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Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells invariably accompanied by Clara-like cells. Together with NEBs, Clara-like cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal live cell imaging of physiological reactions in identified NEBs and surrounding epithelial cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated cells, Clara cells, and Clara-like cells in live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB cells and ciliated cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intracellular free calcium concentration ([Ca2+],) in NEBs and surrounding airway epithelial cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extracellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+], increase in NEB cells, while Clara-like cells displayed a delayed (+/- 4 s) [Ca2+], increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.

Studying depolarisation induced calcium entry in our recently developed in situ lung slice model for molecular live cell imaging of selectively visualised pulmonary neuroepithelial bodies (NEBs), exemplified the need for information on the localisation of voltage-gated calcium channels (Ca(v)) in lungs in general, and related to sensory airway receptors more specifically. The present study therefore aimed at identifying the expression pattern of all major classes and subtypes of Ca(v) channels, using multiple immunostaining of rat lung cryosections. Ca(v) channel antibodies were combined with antibodies that selectively label NEBs, nerve fibre populations, smooth muscle, endothelium and Clara cells. Ca(v)2.1 (P/Q-type) was the only Ca(v) channel expressed in NEB cell membranes, and appeared to be restricted to the apical membrane of the slender NEB cell processes that reach the airway lumen. Subpopulations of the vagal but not the spinal sensory nerve fibres that contact NEBs showed immunoreactivity (IR) for Ca(v)1.2 (L-type) and Ca(v)2.1. Ca(v)2.3 (R-type) was selectively expressed by the so-called Clara-like cells that cover NEBs only, and appears to be a unique marker to discriminate this epithelial cell type from the much more extensive group of Clara cells in rat airways. The laminar nerve endings of smooth muscle-associated airway receptors (SMARs) revealed IR for both Ca(v)2.1 and Ca(v)2.2 (N-type). More generally, Ca(v)1.2 was seen to be expressed in vascular smooth muscle, Ca(v)2.3 and Ca(v)3.1 (T-type) in bronchial smooth muscle, Ca(v)3.1 and Ca(v)3.2 (T-type) in endothelial cells, and Ca(v)1.3 (L-type) in a limited number of epithelial cells. In conclusion, the present immunocytochemical study has demonstrated that the various subtypes of Ca(v) channels have distinct expression patterns in rat lungs. Special focus on morphologically/neurochemically characterised sensory airway receptors learned us that both NEBs and SMARs present Ca(v) channels. Knowledge of the identification and localisation of Ca(v) channels in airway receptors and surrounding tissues provides a solid basis for interpretation of the calcium mediated activation studied in our ex vivo lung slice model.

Today, diagnosis and treatment of chest pain related to pathologic changes in the visceral pleura are often difficult. Data in the literature on the sensory innervation of the visceral pleura are sparse. The present study aimed at identifying sensory end-organs in the visceral pleura, and at obtaining more information about neurochemical coding. The immunocytochemcial data are mainly based on whole mounts of the visceral pleura of control and vagally denervated rats. It was shown that innervation of the rat visceral pleura is characterized by nerve bundles that enter in the hilus region and gradually split into slender bundles with a few nerve fibers. Separate nerve fibers regularly give rise to characteristic laminar terminals. Because of their unique association with the elastic fibers of the visceral pleura, we decided to refer to them as "visceral pleura receptors" (VPRs). Cryostat sections of rat lungs confirmed a predominant location on mediastinal and interlobar lung surfaces. VPRs can specifically be visualized by protein gene product 9.5 immunostaining, and were shown to express vesicular glutamate transporters, calbindin D28K, Na+/K+-ATPase, and P2X3 ATP-receptors. The sensory nerve fibers giving rise to VPRs appeared to be myelinated and to have a spinal origin. Because several of the investigated proteins have been reported as markers for sensory terminals in other organs, the present study revealed that VPRs display the neurochemical characteristics of mechanosensory and/or nociceptive terminals. The development of a live staining method, using AM1-43, showed that VPRs can be visualized in living tissue, offering an interesting model for future physiologic studies.

Descriptions of morphologically well-defined sensory airway receptors are sparse, in contrast to the multiplicity of airway receptors that have been identified electrophysiologically. The present study aimed at further determining the location, morphology and neurochemical coding of subepithelial receptor-like structures that have been sporadically reported in the wall of large diameter airways. The results were compared with those obtained for pulmonary neuroepithelial bodies (NEBs), which are complex intraepithelial sensory airway receptors. Multiple immunocytochemical staining showed branching laminar subepithelial receptor-like endings, which were found to intercalate in the smooth muscle layer of intrapulmonary conducting airways in rats. Because of the consistent intimate association with the airway smooth muscle, the laminar terminals will further be referred to as 'smooth muscle-associated airway receptors (SMARs)'. SMARs were characterised by their Na(+)/K(+)-ATPase alpha3, vesicular glutamate transporter 1 (VGLUT1) and VGLUT2-immunoreactivity, expression of the ATP receptor P2X(3), and the presence of calcium-binding proteins. Nerve fibres giving rise to SMARs were shown to be myelinated and to have a vagal origin. Interestingly, the neurochemical coding and receptor-like appearance of SMARs appeared to be almost identical to at least part of the complex vagal sensory terminals in NEBs. Intraepithelial nerve endings in pulmonary NEBs were indeed also shown to originate from myelinated vagal afferent nerve fibres, and to express Na(+)/K(+)-ATPase alpha3, VGLUT1, VGLUT2, P2X(3) and calcium-binding proteins. Since several of the latter proteins have been reported as markers for mechanoreceptor terminals in other organs, both SMARs and the vagal nodose nerve terminals in NEBs seem good candidates to represent the morphological counterparts of at least subsets of the extensive population of physiologically characterised myelinated vagal airway mechanoreceptors. The observation that SMARs and NEBs are regularly found in each other's immediate neighbourhood, and the very similar characteristics of their nerve terminals, point out that the interpretation of electrophysiological data based on 'local' stimuli should be made with great caution.

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na+ channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 μM l-cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 μM amiloride. Furthermore, neither l-cis-diltiazem nor PsTx affected the β2-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

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 receptoreffector 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.

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a “reporter-patching” method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-like cells. Combined calcium imaging, pharmacology, and immunohistochemistry showed that ligand-binding to functional P2Y2 receptors underpins the activation of Clara-like cells. Hence, NEB cells communicate with their cellular neighbors in the NEB microenvironment by releasing ATP, which rapidly evokes purinergic activation of surrounding Clara-like cells. Besides ATP acting on the P2X3 receptor expressing vagal sensory nerve terminals between NEB cells, local paracrine purinergic signaling within this potential stem cell niche may be important to both normal airway function, airway epithelial regeneration after injury, and/or the pathogenesis of small cell lung carcinomas.—De Proost, I., Pintelon, I., Wilkinson, W. J., Goethals, S., Brouns, I., Van Nassauw, L., Riccardi, D., Timmermans, J.-P., Kemp, P. J., Adriaensen, D. Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging.

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.

The epithelium of intrapulmonary airways in many species harbors diffusely spread innervated groups of neuroendocrine cells, called neuroepithelial bodies (NEBs). Data on the location, morphology, and chemical coding of NEBs in mammalian lungs are abundant, but none of the proposed functions has so far been fully established. Besides C-fiber afferents, slowly adapting stretch receptors, and rapidly adapting stretch receptors, recent reviews have added NEBs to the list of presumed sensory receptors in intrapulmonary airways. Physiologically, the innervation of NEBs, however, remains enigmatic. This short overview summarizes our present understanding of the chemical coding and exact location of the receptor end organs of myelinated vagal airway afferents in intrapulmonary airways. The profuse populations that selectively contact complex pulmonary NEB receptors are compared with the much smaller group of smooth muscle-associated airway receptors. The main objective of our contribution was to stimulate the idea that the different populations of myelinated vagal afferents that selectively innervate intraepithelial pulmonary NEBs may represent subpopulations of the extensive group of known electrophysiologically characterized myelinated vagal airway receptors. Future efforts should be directed toward finding out which airway receptor groups are selectively coupled to the complex NEB receptors.

Mainly due to the lack of conclusive morphological data, correlation between functionally and morphologically defined lung receptors has so far been unsatisfactory. In the present study, multiple immunocytochemical stainings with a panel of markers for (mechanso)sensory nerve fibres were performed in order to visualise putative receptor terminals in rat intrapulmonary airways. We first focussed on determining the location, morphology and neurochemical coding of subepithelial receptor-like structures that have been sporadically reported in the wall of large diameter airways. Immunostaining with antibodies against Na+/K+-ATPase alpha3, vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 revealed branching laminar subepithelial receptor endings associated with airway smooth muscle. The latter nerve terminals appeared to further express calbindin D28k (CB), and the ATP receptor P2X3, but were calcitonin gene-related peptide (CGRP)-negative. The nerve fibres that give rise to these terminals were shown to be myelinated and have a vagal sensory origin. Because of the close association between the laminar terminals of this receptor-like structures and airway smooth muscle, we will further refer to these clearly morphologically identifiable sensory end organs as 'smooth muscle-associated airway receptors (SMARs)'. Secondly, we further explored the sensory innervation of pulmonary neuroepithelial bodies (NEBs). NEBs are intraepithelial groups of neuroendocrine cells, contacted by several nerve fibre populations, at least three of which are sensory. The spinal sensory innervation of NEBs expresses CGRP and substance P, contacts NEBs at their basal pole, and is capsaicin-sensitive. The intraepithelial vagal sensory innervation of NEBs, on the other hand, appears to be myelinated and could be labelled by antibodies against VGLUT1, VGLUT2, CB and P2X3 receptors. Na+/K+-ATPase alpha3 immunostaining additionally labelled part of the vagal sensory innervation of rat pulmonary NEBs. The neurochemical coding and receptor-like appearance of SMARs and of the complex vagal sensory innervation of NEBs appeared to be almost identical and reminiscent of mechanosensors. Both SMARs and vagal nodose nerve terminals in NEBs therefore likely represent the morphological counterparts of subgroups of the extensive population of physiologically characterised myelinated vagal airway receptors, the majority of which are mechanosensitive. Electrophysiological data based on 'local' stimuli should be interpreted with caution, because of the regular close apposition of SMARs and NEBs and the very similar characteristics of their nerve terminals.

reply: Dr. Jerry Yu is a well-respected investigator in the field of sensory airway receptor physiology, and we would like to thank him for his interest in our work and for the valuable expert comments. The Letter to the Editor ([7][1]) mainly relates to items discussed in our recent publications ([