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

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 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.     

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 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 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.     

Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies

Pulmonary neuroepithelial body (NEB) receptors in rats receive at least four different nerve fibre populations. In addition to a spinal sensory innervation that contacts NEBs at their basal side, extensive vagal nodose sensory terminals and separate nitrergic and cholinergic nerve endings protrude between NEB cells. In the present study, antibodies against the vesicular glutamate transporter 2 (VGLUT2), a transmembrane protein responsible for loading glutamate into synaptic vesicles, were used to investigate whether some of the nerve terminals contacting NEBs in rat lungs might use glutamate as a neurotransmitter. VGLUT2 immunoreactivity (IR) was detected in extensive intraepithelial arborising nerve terminals that appeared to contact most of the NEBs. Multiple immunostaining showed VGLUT2 IR in the vagal nodose and spinal sensory nerve terminals contacting NEBs, and in another, most likely sensory, intraepithelial nerve fibre population, the origin and further characteristics of which remain to be elucidated. At least part of the VGLUT2-immunoreactive nerve fibres that contact NEBs were shown to be myelinated. The expression of VGLUT2 indicates that glutamate is stored and released as a neurotransmitter in terminals of several pulmonary (sensory) nerve fibre populations that selectively relate to the complex NEB receptors. The present study strongly suggests an involvement of glutamatergic mechanisms in the peripheral transduction of sensory stimuli from the lungs, via the release of glutamate from nerve terminals, thereby modulating the activity of NEB receptor cells or the excitability of afferent nerves.     

Pulmonary expression of voltage-gated calcium channels: special reference to sensory airway receptors

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.     

Pulmonary intraepithelial vagal nodose afferent nerve terminals are confined to neuroepithelial bodies: an anterograde tracing and confocal microscopy study in adult rats

Our present understanding of the morphology of neuroepithelial bodies (NEBs) in mammalian lungs is comprehensive. Several hypotheses have been put forward regarding their function but none has been proven conclusively. Microscopic data on the innervation that appears to affect the reaction of NEBs to stimuli have given rise to conflicting interpretations. The aim of this study has been to check the validity of the hypothesis that pulmonary NEBs receive an extensive vagal sensory innervation. The fluorescent neuronal tracer DiI was injected into the vagal sensory nodose ganglion and NEBs were visualized in toto by using immunocytochemistry and confocal microscopy on 100-μm-thick frozen sections of the lungs of adult rats. The most striking finding was the extensive intraepithelial terminal arborizations of DiI-labelled vagal afferents in intrapulmonary airways, apparently always co-appearing with calcitonin gene-related peptide (CGRP)-immunoreactive NEBs. Not all NEBs received a traced nerve fibre. Intrapulmonary CGRP-containing nerve fibres, including those innervating NEBs, always appeared to belong to a nerve fibre population different from the DiI-traced fibres and hence did not arise from the nodose ganglion. Therefore, at least some of the pulmonary NEBs in adult rats are supplied with sensory nerve fibres that originate from the vagal nodose ganglion and form beaded ramifications between the NEB cells, thus providing support for the hypothesis of a receptor function for NEBs. Received: 13 November 1997 / Accepted: 17 February 1998     

Differential effects of airway afferent nerve subtypes on cough and respiration in anesthetized guinea pigs

The hypothesis that respiratory reflexes, such as cough, reflect the net and often opposing effects of activation of multiple afferent nerve subpopulations throughout the airways was evaluated. Laryngeal and tracheal mucosal challenge with either citric acid or mechanical probing reliably evoked coughing in anesthetized guinea pigs. No other stimulus reliably evoked coughing in these animals, regardless of route of administration and despite some profound effects on respiration. Selectively activating vagal C-fibers arising from the nodose ganglia with either adenosine or 2-methyl-5-HT evoked only tachypnea. Selectively activating vagal afferents arising from the jugular ganglia induced respiratory slowing and apnea. Nasal afferent nerve activation by capsaicin, citric acid, hypertonic saline, or histamine evoked only respiratory slowing. Histamine, which activates intrapulmonary rapidly adapting receptors but not airway or lung C-fibers or tracheal bronchial cough receptors induced bronchospasm and tachypnea, but no coughing. The results indicate that the reflexes initiated by stimuli thought to be selective for some afferent nerve subtypes will likely depend on the net and potentially opposing effects of multiple afferent nerve subpopulations throughout the airways. The data also provide further evidence that the afferent nerves regulating cough in anesthetized guinea pigs are distinct from either C-fibers or intrapulmonary rapidly adapting receptors.     

Tracheobronchial muscle tonus and its reflex control

Airway smooth muscle tone is reinforced during the inspiratory phase of the breathing cycle and depends largely from neurogenic motor drive carried by the vagus nerve. This muscle tone seems to be produced mostly by a vago-vagal reflex loop initiated by the tonic discharge of tracheo-bronchial and/or alveolar receptors connected to thin sensory vagal fibres (non-myelinated or C-fibres). Inhibitory influences carried by large myelinated vagal fibres connected to tracheobronchial stretch receptors and also numerous afferents from the upper airways, systemic and pulmonary circulation, digestive tract and skeletal and respiratory muscles participate to the modulation of airway tone. The identification of neurotransmitters specific of the motor or sensory pathways helps to understand the peripheral modulation of airway motor drive and also the central integration of some peripheral informations.     

Localization of serotonin, cholecystokinin and somatostatin immunoreactivity in the lower respiratory tract of embryonic, foetal and postnatal sheep.

The lower respiratory tract of the sheep was studied by light-microscopical immunocytochemistry for serotonin, cholecystokinin, somatostatin, bombesin and calcitonin during different periods of lung development; embryonic, foetal and postnatal. At embryonic period only intraepithelial serotonin-containing cells as solitary neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) were found. At foetal stages, immunoreactive cells to serotonin, cholecystokinin and somatostatin were observed in airway epithelium, as solitary NEC and NEBs, and in autonomic intrapulmonary ganglia as single or clusters of small intensely-fluorescent (SIF) cells. In postnatal sheep, serotonin- and cholecystokinin-containing cells were found within airway mucosa as solitary NECs and NEBs. No immunoreactive cells were observed with antiserum to bombesin and calcitonin. Quantitative studies showed that serotonin was the predominant substance, and that solitary neuroendocrine cells were more numerous in distal conducting airways and at foetal stages.     

Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species

Pulmonary neuroepithelial bodies (NEBs) are extensively innervated organoid groups of neuroendocrine cells that lie in the epithelium of intrapulmonary airways. Our present understanding of the morphology of NEBs is comprehensive, but direct physiological studies have so far been challenging because the extremely diffuse distribution of NEBs makes them inaccessible in vivo and because a reliable in vitro model is lacking. Our aim has been to optimise an in vitro method based on vibratome slices of living lungs, a model that includes NEBs, the surrounding tissues and at least part of their complex innervation. This in vitro model offers satisfactory access to pulmonary NEBs, provided that they can be differentiated from other tissue elements. The model was first optimised for living rat lung slices. Neutral red staining, reported to stain rabbit NEBs, proved unsuccessful in rat slices. On the other hand, the styryl pyridinium dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), showed brightly fluorescent cell groups, reminiscent of NEBs, in the airway epithelium of living lung slices from rat. In addition, nerve fibres innervating the NEBs were labelled. The reliable and specific labelling of pulmonary NEBs by 4-Di-2-ASP was corroborated by immunostaining for protein gene-product 9.5. Live cell imaging and propidium iodide staining further established the acceptable viability of 4-Di-2-ASP-labelled NEB cells in lung slices, even over long periods. Importantly, the in vitro model and 4-Di-2-ASP staining procedure for pulmonary NEBs appeared to be equally reproducible in mouse, hamster and rabbit lungs. Diverse immunocytochemical procedures could be applied to the lung slices providing an opportunity to combine physiological and functional morphological studies. Such an integrated approach offers additional possibilities for elucidating the function(s) of pulmonary NEBs in health and disease. This work was supported by the following research grants: Fund for Scientific Research Flanders (G.0155.01 to D.A.), NOI-BOF (to D.A.) and BOF-RUCA Small Projects (KPO2 to D.A., I.B. and F.V.M.) from the University of Antwerp.     

Colitis decreases mechanosensitive K2P channel expression and function in mouse colon sensory neurons

TREK-1, TREK-2 and TRAAK are mechanosensitive two-pore domain K(+) (K(2P)) channels thought to be involved in the attenuation of mechanotransduction. Because colon inflammation is associated with colon mechanohypersensitivity, we hypothesized that the role of these channels in colon sensory (dorsal root ganglion, DRG) neurons would be reduced by colon inflammation. Accordingly, we studied the functional expression of mechanosensitive K(2P) channels in colon sensory neurons in both thoracolumbar (TL) and lumbosacral (LS) DRG that represent the splanchnic and pelvic nerve innervations of the colon, respectively. In colon DRG neurons identified by retrograde tracer previously injected into the colon wall, 62% of TL neurons and 83% of LS neurons expressed at least one of three K(2P) channel mRNAs; the proportion of neurons expressing the TREK-1 gene was greater in LS than in TL DRG. In electrophysiological studies, single-channel activities of TREK-1a, TREK-1b, TREK-2, and TRAAK-like channels were detected in cultured colon DRG neuronal membranes. After trinitrobenzene sulfonic acid-induced colon inflammation, we observed significant decreases in the amount of TREK-1 mRNA, in the response of TREK-2-like channels to membrane stretch, and in the whole cell outward current during osmotic stretch in LS colon DRG neurons. These findings document that the majority of DRG neurons innervating the mouse colon express mechanosensitive K(2P) channels and suggest that a decrease in their expression and activities contributes to the increased colon mechanosensitivity that develops in inflammatory bowel conditions.     

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 GABA_A and GABA_B (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.     

Innervation of pulmonary neuroendocrine cells and neuroepithelial bodies in developing rabbit lung.

We investigated the development of innervation of the pulmonary neuroendocrine cell (PNEC) system composed of single cells and organoid cell clusters, neuroepithelial bodies (NEB) in rabbit fetal and neonatal lungs. To visualize the nerve fibers and their contacts with PNECs/NEBs, we used confocal microscopy and multilabel immunohistochemistry (IHC) with pan-neural marker, synaptic vesicle protein 2 (SV2), and serotonin (5-HT) as markers for PNECs/NEBs, and smooth muscle actin or cytokeratin to identify airway landmarks. The numbers and distribution of PNEC/NEB at different stages of lung development (E16, 18, 21, 26, and P2) and the density of innervation were quantified. First PNECs immunoreactive for 5-HT were identified in primitive airway epithelium at E18 as single cells or as small cell clusters with or without early nerve contacts. At E21 a significant increase in the number of PNECs with formation of early innervated NEB corpuscules was observed. The overall numbers of PNECs/NEBs and the density of mucosal, submucosal, and intercorpuscular innervation increased with progressing gestation and peaked postnatally (P2). At term, the majority of NEBs and single PNECs within airway mucosa possessed neural contacts. Such an extensive and complex innervation of the PNEC system indicates a multifunctional role in developing lung and during neonatal adaptation.     

Innervation and cytochemistry of the neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus)

Summary Neuroepithelial bodies (NEB) were identified in the lung of Bufo marinus. The characteristics of the cells and their innervation were studied with electron and fluorescence microscopy before and after close vagosympathetic denervation. The bodies consist of low columnar cells which rest on the epithelial basal lamina. The majority of the cells do not reach the lumen of the lung (basal cells); the few which do (apical cells) are bordered by microvilli and possess a single cilium. The neuroepithelial cell cytoplasm contains a variety of organelles the most characteristic of which are dense cored vesicles. Microspectrofluorometry and electron microscopic cytochemistry indicate significant quantities of 5-hydroxytryptamine in these cells. The neuroepithelial bodies could be divided into three groups on the basis of their innervation: 1) About 60% of the NEBs are innervated solely by nerve fibres containing agranular vesicles which form reciprocal synapses; 2) about 20% are innervated solely by adrenergic nerve fibres which form distinct synaptic contacts; and 3) the remaining 20% are innervated by both types of nerve fibres. It is proposed that the NEBs are receptors monitoring intrapulmonary P_{CO 2} and so leading to modulation of activity in afferent nerve fibres (type containing agranular vesicles). The presence of NEBs solely with an adrenergic (efferent) innervation poses a problem with this interpretation.     

Pulmonary sensory and reflex responses in the mouse.

Mouse model research is proliferating because of its readiness for genetic manipulation. Little is known about pulmonary vagal afferents in mice, however. The purpose of this study was to determine whether their pulmonary afferents are similar to those in large animals. Single-unit activity was recorded in the cervical vagus nerve of anesthetized, open-chest, and mechanically ventilated mice. We evaluated airway sensory activity in 153 single units; 141 were mechanosensitive, with 134 inflation receptors and 7 deflation receptors. The remaining 12 receptors were chemosensitive and mechanically insensitive, showing low basal firing frequency and behaving like C-fiber or high-threshold Adelta-receptors. In separate studies, phrenic activity was recorded as an index of respiratory drive to assess pulmonary reflexes. Lung inflation produced a typical Hering-Breuer reflex, and intravenous injection of phenylbiguanide produced the typical chemoreflex resulting in apnea, bradycardia, and hypotension. These reflexes were blocked by bilateral vagotomy. We conclude that mice possess a similar set of airway sensors and pulmonary reflexes as typically found in larger animals.     

Development of neuroepithelial bodies in intact and cultured lungs of fetal rats

Intact, 14- to 21-day fetal rat lung pairs, neonatal lungs, and cultured 15- and 16-day lung explants were examined in 2-micron-thick glycol methacrylate sections stained by PAS-lead hematoxylin. Selected stages were also studied in histochemical preparations for aliesterase and formaldehyde-induced monoamine fluorescence, as well as by scanning and transmission electron microscopy. Neuroepithelial bodies (NEBs) first appear in pseudoglandular lungs at 15 days in vivo as pyramidal groups of basal, diffusely lead-hematoxylin-positive cells in glycogen-depleted epithelium of main and lobar bronchi. By day 16, primitive NEBs occur within three to four generations of the terminal buds, and older, proximal bodies are larger and more distinctive than at 15 days. Aliesterase activity is first detected in basally located, developing NEBs on day 16. During the canalicular and alveolar sac periods, NEBs appear and mature on a proximal-to-distal gradient along the airway, as they do in developing rabbit and human lungs. As earlier-formed airways elongate, additional NEBs appear and supplement the population already present. By days 20-21, NEBs occur at all airway levels down to the bronchiolo-alveolar junctions, and many of the cells have discrete PAS- and lead-hematoxylin-positive, infranuclear granules. Near term some NEBs exhibit serotonin fluorescence after incubation in 5-hydroxytryptophan and have abundant, ca. 100-nm, electron-dense granules. These are concentrated toward the cell base like the stained granules visualized by light microscopy. Similar results were obtained from lungs placed in organ culture. From 2 days in culture to a time equivalent to term, NEB formation parallels that in vivo, indicating that developmental requirements are met in in vitro. Taken altogether, morphologic and cytochemical evidence suggests that NEBs of rats are functional in late fetal life and that their development is relatively independent of extrapulmonary influences and of the intraepithelial ingrowth of sensory nerve endings.     

Effects of unilateral vagal stimulation on intrapulmonary neuroepithelial bodies

We studied the influence of unilateral vagal stimulation on intrapulmonary neuroepithelial bodies (NEB) in rabbits. The left vagus nerve was cut and electrically stimulated for 10 min. Animals were killed and the lungs studied with fluorescence and electron microscopy. Intensity of formaldehyde-induced fluorescence, which reflects the serotonin content in NEB, was higher on the stimulated side than on the nonstimulated side (118 +/- 7 vs. 100%, n = 8, P less than 0.001). The latter difference was found to correlate with the stimulus amplitude (r = 0.9, P less than 0.05). Ultrastructurally a decrease in the number of exocytotic dense-cored vesicle (DCV) profiles at the level of the NEB basal epithelial cell membrane was found on the stimulated side (0.32 +/- 0.10 vs. 0.45 +/- 0.16 DCV/micron of basal epithelial cell membrane, n = 8, P less than 0.05). Section of the left vagus nerve without electrical stimulation affected neither the fluorescence intensity nor the number of exocytotic DCV profiles. In animals with supranodosal or infranodosal chronic vagotomy the observed effects of unilateral vagal stimulation were no longer present. We conclude that 1) vagal stimulation increases the serotonin content of NEB; 2) it decreases the number of exocytotic DCV profiles; 3) this effect depends on the amplitude of the stimulus; 4) it is obtained through efferent vagal fibers; 5) these results are the opposite of the effects seen after exposing normal NEB to acute hypoxia; and 6) these physiological experiments corroborate a vagal innervation of NEB, which may play an important role in modulating the sensitivity and reaction of NEB to various stimuli.     

Chemical profile of vagal preganglionic motor cells innervating the airways in ferrets: the absence of noncholinergic neurons.

In ferrets, we investigated the presence of choline acetyltransferase (ChAT), vasoactive intestinal peptide (VIP), and markers for nitric oxide synthase (NOS) in preganglionic parasympathetic neurons innervating extrathoracic trachea and intrapulmonary airways. Cholera toxin beta-subunit, a retrograde axonal transganglionic tracer, was used to identify airway-related vagal preganglionic neurons. Double-labeling immunohistochemistry and confocal microscopy were employed to characterize the chemical nature of identified airway-related vagal preganglionic neurons at a single cell level. Physiological experiments were performed to determine whether activation of the VIP and ChAT coexpressing vagal preganglionic neurons plays a role in relaxation of precontracted airway smooth muscle tone after muscarinic receptor blockade. The results showed that 1) all identified vagal preganglionic neurons innervating extrathoracic and intrapulmonary airways are acetylcholine-producing cells, 2) cholinergic neurons innervating the airways coexpress ChAT and VIP but do not contain NOS, and 3) chemical stimulation of the rostral nucleus ambiguus had no significant effect on precontracted airway smooth muscle tone after muscarinic receptor blockade. These studies indicate that vagal preganglionic neurons are cholinergic in nature and coexpress VIP but do not contain NOS; their stimulation increases cholinergic outflow, without activation of inhibitory nonadrenergic, noncholinergic ganglionic neurons, stimulation of which induces airway smooth muscle relaxation. Furthermore, these studies do not support the possibility of direct inhibitory innervation of airway smooth muscle by vagal preganglionic fibers that contain VIP.     

KCa1.1 channel contributes to cell excitability in unmyelinated but not myelinated rat vagal afferents

High conductance calcium-activated potassium (BK(Ca)) channels can modulate cell excitability and neurotransmitter release at synaptic and afferent terminals. BK(Ca) channels are present in primary afferents of most, if not, all internal organs and are an intriguing target for pharmacological manipulation of visceral sensation. Our laboratory has a long-standing interest in the neurophysiological differences between myelinated and unmyelinated visceral afferent function. Here, we seek to determine whether there is a differential distribution of BK(Ca) channels in myelinated and unmyelinated vagal afferents. Immunocytochemistry studies with double staining for the BK-type K(Ca)1.1 channel protein and isolectin B4 (IB4), a reliable marker of unmyelinated peripheral afferents, reveal a pattern of IB4 labeling that strongly correlates with the expression of the K(Ca)1.1 channel protein. Measures of cell size and immunostaining intensity for K(Ca)1.1 and IB4 cluster into two statistically distinct (P < 0.05) populations of cells. Smaller diameter neurons most often presented with strong IB4 labeling and are presumed to be unmyelinated (n = 1,390) vagal afferents. Larger diameter neurons most often lacked or exhibited a very weak IB4 labeling and are presumed to be myelinated (n = 58) vagal afferents. Complimentary electrophysiological studies reveal that the BK(Ca) channel blockers charybdotoxin (ChTX) and iberiotoxin (IbTX) bring about a comparable elevation in excitability and action potential widening in unmyelinated neurons but had no effect on the excitability of myelinated vagal afferents. This study is the first to demonstrate using combined immunohistochemical and electrophysiological techniques that K(Ca)1.1 channels are uniquely expressed in unmyelinated C-type vagal afferents and do not contribute to the dynamic discharge characteristics of myelinated A-type vagal afferents. This unique functional distribution of BK-type K(Ca) channels may provide an opportunity for afferent selective pharmacological intervention across a wide range of visceral pathophysiologies, particularly those with a reflexogenic etiology and pain.     

Functional localization of pulmonary stretch receptors in the airways of the cat

During a respiratory effort against a closed airway the afferent activity of vagal fibres from pulmonary stretch receptors does not appreciably increase during the inspiratory phase because the lung is prevented from expanding. The possibility to perform occlusions at different levels of the airways allows the localization of pulmonary stretch receptors in the tracheo-bronchial tree. 100 fibres from pulmonary stretch receptors of the left and right sides of the tracheo-bronchial tree have been studied in 3 cats and their localization found as follows: 10% in the higher half of the intrathoracic trachea, 22% in the lower half of the intrathoracic trachea and the carina, 7% in the main bronchus and 61% in the intrapulmonary airways. Knowing the surface area of the tracheo-bronchial tree at different levels and assuming total of 1200 stretch receptors from each side their average concentration resulted as follows: 50.0 receptors/cm2 in the higher half of the intrathoracic trachea, 108.0/cm2 in the lower half of the intrathoracic trachea and the carina, 213.0/cm2 in the main bronchus and 1.3/cm2 in the intrapulmonary airways.