Pulmonary neuroepithelial bodies (NEBs) express GABAergic markers: a new way of identifying NEBs for live cell imaging

Gamma-aminobutyric acid (GABA) is an important signaling molecule, both inand outside of the central nervous system (CNS), and serves a multitude of known physiological functions. GABA is synthesized from glutamic acid by a decarboxylation reaction that is catalyzed by the enzyme glutamic acid decarboxylase (GAD). In mammals, two isoforms of GAD are expressed, GAD65 and GAD67, which are encoded by two distinct genes. Recently, a GABAergic signaling system has been proposed in pulmonary „neuroepithelial bodies (NEBs)‟ in monkey lungs. Pulmonary NEBs are organizations of densely innervated groups of neuroendocrine cells present in the airway epithelium, and invariably shielded from the airway lumen by so-called Clara-like cells, all together forming a complex referred to as the NEB microenvironment [1]. At the moment, however, very little is known about the possible involvement of GABAergic signaling pathways in the lungs in health and disease. The presented study intended to further elaborate the idea using mouse models that offer more extensive possibilities for fundamental experimental research. Interest was focused on the value of models for confocal live cell imaging of the NEB microenvironment. Immunofluorescence, combined with spinning disk confocal microscopy, of lung cryostat sections of prenatal (gestational day 17-20), three-week-old and adult mice, revealed that in mouse lungs GAD was expressed in highly selective cell groups of the airway epithelium. Double staining with calcitonin gene-related peptide (CGRP), a marker for NEBs in several species including mice, identified all GAD-expressing cells as NEB cells. Multilabel immunostaining with markers for NEB-associated nerve fiber populations and Clara cells clearly demonstrated that in mouse lungs GAD is present in NEB cells only. Recently, we adopted a GAD67-green fluorescent protein (GFP) knock-in mouse that was developed to study GABAergic systems in the CNS, studied the GAD67-GFP expression and distribution in the lungs, and found distinct GAD67-GFP expressing cell groups in the airway epithelium. Double staining with CGRP showed that all CGRP-ir NEBs expressed GAD67-GFP, and vice versa. Although the functional significance of GABA synthesis in NEBs remains to be determined, the identification of GFP-fluorescent NEBs in the GAD67-GFP mouse model will certainly boost future functional NEB studies using our recently established ex vivo lung slice model for confocal molecular live cell imaging of NEBs. Support: IWT fellowship SB/81162 (R.L.); FWO grant G.0081.08 (D.A., I.B.); UA grant GOA BOF 2007 (D.A.)