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GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice

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Abstract

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates, but has also been reported in multiple cell types outside the CNS. A GABAergic system has been proposed in neuroepithelial bodies (NEBs) in monkey lungs. Pulmonary NEBs are known as complex intraepithelial sensory airway receptors and are part of the NEB microenvironment. Aim of the present study was to unravel a GABAergic signaling system in the NEB microenvironment in mouse lungs, enabling the use of genetically modified animals for future functional studies. Immunostaining of mouse lungs revealed that glutamic acid decarboxylase 65/67 (GAD65/67), a rate-limiting enzyme in the biosynthesis of GABA, and the vesicular GABA transporter (VGAT) were exclusively expressed in NEB cells. In GAD67-green fluorescent protein (GFP) knock-in mice, all pulmonary NEBs appeared to express GFP. For confocal live cell imaging, ex vivo vibratome lung slices of GAD67-GFP mice can be directly loaded with fluorescent functional probes, e.g. a red-fluorescent calcium dye, without the necessity of time-consuming prior live visualization of NEBs. RT-PCR of the NEB microenvironment obtained by laser microdissection revealed the presence of both GABAA and GABAB (R1 and R2) receptors, which was confirmed by immunostaining. In conclusion, the present study not only revealed the presence of a GABAergic signaling pathway, but also the very selective expression of GFP in pulmonary NEBs in a GAD67-GFP mouse model. Different proof of concept experiments have clearly shown that adoption of the GAD67-GFP mouse model will certainly boost future functional imaging and gene expression analysis of the mouse NEB microenvironment.

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Acknowledgments

This study was supported by grants of the Fund for Scientific Research-Flanders (FWO; G.0589.11 to D.A. and J-P.T.), the Hercules Foundation (AUAH-09-001 to D.A.) and the University of Antwerp (GOA BOF 2007 to D.A. and KP BOF 2011 to I.B.). We thank Prof. Dr. L.-Y. Lee (University of Kentucky, Lexington, KY, USA) for his initial help with introducing GAD67-GFP mice into our research, F. Terloo for technical assistance, D. De Rijck for help with imaging and illustrations, D. Vindevogel for aid with the manuscript, and S. Kockelberg for administrative help.

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

    Kathy Schnorbusch, Robrecht Lembrechts, Isabel Pintelon, Jean-Pierre Timmermans, Inge Brouns & Dirk Adriaensen

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  1. Kathy Schnorbusch
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  2. Robrecht Lembrechts
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  3. Isabel Pintelon
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  4. Jean-Pierre Timmermans
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  5. Inge Brouns
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  6. Dirk Adriaensen
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Correspondence to Dirk Adriaensen.

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Schnorbusch, K., Lembrechts, R., Pintelon, I. et al. GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice. Histochem Cell Biol 140, 549–566 (2013). https://doi.org/10.1007/s00418-013-1093-x

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