Selective gene expression analysis of the neuroepithelial body microenvironment in postnatal lungs with special interest for potential stem cell characteristics.

BACKGROUND: The pulmonary neuroepithelial body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. METHODS: A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. RESULTS: Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. CONCLUSIONS: This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular.

Directed Migration of Pulmonary Neuroendocrine Cells toward Airway Branches Organizes the Stereotypic Location of Neuroepithelial Bodies.

The airway epithelium consists of diverse cell types, including neuroendocrine (NE) cells. These cells are thought to function as chemoreceptors and as a component of the stem cell niche as well as the cells of origin in small-cell lung cancer. NE cells often localize at bifurcation points of airway tubes, forming small clusters called neuroepithelial bodies (NEBs). To investigate NEB development, we established methods for 3D mapping and ex vivo 4D imaging of developing lungs. We found that NEBs localize at stereotypic positions in the bifurcation area irrespective of variations in size. Notch-Hes1 signaling contributes to the differentiation of solitary NE cells, regulating their number but not localization. Live imaging revealed that individual NE cells migrate distally to and cluster at bifurcation points, driving NEB formation. We propose that NEB development is a multistep process involving differentiation of individual NE cells and their directional migration to organize NEBs.

Epithelial Morphogenesis: Stage Diving with Purpose.

In a new paper in the October 8(th) issue of Cell, Kuo and Krasnow (2015) report a previously undescribed mechanism for cell sorting and reveal a dynamic, daredevil behavior of epithelial cells.

Formation of a Neurosensory Organ by Epithelial Cell Slithering.

Epithelial cells are normally stably anchored, maintaining their relative positions and association with the basement membrane. Developmental rearrangements occur through cell intercalation, and cells can delaminate during epithelial-mesenchymal transitions and metastasis. We mapped the formation of lung neuroepithelial bodies (NEBs), innervated clusters of neuroendocrine/neurosensory cells within the bronchial epithelium, revealing a targeted mode of cell migration that we named "slithering," in which cells transiently lose epithelial character but remain associated with the membrane while traversing neighboring epithelial cells to reach cluster sites. Immunostaining, lineage tracing, clonal analysis, and live imaging showed that NEB progenitors, initially distributed randomly, downregulate adhesion and polarity proteins, crawling over and between neighboring cells to converge at diametrically opposed positions at bronchial branchpoints, where they reestablish epithelial structure and express neuroendocrine genes. There is little accompanying progenitor proliferation or apoptosis. Activation of the slithering program may explain why lung cancers arising from neuroendocrine cells are highly metastatic.

Immunohistochemical characterization of the chemosensory pulmonary neuroepithelial bodies in the naked mole-rat reveals a unique adaptive phenotype.

The pulmonary neuroepithelial bodies (NEBs) constitute polymodal airway chemosensors for monitoring and signaling ambient gas concentrations (pO2, pCO2/H+) via complex innervation to the brain stem controlling breathing. NEBs produce the bioactive amine, serotonin (5-HT), and a variety of peptides with multiple effects on lung physiology and other organ systems. NEBs in mammals appear prominent and numerous during fetal and neonatal periods, and decline in the post-natal period suggesting an important role during perinatal adaptation. The naked mole-rat (NMR), Heterocephalus glaber, has adapted to the extreme environmental conditions of living in subterranean burrows in large colonies (up to 300 colony mates). The crowded, unventilated burrows are environments of severe hypoxia and hypercapnia. However, NMRs adjust readily to above ground conditions. The chemosensory NEBs of this species were characterized and compared to those of the conventional Wistar rat (WR) to identify similarities and differences that could explain the NMR's adaptability to environments. A multilabel immunohistochemical analysis combined with confocal microscopy revealed that the expression patterns of amine, peptide, neuroendocrine, innervation markers and chemosensor component proteins in NEBs of NMR were similar to that of WR. However, we found the following differences: 1) NEBs in both neonatal and adult NMR lungs were significantly larger and more numerous as compared to WR; 2) NEBs in NMR had a more variable compact cell organization and exhibited significant differences in the expression of adhesion proteins; 3) NMR NEBs showed a significantly greater ratio of 5-HT positive cells with an abundance of 5-HT; 4) NEBs in NMR expressed the proliferating cell nuclear antigen (PCNA) and the neurogenic gene (MASH1) indicating active proliferation and a state of persistent differentiation. Taken together our findings suggest that NEBs in lungs of NMR are in a hyperactive, functional and developmental state, reminiscent of a persistent fetal state that extends postnatally.

[Pulmonary neuroepithelial bodies in the rat].

This paper describes the study of the neuroepithelial bodies (NEB) in the lungs of adult healthy Wistar rats (n = 12). Using the immunocytochemical reaction demonstrating synaptophysin, NEB and immunopositive nerve terminals approaching them, were visualized. It was found that NEB were the structures constantly presented in the rat lung. In contrast to the diffuse neuroendocrine elements, NEB are characterized by grouped distribution of cells. It was found that some part of NEB had no efferent innervation.

Bromodeoxyuridine (BrdU)-label-retaining cells in mouse terminal bronchioles.

Adult male mice were continuously treated with bromodeoxyuridine (BrdU) for 1, 2, or 4 weeks by an osmotic pump. To detect BrdU-label-retaining cells (LRCs), putative progenitor/stem cells, other animals were continuously treated with BrdU for 2 weeks, and were then kept without any treatments for 2, 6, or 18 months. The lungs were fixed with 4% paraformaldehyde, and were paraffin-embedded. We observed terminal bronchioles with BrdU immunostaining alone or with BrdU immunostaining accompanying immunostaining for Clara cell secretory protein (CCSP), forkhead box protein J1 (FoxJ1), or calcitonin gene-related peptide (CGRP). The average incidences of BrdU-incorporated cells in the terminal bronchioles after 1, 2, and 4 weeks of continuous BrdU infusion were 6.2%, 11.9%, and 23.1%, respectively. Most BrdU-incorporated cells in these periods were CCSP-immunoreactive (91.7%, 91.3%, and 88.2%, respectively), which means progenitor function of Clara cells. FoxJ1-immunoreactive BrdU-incorporated cells were fewer (5.4%, 3.0%, 2.7%, respectively). The average incidences of BrdU-LRCs in the terminal bronchioles after 2, 6, and 18 months were 7.2%, 4.3, and 2.7%, respectively. Most BrdU-LRCs were CCSP-immunoreactive (91.0%, 92.7%, and 89.6%, respectively), and FoxJ1-immunoreactive BrdU-LRCs were fewer (6.0%, 5.7%, and 2.1%, respectively). CGRP-positive BrdU-incorporated cells were occasional. CGRP-positive BrdU-LRCs were detected in 17.6% of neuroepithelial bodies (NEBs) at 2 months, but disappeared at 6 months. BrdU-positive stem cell candidates, which locate at the brochiolo-alveolar duct junction or cover NEB, were few throughout this study. In conclusion, in the lungs treated only with BrdU, CCSP-immunoreactive cells are important to maintain homeostasis in the terminal bronchiolar epithelium.

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

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.

Precision-cut vibratome slices allow functional live cell imaging of the pulmonary neuroepithelial body microenvironment in fetal mice.

We recently developed an ex vivo lung slice model that allows for confocal live cell imaging (LCI) of neuroepithelial bodies (NEBs) in postnatal mouse lungs (postnatal days 1-21 and adult). NEBs are morphologically well-characterized, extensively innervated groups of neuroendocrine cells in the airway epithelium, which are shielded from the airway lumen by 'Clara-like' cells. The prominent presence of differentiated NEBs from early embryonic development onwards, strongly suggests that NEBs may exert important functions during late fetal and neonatal life. The main goal of the present study was to adapt the current postnatal LCI lung slice model to enable functional studies of fetal mouse lungs (gestational days 17-20).In vibratome lung slices of prenatal mice, NEBs could be unequivocally identified with the fluorescent stryryl pyridinium dye 4-Di-2-ASP. Changes in the intracellular free calcium concentration and in mitochondrial membrane potential could be monitored using appropriate functional fluorescent indicators (e.g. Fluo-4).It is clear that the described fetal mouse lung slice model is suited for LCI studies of Clara cells, ciliated cells, and the NEB microenvironment, and offers excellent possibilities to further unravel the significance of NEBs during the prenatal and perinatal period.

Neuroepithelial body microenvironment is a niche for a distinct subset of Clara-like precursors in the developing airways.

Clara cells of mammalian airways have multiple functions and are morphologically heterogeneous. Although Notch signaling is essential for the development of these cells, it is unclear how Notch influences Clara cell specification and if diversity is established among Clara cell precursors. Here we identify expression of the secretoglobin Scgb3a2 and Notch activation as early events in a program of secretory cell fate determination in developing murine airways. We show that Scgb3a2 expression in vivo is Notch-dependent at early stages and ectopically induced by constitutive Notch1 activation, and also that in vitro Notch signaling together with the pan-airway transcription factor Ttf1 (Nkx2.1) synergistically regulate secretoglobin gene transcription. Furthermore, we identified a subpopulation of secretory precursors juxtaposed to presumptive neuroepithelial bodies (NEBs), distinguished by their strong Scgb3a2 and uroplakin 3a (Upk3a) signals and reduced Ccsp (Scgb1a1) expression. Genetic ablation of Ascl1 prevented NEB formation and selectively interfered with the formation of this subpopulation of cells. Lineage labeling of Upk3a-expressing cells during development showed that these cells remain largely uncommitted during embryonic development and contribute to Clara and ciliated cells in the adult lung. Together, our findings suggest a role for Notch in the induction of a Clara cell-specific program of gene expression, and reveals that the NEB microenvironment in the developing airways is a niche for a distinct subset of Clara-like precursors.

Novel insights in the neurochemistry and function of pulmonary sensory receptors.

Afferent nerves in the airways and lungs contribute to optimisation of the breathing pattern, by providing local pulmonary information to the central nervous system. Airway sensory nerve terminals are consequently tailored to detect changes readily in the physical and chemical environment, thereby leading to a variety of respiratory sensations and reflex responses. Most intrapulmonary nerve terminals arise from fibres travelling in the vagal nerve, allowing a classification of "sensory airway receptors", based on their electrophysiologically registered action potential characteristics. Nowadays, at least six subtypes of electrophysiologically characterised vagal sensory airway receptors have been described, including the classical slowly and rapidly adapting (stretch) receptors and C-fibre receptors. The architecture of airways and lungs makes it, however, almost impossible to locate functionally the exact nerve terminals that are responsible for transduction of a particular intrapulmonary stimulus. With the advances in immunohistochemistry in combination with confocal microscopy, airway sensory receptor end organs can now be examined and evaluated objectively. Based on their "neurochemical coding", morphology, location and origin, three sensory receptor end organs are currently morphologically well characterised: smooth muscle-associated airway receptors (SMARs), neuroepithelial bodies (NEBs) and visceral pleura receptors (VPRs). The present information on the functional, morphological and neurochemical characteristics of these sensory receptors leads to important conclusions about their (possible) function. Currently, ex vivo lung models are developed that allow the selective visualisation of SMARs, NEBs and VPRs by vital staining. The described ex vivo models will certainly facilitate direct physiological studies of the morphologically and neurochemically identified airway receptors, thereby linking morphology to physiology by identifying in situ functional properties of a given receptor end organ.

Recruitment of GABA(A) receptors in chemoreceptor pulmonary neuroepithelial bodies by prenatal nicotine exposure in monkey lung.

Pulmonary neuroepithelial bodies (NEB) act as airway oxygen sensors and produce serotonin, a variety of neuropeptides and are involved in autonomic nervous system control of breathing, especially during the neonatal period. We now report that NEB cells also express a GABAergic signaling loop that is increased by prenatal nicotine exposure. In this study, cultured monkey NEB cells show hypoxia-evoked action potentials and hypoxia-sensitive K(+) current. As shown by both immunofluorescence and RT-PCR, monkey NEB cells synthesize and contain serotonin. The monkey NEB cells express the beta2 and beta3 GABA_A receptor subunits, GAD and also express alpha7, alpha4 and beta4 nicotinic receptor (nAChR) subunits. The alpha7 nAChR is co-expressed with GAD in NEB. The numbers of NEB and beta3 GABA_A receptor subunits expressed in NEB cells in lungs from control newborn monkeys were compared to lungs from animals that received nicotine during gestation. Prenatal nicotine exposure increased the numbers of NEB by 46% in lung and the numbers of NEB cells expressing GAD and GABA_A beta3 receptors increased by 67% and 66%, respectively. This study suggests that prenatal nicotine exposure can modulate NEB function by increasing the numbers of NEB cells and by increasing both GAD expression and beta3 GABA_A receptor subunit expression. The interaction of the intrinsic GABAergic system in the lung with nicotinic receptors in PNEC/NEB may provide a mechanism to explain the link between smoking during pregnancy and SIDS.

Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs.

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 P2X(2) and P2X(3) 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.

Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging.

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 P2Y(2) 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 P2X(3) 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.

Pulmonary neuroendocrine cell system in pediatric lung disease-recent advances.

The airway epithelium of human and animal lungs contains highly specialized pulmonary neuroendocrine cells (PNEC), distributed as solitary cells and as innervated clusters, neuroepithelial bodies (NEB). The designation "PNEC system" stems from the expression of both neural and endocrine cell phenotypes, including the synthesis and release of amine (serotonin, 5-HT) and a variety of neuropeptides (that is, bombesin). The role and function of PNEC in the lung have remained a subject of speculation for many years. During the last decade, studies using modern techniques of cellular and molecular biology revealed a complex functional role for PNEC, beginning during the early stages of lung development as modulators of fetal lung growth and differentiation and at the time of birth as airway O2 sensors involved in neonatal adaptation. Postnatally and beyond, PNEC/NEB are providers of a lung stem cell niche that is important in airway epithelial regeneration and lung carcinogenesis. The focus of this review is to present and discuss recent findings pertaining to the responses of PNEC to intrauterine environmental stimuli, ontogeny and molecular regulation of PNEC differentiation, innervation of NEB, and their role as airway chemoreceptors, including mechanisms of O2 sensing and chemotransmission of hypoxia stimulus. Abnormalities of PNEC/NEB have been reported in a variety of pediatric pulmonary disorders but the clinical significance or the mechanisms involved are unknown. The discussion on the possible role of PNEC/NEB in the pathogenesis and pathobiology of pediatric lung diseases includes congenital lung disorders, bronchopulmonary dysplasia, disorders of respiratory control, neuroendocrine hyperplasia of infancy, cystic fibrosis, bronchial asthma, and pulmonary hypertension.

Sensory receptors in the airways: neurochemical coding of smooth muscle-associated airway receptors and pulmonary neuroepithelial body innervation.

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.

Functional facets of the pulmonary neuroendocrine system.

Pulmonary neuroendocrine cells (PNECs) have been around for 60 years in the scientific literature, although phylogenetically they are ancient. Their traditionally ascribed functions include chemoreception and regulation of lung maturation and growth. There is recent evidence that neuroendocrine (NE) differentiation in the lung is regulated by genes and pathways that are conserved in the development of the nervous system from Drosophila to humans (such as achaete-scute homolog-1), or implicated in the carcinogenesis of the nervous or NE system (such as the retinoblastoma tumor suppressor gene). In addition, complex neural networks are in place to regulate chemosensory and other functions. Even solitary PNECs appear to be innervated. For the first time ever, we have mouse models for lung NE carcinomas, including the most common and virulent small cell lung carcinoma. Moreover, PNECs may be important for inflammatory responses, and pivotal for lung stem cell niches. These discoveries signify an exciting new era for PNECs and are likely to have therapeutic and diagnostic applications.

Neurochemical characterisation of sensory receptors in airway smooth muscle: comparison with pulmonary neuroepithelial bodies.

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.