamate transporters. Similar to the intrapulmonary location of NEBs, these SMARs appeared to be even more explicitly located near airway bifurcations. Both the trachealis muscle and the smooth-muscle bundles of extrapulmonary bronchi were also shown to contain laminar nerve terminals that were morphologically similar to the SMARs reported in the intrapulmonary airways. Thus, this study provides an in-vitro model enabling, for the first time, the fast and reliable visualisation of SMARs and the myelinated nerve fibres from which they originate in airway whole-mount preparations ex vivo. As such, this model opens up further perspectives and creates a valid basis for direct physiological measurement and manipulation of the individually identified airway receptors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Adcock JJ (2002) Airway nerves: in vitro electrophysiology. Curr Opin Pharmacol 2:280–282

    Article  PubMed  CAS  Google Scholar 

  • Adriaensen D, Scheuermann DW (1993) Neuroendocrine cells and nerves of the lung. Anat Rec 236:70–85

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Avadhanam KP, Plopper CG, Pinkerton KE (1997) Mapping the distribution of neuroepithelial bodies of the rat lung. A whole-mount immunohistochemical approach. Am J Pathol 150:851–859

    PubMed  CAS  Google Scholar 

  • Berthoud H-R, Patterson LM, Neumann F, Neuhuber WL (1997) Distribution and structure of vagal afferent intraganglionic laminar endings (IGLEs) in the rat gastrointestinal tract. Anat Embryol 195:183–191

    Article  PubMed  CAS  Google Scholar 

  • Brouns I, Van Nassauw L, Van Genechten J, Majewski M, Scheuermann DW, Timmermans J-P, Adriaensen D (2002) 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Brumback AC, Lieber JL, Angleson JK, Betz WJ (2004) Using FM1-43 to study neuropeptide granule dynamics and exocytosis. Methods 33:287–294

    Article  PubMed  CAS  Google Scholar 

  • Canning BJ, Mori N, Mazzone SB (2006) Vagal afferent nerves regulating the cough reflex. Respir Physiol Neurobiol 152:223–242

    PubMed  Google Scholar 

  • Cho T, Chan W, Cutz E (1989) Distribution and frequency of neuro-epithelial bodies in post-natal rabbit lung: quantitative study with monoclonal antibody against serotonin. Cell Tissue Res 255:353–362

    Article  PubMed  CAS  Google Scholar 

  • Cochilla AJ, Angleson JK, Betz WJ (1999) Monitoring secretory membrane with FM1-43 fluorescence. Annu Rev Neurosci 22:1–10

    Article  PubMed  CAS  Google Scholar 

  • Fu XW, Nurse CA, Wang YT, Cutz E (1999) Selective modulation of membrane currents by hypoxia in intact airway chemoreceptors from neonatal rabbit. J Physiol (Lond) 514:139–150

    Article  CAS  Google Scholar 

  • Fu XW, Nurse CA, Wong V, Cutz E (2002) Hypoxia-induced secretion of serotonin from intact pulmonary neuroepithelial bodies in neonatal rabbit. J Physiol (Lond) 539:503–510

    Article  CAS  Google Scholar 

  • Fukuda J, Ishimine H, Masaki Y (2003) Long-term staining of live Merkel cells with FM dyes. Cell Tissue Res 311:325–332

    PubMed  Google Scholar 

  • Kannari K, Sato O, Maeda T, Iwanaga T, Fujita T (1991) A possible mechanism of mechanoreception in Ruffini endings in the peridontal ligament of hamster incisors. J Comp Neurol 313:368–376

    Article  PubMed  CAS  Google Scholar 

  • Larson SD, Schelegle ES, Hyde DM, Plopper CG (2003) The three-dimensional distribution of nerves along the entire intrapulmonary airway tree of the adult rat and the anatomical relationship between nerves and neuroepithelial bodies. Am J Respir Cell Mol Biol 28:592–599

    Article  PubMed  CAS  Google Scholar 

  • Lauweryns JM, Peuskens JC (1972) Neuro-epithelial bodies (neuroreceptor or secretory organs?) in human infant bronchial and bronchiolar epithelium. Anat Rec 172:471–481

    Article  PubMed  CAS  Google Scholar 

  • Mazzone SB (2004) Sensory regulation of the cough reflex. Pulm Pharmacol Ther 17:361–368

    Article  PubMed  CAS  Google Scholar 

  • Mazzone SB (2005) An overview of the sensory receptors regulating cough. Cough 1:2

    Article  PubMed  Google Scholar 

  • Mazzone SB, Canning BJ (2003) Identification of the afferent nerves mediating cough in guinea pigs. FASEB J 17:A822

    Google Scholar 

  • Meyers JR, MacDonald RB, Duggan A, Standaert DG, Corwin JT, Corey DP (2003) Lighting up the senses: FM1-43 loading of sensory cells through nonselective ion channels. J Neurosci 23:4054–4065

    PubMed  CAS  Google Scholar 

  • Phillips RJ, Powley TL (2000) Tension and stretch receptors in gastrointestinal smooth muscle: re-evaluating vagal mechanoreceptor physiology. Brain Res Rev 34:1–26

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Ryan TA (2001) Presynaptic imaging techniques. Curr Opin Neurobiol 11:544–549

    Article  PubMed  CAS  Google Scholar 

  • Scheuermann DW (1987) Morphology and cytochemistry of the endocrine epithelial system in the lung. Int Rev Cytol 106:35–88

    Article  PubMed  CAS  Google Scholar 

  • Smiley-Jewell SM, Tran MU, Weir AJ, Johnson ZA, Van Winkle LS, Plopper CG (2002) Three-dimensional mapping of smooth muscle in the distal conducting airways of mouse, rabbit, and monkey. J Appl Physiol 93:1506–1514

    PubMed  CAS  Google Scholar 

  • Sorokin SP, Hoyt RF (1989) Neuroepithelial bodies and solitary small-granule cells. In: Massaro D (ed) Lung cell biology. Dekker, New York, pp 191–344

    Google Scholar 

  • Weichselbaum M, Everett AW, Sparrow MP (1996) Mapping the innervation of the bronchial tree in fetal and postnatal pig lung using antibodies to PGP9.5 and SV2. Am J Respir Cell Mol Biol 15:703–710

    PubMed  CAS  Google Scholar 

  • Widdicombe JG (2001) Airway receptors. Respir Physiol 125:3–15

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The skilful technical assistance of G. Svensson is gratefully acknowledged. We are especially grateful to J. Van Daele and D. De Rijck for help with microscopy, imaging and illustrations, to D. Vindevogel for aid with the manuscript and to S. Kockelberg and H. De Pauw for administrative assistance.

Author information

Authors and Affiliations

  1. Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Groenenborgerlaan 171, BE-2020, Antwerp, Belgium

    Ian De Proost, Isabel Pintelon, Inge Brouns, Jean-Pierre Timmermans & Dirk Adriaensen

Authors
  1. Ian De Proost
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Isabel Pintelon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Inge Brouns
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean-Pierre Timmermans
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dirk Adriaensen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dirk Adriaensen.

Additional information

This work was supported by the Fund for Scientific Research-Flanders (G.0155.01 and G.0085.04 to D.A.) and by NOI-BOF 2003 (to D.A.) and KP-BOF 2006 (to I.B.) from the University of Antwerp.

Rights and permissions

Reprints and permissions

About this article

Cite this article

De Proost, I., Pintelon, I., Brouns, I. et al. 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 (2007). https://doi.org/10.1007/s00441-007-0431-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-007-0431-5

Keywords

Search

Navigation