Functional morphology of pulmonary neuroepithelial bodiesExtremely complex airway receptors.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 270A:25– 40 (2003) Functional Morphology of Pulmonary Neuroepithelial Bodies: Extremely Complex Airway Receptors DIRK ADRIAENSEN,* INGE BROUNS, JEROEN VAN GENECHTEN, AND JEAN-PIERRE TIMMERMANS Laboratory of Cell Biology and Histology, Department of Biomedical Sciences, University of Antwerp–RUCA, Antwerp, Belgium ABSTRACT Innervated groups of neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium of intrapulmonary airways in many species. Our present understanding of the morphology of NEBs in mammalian lungs is comprehensive, but none of the proposed functional hypotheses have been proven conclusively. In recent reviews on airway innervation, NEBs have been added to the list of presumed physiological lung receptors. Microscopic data on the innervation of NEBs, however, have given rise to conﬂicting interpretations. Using neuronal tracing, denervation, and immunostaining, we recently demonstrated that the innervation of NEBs is much more complex than the almost unique vagal nodose sensory innervation suggested by other authors. The aim of the present work is to summarize our present understanding about the origin and chemical coding of the profuse nerve terminals that selectively contact pulmonary NEBs. A thorough knowledge of the complex interactions between the neuroendocrine cells and at least ﬁve different nerve ﬁber populations is essential for deﬁning the position(s) of NEBs among the many pulmonary receptors characterized by lung physiologists. Anat Rec Part A 270A:25– 40, 2003. © 2003 Wiley-Liss, Inc. Key words: NEBs; neuroepithelial bodies; innervation; airway receptors; lung; rat Highly specialized neuroepithelial bodies (NEBs) (Lauweryns et al., 1972), which consist of extensively innervated groups of pulmonary neuroendocrine cells (PNECs), are normal components of the epithelium of intrapulmonary airways in humans, mammals, and all air-breathing vertebrate species investigated so far. The pulmonary neuroendocrine system was ﬁrst reported more than 50 years ago (Fröhlich, 1949), but especially over the last 25 years detailed information has been provided about the distribution, ontogeny, and microscopic morphology of NEBs (for reviews see Scheuermann, 1987; Sorokin and Hoyt, 1989; Adriaensen and Scheuermann, 1993; Sorokin et al., 1997). PNECs belong to the diffuse neuroendocrine system (DNES) (Pearse and Takor Takor, 1979), members of which have been assigned important roles in the peripheral control of various organs. The pulmonary DNES in healthy lungs appears to be characterized by the production of amines and several neuropeptides, including serotonin (5-HT), bombesin (gastrin-releasing peptide), calci© 2003 WILEY-LISS, INC. tonin gene-related peptide (CGRP), calcitonin, enkephalin, somatostatin, cholecystokinin, and substance P (SP) (for reviews see Sorokin and Hoyt, 1989; Scheuermann et al., 1992; Adriaensen and Scheuermann, 1993). These bioactive substances are stored in secretory granules (60 –200 nm diameter) with typical endocrine-like characteristics, the so-called dense-cored vesicles (DCVs). Interestingly, PNECs are by far the ﬁrst cell type to fully differentiate in the human airway epithelium (before *Correspondence to: Dirk Adriaensen, Laboratory of Cell Biology and Histology, University of Antwerp–RUCA, Groenenborgerlaan 171, B-2020 Antwerp, Belgium. Fax: ⫹32-3-218-0301. E-mail: email@example.com Received 22 May 2002; Accepted 5 September 2002 DOI 10.1002/ar.a.10007 26 ADRIAENSEN ET AL. the 8th week of gestation), and changes in PNECs/NEBs have been associated with many perinatal, neonatal, and adult lung diseases and disorders. NEBs may have important functions in the regulation of physiological processes in lungs at all ages, but their exact role is poorly understood. Mainly on the basis of incomplete morphological data, several hypotheses about the function of pulmonary NEBs have been proposed (Sorokin and Hoyt, 1990). NEBs are now believed to have different functions during speciﬁc periods in prenatal, early postnatal, and adult life. Oxygen-sensing (Youngson et al., 1993; Cutz and Jackson, 1999; Peers and Kemp, 2001) and effector (O’Kelly et al., 1998, 1999) mechanisms have been identiﬁed in pulmonary neuroendocrine cells. Recent reviews on afferent receptors in the lower airways have added NEBs to the list of presumed receptor types, which included slowly (SARs) and rapidly adapting stretch receptors (RARs) and C-ﬁber receptors (Widdicombe, 2001). However, Widdicombe (2001) also stated that so far there have been no claims for recordings from single afferent ﬁbers from NEBs. In our opinion, the most likely reason for that is a lack of knowledge of the functional morphology and the nervous connections of these NEB “receptors.” Because the present review is part of a special issue on airway receptors, it mainly focuses on the innervation of pulmonary NEBs in general, and on the sensory components in particular. Our recent neuronal tracing, immunocytochemistry, and denervation experiments in rats, which provide good evidence for an extremely complex innervation pattern of NEBs (Adriaensen et al., 1998, 2001; Brouns et al., 2000, 2002a, b, 2003), are summarized, illustrated, and discussed in the light of the possible receptor function(s) of pulmonary NEBs. Literature Data and Classical Concepts About the Innervation of Pulmonary NEBs A selective innervation of NEBs was probably described long before the existence of the pulmonary neuroendocrine system itself was known. Several authors described intraepithelial varicose nerve terminals that were concentrated in groups and irregularly distributed along the airways of different species, including humans (Berkley, 1894; Larsell, 1921; Larsell and Dow, 1933; Elftman, 1943). It was Fröhlich (1949) who described delicate nerve terminals at the surface of, or between the cells of grouped neuroendocrine cells in the bronchial epithelium of rabbits and cats. Since that time, many researchers have observed an indisputable innervation of NEBs in both light and electron microscopic investigations. Organ culture studies suggested that the nerve terminals contacting NEBs rapidly degenerate, but that denervated NEBs survive (Sonstegard et al., 1979), and that solitary PNECs may give rise to structured organoid bodies in vitro (Carabba et al., 1985). Three-dimensional reconstruction of a single hamster NEB using electron microscopic images of serial sections showed that also in intact animals, not every NEB is necessarily innervated (Pearsall et al., 1985). A number of methods have been used to visualize nerve ﬁbers that contact NEBs. To this end, an unambiguous and simultaneous identiﬁcation of PNECs and nerves was considered essential. With the use of different silver-staining techniques, nonmyelinated nerve ﬁbers that contact the basement membrane at the level of NEBs or surround the neuroendocrine cells were described in the lungs of human newborns (Fröhlich, 1949; Lauweryns and Peuskens, 1972), rabbits (Lauweryns et al., 1972; Hung, 1980), rats (Wasano, 1977), and mice (Hung, 1984), as well as in lower vertebrates such as turtles (Scheuermann et al., 1983). Silver impregnation of the NEB cells, however, almost always hampers a clear view of the nerve terminals contacting the NEB cells. A regularly used method for studying lung innervation is the histochemical demonstration of acetylcholinesterase (AChE) activity. Networks of AChE-positive nerve ﬁbers were described in contact with pulmonary NEBs of fetal and newborn rabbits (Sonstegard et al., 1982; Cutz et al., 1985) and sheep (Cutz and Orange, 1977). Unfortunately, this technique produces a blurred image of ﬁne structures and stains the NEB cells, resulting in a poor deﬁnition of nerve endings at the contact sites. Formaldehyde-induced ﬂuorescence resulted in the demonstration of a blue-green nerve plexus, with spectral characteristics of catecholamines, in the lamina propria of airways, close to the base of NEBs in rabbits (Lauweryns et al., 1972; Hung, 1980), turtles (Scheuermann et al., 1983), frogs (Wasano and Yamamoto, 1978), and toads (Rogers and Haller, 1978). NEB cells can be differentiated, displaying a yellow ﬂuorescence with spectral characteristics of serotonin. More recently, immunocytochemical methods that were generally applied for the demonstration of NEBs often also appeared to label contacting nerve ﬁbers. In most cases, the problem of differentiating the nerve terminals at the level of the NEBs remains. In rabbits, hamsters, and rats, pulmonary NEBs and associated nerve ﬁbers were described using antisera against protein gene product 9.5 (PGP9.5) (Lauweryns and Van Ranst, 1988). In newborn cats (Adriaensen and Scheuermann, 1993), NSEimmunoreactive (IR) nerve ﬁbers were observed to contact NSE-positive NEBs. CGRP-IR nerve ﬁbers connected to CGRP-positive NEBs were reported in the lungs of cats (Adriaensen and Scheuermann, 1993) and rats (Cadieux et al., 1986; Van Ranst and Lauweryns, 1990; Terada et al., 1992). In guinea pigs, a double immunoﬂuorescence labeling technique, applying antisera against neuropeptides and general neuroendocrine markers (PGP9.5; chromogranin), resulted in the demonstration of solitary tracheal and bronchial PNECs spirally surrounded by CGRP/ SP-IR nerve terminals (Kummer et al., 1991). Using additional pre-embedding electron microscopic immunocytochemistry, the latter nerve terminals were seen to form direct contacts with the PNECs, but without the presence of synaptic specializations. In the human lung, however, where PNECs and NEBs were shown to be SP-IR, no associated SP-positive nerve ﬁbers have been reported (Gallego et al., 1990). None of the techniques described so far demonstrated unambiguously that the closely associated nerve ﬁbers indeed provide a direct innervation of the neuroendocrine cells in NEBs. Such information could only be obtained using electron microscopy. Nerve ﬁbers associated with NEBs were seen in the mouse lung using scanning electron microscopy (Hung, 1984), but because this technique only reveals surface structure, it was not possible to obtain a clear view of the interactions between neuroendocrine cells and nerves. NEBS: COMPLEX AIRWAY RECEPTORS With the use of transmission electron microscopy (TEM), nerve terminals were demonstrated directly contacting the basal part of PNECs, or running between and looping around the cells in NEBs. The following references were selected based mainly on their descriptions of synapse-like specializations between nerve terminals and NEB cells. Direct innervation of pulmonary NEBs was observed in humans (Lauweryns et al., 1970) and many mammalian species, such as mice (Wasano, 1977; Wasano and Yamamoto, 1981), rats (Van Lommel and Lauweryns, 1993a), hamsters (Sorokin and Hoyt, 1989), rabbits (Lauweryns et al., 1972; Lauweryns and Van Lommel, 1987; Sorokin and Hoyt, 1989; Adriaensen and Scheuermann, 1993), and cats (Van Lommel and Lauweryns, 1993b). In birds, similar NEB/nerve contacts were reported in the lungs of chickens (Cook and King, 1969; Walsh and McLelland, 1978; Wasano and Yamamoto, 1979; López et al., 1983), pigeons (McLelland and MacFarlane, 1986), and quails (Adriaensen et al., 1994). In lower vertebrates, direct innervation was shown in reptiles, such as turtles (Scheuermann et al., 1983), lizards (Ravazzola and Orci, 1981), and snakes (Wasano and Yamamoto, 1976); in amphibia, such as newts (Scheuermann et al., 1989; Goniakowska-Witalinska et al., 1992; Adriaensen and Scheuermann, 1993), frogs (Wasano and Yamamoto, 1978; Goniakowska-Witalinska, 1981), and toads (Rogers and Haller, 1980; Goniakowska-Witalinska et al., 1990); and in lungﬁsh (Dipnoi) (Adriaensen et al., 1990). Nerve ﬁbers have been reported to contact solitary PNECs and NEB cells, although by far the most reports concern NEBs. In several species, solitary PNECs apparently do not show direct contacts with nerve ﬁbers, which was reason enough for some authors to regard them as different entities from NEBs. It is clear, however, that the surface membrane of all PNECs is functionally divided into basolateral and apical regions. Possible nerve contacts in this respect provide further possibilities for functional integration. Using TEM, different types of morphologically characterized nerve terminals have been described in contact with PNECs. The type of nerve terminal most often reported in contact with NEBs, in many species, is packed with mitochondria and some small clear vesicles. They penetrate deep between the NEB cells, often close to the luminal surface. The nerve terminals reveal asymmetric synaptic contacts, with an accumulation of DCVs near electron-dense coneshaped thickenings of the surface membrane of PNECs, and are generally believed to be afferent (sensory) (Cook and King, 1969; Wasano and Yamamoto, 1981; Lauweryns et al., 1985; Lauweryns and Van Lommel, 1986, 1987; Adriaensen and Scheuermann, 1993; Van Lommel and Lauweryns, 1993a, b). A second frequently reported type of nerve terminal appears to be loaded with small clear vesicles (40 – 60 nm diameter) and shows a few larger DCVs and mitochondria. In several species they reveal asymmetric synaptic contacts with NEB cells, with an accumulation of the clear vesicles near cone-shaped electron-dense specializations of the axolemma of the nerve terminal, while the surface membrane of the PNEC shows a uniform thickening; these nerve terminals are generally considered efferent (cholinergic-like) (Walsh and McLelland, 1978; Sonstegard et al., 1982; Stahlman and Gray, 1984; Adriaensen and Scheuermann, 1993). 27 In some mammalian species, it has been demonstrated that both morphologically afferent and efferent contacts may be peripheral specializations of the same (sensory?) nerve ﬁber (Lauweryns and Van Lommel, 1987; Van Lommel and Lauweryns, 1993a, b). A few reports mention nerve endings ﬁlled with small dense-cored granules (about 60 nm diameter) (Rogers and Haller, 1978), typical of adrenergic nerves, and terminals containing larger (80 –225 nm), moderately dense DCVs (Stahlman and Gray, 1984), believed to belong to a peptidergic pathway, in close apposition to PNECs. Although TEM probably offers a rather good morphological characterization of the direct innervation of a limited number of pulmonary NEBs in several species, it leaves another important question unanswered, i.e., that of the origin of the nerve ﬁber population(s) that selectively innervate(s) NEBs. To date, most of the studies dealing with this aspect have combined electron microscopy, for the evaluation of nerve terminals contacting NEBs, and experimental vagotomy with or without an additional hypoxic stimulus (in rabbits (Lauweryns et al., 1985; Lauweryns and Van Lommel, 1986) and rats (Van Lommel and Lauweryns, 1993a)). It was reported that, after unilateral infranodosal vagotomy, the numbers of both morphologically afferent and efferent nerve terminals in NEBs were reduced by about 70% in lungs ipsilateral to the denervation, while no changes were seen in the contralateral lungs. The NEB innervation appeared to be intact after supranodosal vagotomy. Based on these ﬁndings, it was suggested that NEBs are predominantly innervated by sensory nerve ﬁbers originating from neurons located in the vagal ganglion nodosum. The remaining 30% of nerve terminals in the investigated NEBs were thought to be the result of cross innervation from the contralateral vagus or of the presence of another unknown nerve ﬁber population (e.g., sympathetic) contacting NEBs. Electrical stimulation of the sectioned vagal nerve revealed a possible vagal inhibitory secretomotor inﬂuence on rabbit pulmonary NEBs, since the basal secretion of DCVs was apparently decreased (Lauweryns et al., 1985). More speciﬁcally for rat lung, the very extensive innervation of NEBs was suggested to be exclusively sensory, originating from the vagal nodose ganglion (Van Lommel and Lauweryns, 1993a). The electron microscopically quantiﬁed efferent- and afferent-like terminals were interpreted as belonging to the same population of sensory ﬁbers, since vagotomy experiments resulted in a clearly reduced number of nerve terminals in NEBs, while the ratio between efferent- and afferent-like terminals appeared unchanged in the selected NEBs. It was further concluded that, unlike in rabbits, a separate motor innervation of NEBs is probably absent in rat lungs. However, given our own more recent data on the innervation of rat NEBs (see below), the question could be raised as to whether the combination of experimental denervation and electron microscopy, which is very labor-intensive and therefore necessarily limited by the number of fully quantiﬁable specimens, is a reliable method by which to draw conclusions on such a complex and inhomogeneous diffuse population of NEBs. The latter point is strengthened by the observation that NEBs in organ-cultured rat lungs apparently receive an innervation from neurons located in intrinsic airway ganglia (Sorokin et al., 1993). 28 ADRIAENSEN ET AL. To date, we know very little about the presence or origin of different nerve ﬁber populations selectively innervating NEBs in the human lung. To conclude this historical perspective and at the same time come back to the main point of the present short review, i.e., the characterization of pulmonary NEBs as putative airway receptors, it may be of interest to revisit the scheme that was presented in our 1993 NEB review (Adriaensen and Scheuermann, 1993) (Fig. 1). Essentially, what is represented is the best possible view we had at that time of the morphological organization of NEBs and directly related nerve terminals. It further aimed at explaining a possible mechanism for the assumed reaction of NEBs to airway hypoxia. Most of the information represented in the scheme was based on electron microscopic literature data. It may be stressed that several more recent reviews on pulmonary NEBs still come to the same conclusion, i.e., NEB innervation is mainly vagal nodose and NEBs may act as hypoxia sensors via a vagal afferent pathway (Cutz and Jackson, 1999; Van Lommel et al., 1999), and that the same scheme was used as representative for pulmonary NEBs in a recent review on airway receptors (Widdicombe, 2001). The interest in this concept has been recently strengthened by the characterization of a complete, carotid body-like, oxygen-sensing mechanism in NEBs, which implicates exocytosis of transmitters as a result of hypoxia (for review see Peers and Kemp, 2001). In summary, many researchers in the ﬁeld today believe that if the stimulus is strong enough, hypoxia may cause, in addition to local reﬂex actions, an afferent signal to travel toward the central nervous system (CNS) via the vagus nerve. However, this leaves us with the essential question of why lung physiologists have been unable to conﬁrm this apparently rather simple mechanism. Are NEBs really the lung receptors we would like them to be? If so, did we overlook something? Having many years of experience looking at the functional morphology of NEBs, we evidently went for the latter possibility and tried to ﬁnd out if it is really that simple. We realized that if NEBs are indeed airway receptors, the best way to try to understand them would be to have a good look at their nervous connections, and we decided to focus on the rat lung. Functional Morphological Characterization of the Selective Innervation of NEBs in Rat Lungs Vagal nodose sensory component of the selective innervation of pulmonary NEBs. After we considered all available literature data, it was clear that an essential factor in the recognition of NEBs as airway receptors would be the full conﬁrmation and characterization of their vagal nodose innervation. The following questions arose: Is the vagal nodose innervation of NEBs really there? How can it unambiguously be identiﬁed in the light microscope? What are the neurochemical characteristics? How does this vagal nodose connection relate to what lung physiologists know, or thought they knew? Since several authors reported that in rat lungs CGRP-IR NEBs at all levels of the intrapulmonary airways are contacted by CGRP-positive nerve ﬁbers (Cadieux et al., 1986; Shimosegawa and Said, 1991; Terada et al., 1992; Sorokin et al., 1997) (personal observations), it was generally believed that they represented the long-predicted vagal sensory ﬁbers. However, after performing some vagotomy experiments we realized that this CGRP-IR nerve ﬁber population is nonvagal, as is further characterized in the next section. The problem of identifying a vagal nodose component of the innervation of NEBs was then addressed in the most direct way possible: the red ﬂuorescent neuronal tracer DiI, a lipophilic carbocyanine dye (Honig, 1993), was injected unilaterally into rat nodose ganglia, and NEBs were visualized in toto by using immunocytochemistry and confocal microscopy on thick frozen sections (Adriaensen et al., 1998). The most striking ﬁnding was the extensive intraepithelial terminal arborizations of DiI-labeled vagal nodose afferents in intrapulmonary airways, often located close to the luminal surface of the airways and apparently always co-appearing with CGRP-IR NEBs (Fig. 2). Not all NEBs received a traced nerve ﬁber. Intrapulmonary CGRP-containing nerve ﬁbers, including those innervating NEBs, always appeared to belong to a nerve ﬁber population different from the DiI-traced ﬁbers. This was the ﬁrst hard evidence demonstrating at the light microscopic level that at least some of the pulmonary NEBs in rats are indeed supplied with sensory nerve ﬁbers that originate in the vagal nodose ganglion, and that form beaded ramiﬁcations between the NEB cells. However, neuronal tracing is a labor-intensive and time-consuming technique, and is hardly compatible with a routine application. Therefore, we tried to further characterize this vagal nodose population of nerve terminals in NEBs. Combinations of neuronal tracing and/or vagal denervation experiments with immunolabeling taught us that the vagal nodose ﬁbers contacting pulmonary NEBs can be labeled selectively with antibodies against the calciumbinding protein calbindin D28k (Adriaensen et al., 1999; Brouns et al., 2000). Calbindin (CB) appears to label NEB cells, as well as the nodose ﬁbers contacting them (Fig. 3), and hence can be regarded as a very interesting routine marker for NEBs in the rat lungs. Apparently, a little less than half of the NEBs are contacted by such CB-IR nerve ﬁbers in control animals. The ﬁrst NEBs contacted by CB-IR nerve ﬁbers could be detected at gestational day (GD) 17. After unilateral infranodose vagotomy, no CB-IR nerves contacting NEBs were left in the ipsilateral lung, while no changes were observed in the contralateral lung. Because both NEBs and contacting nerve ﬁbers are stained, this marker does not allow an evaluation of the intraepithelial terminals. Double immunocytochemical staining with antibodies against CB and CGRP clearly revealed that both substances mark a different nerve ﬁber population, often contacting the same NEBs (Fig. 4). To further characterize the different nerve ﬁber populations related to NEBs in the rat lung, we performed a systemic capsaicin treatment (Brouns et al., 2003). The results revealed no changes in the CB-IR vagal nodose innervation of NEBs as compared to control rats, strongly suggesting that a capsaicin-insensitive population is involved. The method of capsaicin treatment is time-consuming, destroys certain nerve ﬁber populations, and is obviously not always the method of choice for the selective demonstration of nerve ﬁber populations in routine applications. Therefore, we tried to conﬁrm the results of the capsaicin treatment using antibodies against the capsaicin receptor (vanilloid receptor 1 (VR1)). Double staining for VR1 and CB indeed conﬁrmed that the vagal nodose component of the innervation of NEBs does not express Fig. 1. Conceptual scheme summarizing ﬁndings on the innervation of mammalian NEBs and their possible reaction to airway hypoxia. Hypoxic air stimulates (red dotted arrows) the NEB cells (yellow) to discharge the contents of their DCVs at afferent synaptic sites of intracorpuscular sensory nerve endings (white arrowheads). This could result in depolarization and the development of a generator potential that spreads over the nerve ﬁber (white arrows). Given the possibility that afferent and efferent nerve terminals might occur along the same nerve process, as was indeed demonstrated in serial sections of newborn rabbit NEBs, the efferent endings would develop synaptic activity and discharge neurotransmitter (black arrowheads). This may in turn inﬂuence the physiological state of the receptor cell (green arrows), thus modulating the transduction of stimuli or regulating local paracrine secretion acting on nearby blood vessels (red arrows), or smooth muscle bundles (blue arrows). The mechanism of local modulation of a neuroreceptor by efferent nerve endings formed at the periphery of sensory nerves could be called the “axon reﬂex.” When the generator potential reaches a threshold value, an action potential is triggered (white arrows with double arrowheads). Some nerve terminals appear to be exclusively efferent (double black arrowheads). Clara-like cells (CC); capillary (C); smooth muscle bundle (SM); airway lumen (L). Modiﬁed from Adriaensen and Scheuermann (1993). Fig. 2. a and b: Detail of DiI-traced (red ﬂuorescence) vagal sensory nerve terminals contacting a pulmonary NEB (calcitonin gene-related peptide (CGRP)-immunoﬂuorescence; FITC ﬂuorescence) at the alveolar level in the adult rat lung. Maximum value projections (MVPs) of 36 optical sections (1-m interval). a: Red channel clearly demonstrating the DiI-traced nerve ﬁbers approaching the epithelium (arrows) and forming intraepithelial terminals (arrowheads). b: Combination of the red and green channels, showing the complete NEB and all of the labeled nerve endings. The traced nerve endings run along a bronchiole (B) and enter the epithelium at the base of the NEB (open arrowheads). Fig. 3. MVP of 14 confocal optical sections (2-m interval) of an NEB in a bronchus of a neonatal rat. Thick calbindin D28k (CB)-IR (red Cy3 ﬂuorescence) nerve ﬁbers (arrows) contact the CB-IR NEB cells. 30 ADRIAENSEN ET AL. capsaicin receptors (Fig. 5), while often another population that is VR1-IR appears to co-innervate NEBs (see below). In contrast to some literature data claiming that capsaicin treatment causes a depletion of CGRP from rat NEBs (Tjen-A-Looi et al., 1998), quantiﬁcation of the number of CGRP- and PGP9.5-IR NEBs after capsaicin treatment did not reveal signiﬁcant differences with control lungs in our study. The latter observation was strengthened by the absence of VR1 expression from NEBs (Figs. 5 and 12), making a direct effect of capsaicin on the CGRP content of NEBs highly unlikely. Another interesting feature of the vagal sensory component of the innervation of pulmonary NEBs, especially in the light of the present efforts to characterize NEBs as airway receptors, can be visualized using antibodies against the myelin basic protein (MBP), as a marker for myelinated nerve ﬁbers in the lung. It was observed that combined with CB staining, from postnatal day 10 on, the vagal nodose ﬁbers contacting NEBs are invariably myelinated (Fig. 6) (Brouns et al., 2003). Myelinated nerve ﬁbers have been observed electron microscopically in the vicinity of NEBs (Van Lommel and Lauweryns, 1993a), Figures 4 –10. NEBS: COMPLEX AIRWAY RECEPTORS 31 but no evidence or suggestions have linked these myelinated ﬁbers to the vagal nodose intraepithelial nerve terminals situated between NEB cells. Examination of rat lungs using speciﬁc antibodies against P2X3 purinoreceptors (ATP receptors) revealed intraepithelial arborizations of P2X3 receptor-IR nerve terminals, which in all cases appeared to ramify between CGRP- or CB-labeled NEB cells (Brouns et al., 2000) (Figs. 7 and 8). The ﬁrst NEBs contacted by P2X3 receptor-IR nerve terminals could be detected at GD 17. NEB cells did not express P2X3 receptors. It was further demonstrated that P2X3 receptor and CB IR completely colocalize in the vagal nodose population of nerve ﬁbers that selectively contacts NEBs (Fig. 8), whereas CGRP-IR ﬁbers clearly form a different population (Fig. 7). The disappearance of characteristic P2X3 receptor-IR nerve terminals in contact with NEBs after infranodosal vagal denervation, and the colocalization of tracer and P2X3 receptor-labeling in vagal nodose neuronal cell bodies in retrograde tracing experiments from the lungs, support our hypothesis that the P2X3 receptor-expressing nerve ﬁbers contacting NEBs have their origin in the vagal nodose ganglia. The combination of MBP and P2X3 receptor immunostaining again clearly pointed to a myelinated population (Fig. 9) (Brouns et al., 2003). Further concentrating on ATP and ATP receptors, we applied quinacrine histochemistry, a method that has been shown to selectively label high concentrations of ATP, if accumulated together with proteins in secretory granules. In rat lungs, the latter method has been shown to selectively label NEBs (Brouns et al., 2000). Combination of quinacrine histochemistry and P2X3 receptor-staining showed that the ATP receptor-expressing nerve terminals in rat lungs are exclusively associated with quinacrine-stained NEBs (Fig. 10). ATP may, therefore, act as a neurotransmitter in the vagal sensory innervation of NEBs via a P2X3 receptor-mediated pathway. Given the extensive data obtained in other systems (Burnstock, 1999a, b), the possibility that at least some of the NEBs may be involved in vagal afferent respiratory mechanosensory transduction should be considered. All available data on the vagal nodose sensory component of the innervation of NEBs are summarized in the scheme of Figure 20 (nerve ﬁber population shown in blue). Essentially, the sensory nerve ﬁber population involved reveals extensive intraepithelial terminals, has a vagal nodose origin, can be marked by its CB IR, expresses P2X3 ATP receptors but no VR1 capsaicin receptors, and is myelinated. Consequently, as predicted in the initial scheme (Fig. 1), NEBs are indeed supplied by a population of vagal nodose sensory ﬁbers that have now been fully characterized. However, the question remains: why has it so far been impossible for lung physiologists to link NEBs to measurable activities in vagal afferents? Is it perhaps more complicated than was predicted? Fig. 4. Immunocytochemical double-staining of an NEB in a bronchiole of a 10-day-old rat labeled for CGRP (green FITC ﬂuorescence) and CB (red Cy3 ﬂuorescence). CGRP/CB-IR NEB cells are contacted by a nerve bundle composed of CB-IR nerve ﬁbers (arrows) and thin varicose CGRP-IR nerve terminals (arrowheads). Note that in the NEB cells, CGRP labeling is strongest at the basal side while CB labeling appears to be more uniform throughout the cells. MVP of 27 optical sections (1-m interval). Fig. 5. Immunocytochemical double-staining for capsaicin receptors (VR1; green FITC ﬂuorescence) and CB (red Cy3 ﬂuorescence), as a marker for the vagal sensory subpopulation of nerve terminals contacting pulmonary NEBs. High magniﬁcation detail of the basal part of a CB-IR NEB, revealing that VR1 IR (open arrows) and CB IR (arrows) are expressed by separate nerve ﬁber populations. MVP of six confocal optical sections (1-m interval). Fig. 6. Immunocytochemical staining for CB (green FITC ﬂuorescence) and myelin basic protein (MBP; red Cy3 ﬂuorescence) of a bronchiolar NEB in a 21-day-old rat. Combination of the red and green channel shows that the CB-IR nerve ﬁbers (arrows) are surrounded by MBP-IR myelin sheaths (open arrows), that are lost (arrowheads) just before the CB-IR ﬁbers branch and contact the CB-IR NEB. MVP of 25 confocal optical sections (1-m interval). Fig. 7. a and b: Immunocytochemical double-staining for the ATP receptor P2X3 (green FITC-ﬂuorescence) and CGRP (red Cy3-ﬂuorescence) revealing pulmonary NEBs in a rat bronchiole contacted by P2X3 receptor- and CGRP-expressing nerve ﬁbers. MVPs of 16 confocal optical sections (1-m interval). a: Green channel showing two neighboring intraepithelial complexes of P2X3 receptor-IR nerve terminals. Note an approaching nerve ﬁber (arrows). b: Combination of the red and green channels, showing two CGRP-IR NEBs contacted by separate populations of CGRP-containing (open arrows) and P2X3 receptor-expressing (arrows) nerve ﬁbers. Fig. 8. a– c: Bronchial CB-IR (red Cy-3 ﬂuorescence) NEB contacted by a complex network of P2X3 receptor-(green FITC-ﬂuorescence) and CB-IR nerve terminals. MVP of 10 confocal optical sections (1.5-m interval). a: Green channel showing an intraepithelial P2X3 receptorexpressing arborization originating from multiple nerve ﬁber endings (arrows). b: Red channel showing CB-IR nerve ﬁbers (open arrowheads) in contact with the CB-IR NEB. c: Combination of both channels revealing that nerve ﬁbers in contact with the NEB express both P2X3 receptors and CB, although the staining intensity varies along the nerve ﬁbers. Fig. 9. a and b: Confocal microscopic images of immunocytochemical double-staining for MBP (red Cy3 ﬂuorescence) and P2X3 receptors (green FITC ﬂuorescence) in the bronchus of a 21-day-old rat. MVP of 30 optical sections (0.8-m interval). a: MBP IR can be detected in myelin sheaths (open arrows) of nerve ﬁbers in close proximity to the bronchial epithelium (E). b: Combination of the red and green channels, showing that FITC-labeled vagal afferent nerve ﬁbers expressing P2X3 receptors (arrows) approach the epithelium, branch, protrude between the epithelial cells, and give rise to intraepithelial nerve terminals, many of which (open arrowheads) are seen close to the luminal surface (L, lumen of the bronchus). The latter intraepithelial structure represents an NEB. The P2X3 receptor-IR nerve ﬁbers are myelinated and the MBP-IR myelin sheets are lost in close proximity to the target (arrowhead). Fig. 10. a– c: Consecutive quinacrine histochemistry and P2X3 receptor staining in a rat bronchus. a: Fluorescence micrograph of quinacrine accumulation in an intraepithelial cell group (arrowhead), indicating the presence of an ATP-storing NEB. b and c: P2X3 receptor immunocytochemistry revealing the presence of extensive receptorexpressing nerve terminals at exactly the same spot as marked in part a (arrowhead). Note the P2X3 receptor IR in bronchial smooth muscle bundles (SM). Single confocal optical section. c: High magniﬁcation detail of part b, clearly showing the P2X3 receptor-IR nerve ﬁber (arrow) that loops through the SM, branches (arrowheads), and gives rise to a terminal intraepithelial arborization. MVP of 20 confocal optical sections (0.95-m interval). Calcitonin gene-related peptide-immunoreactive component of the selective innervation of pulmonary NEBs. As noted above, the vagal sensory component of the innervation of rat pulmonary NEBs appeared to form a clearly different nerve ﬁber population than the sensory CGRP-IR innervation that contacts 32 ADRIAENSEN ET AL. NEBs (Shimosegawa and Said, 1991; Terada et al., 1992; Sorokin et al., 1997). Denervation studies and retrograde tracing from the lung (Springall et al., 1987) (personal observations) strongly indicate that the CGRP-IR nerve ﬁbers that selectively contact NEBs in the rat lung belong to a spinal sensory population, originating in dorsal root ganglia (DRG) T1–T6. The ﬁrst NEBs contacted by CGRP-IR nerve ﬁbers could be detected at GD 19. We then tried to further characterize this CGRP-IR population (Brouns et al., 2003). The ﬁrst observation was that CGRP-positive terminals contacting NEBs invariably colocalize SP (Fig. 11). Using CGRP IR alone, it was impossible to visualize the real contact sites between nerve terminals and the NEB cells that also show a strong CGRP staining. SP does not label rat NEBs. Therefore, CGRP/SP double labeling allows the differentiation of in- Figures 11–19. NEBS: COMPLEX AIRWAY RECEPTORS 33 dividual nerve terminals at the level of NEB cells. It turned out that the spinal sensory CGRP⫹/SP⫹ nerve terminals do not penetrate the epithelium, as was shown for the vagal sensory endings, but that they form a plexus that preferentially contacts the basal surface of NEBs (Figs. 11 and 12), clearly a population that was not present in the initial scheme (Fig. 1). Obviously, airways also harbor a vagal CGRP/SP-IR nerve ﬁber population, but evidence suggests that in rat lungs these vagal ﬁbers mainly originate from the jugular ganglia, and terminals can be found in the epithelium of large-diameter bronchi only—apparently without any speciﬁc relationship with NEBs (personal observations). This population is not discussed further. We then studied the selective CGRP/SP-IR innervation of NEBs in lungs of capsaicin-treated rats (Brouns et al., 2003). After capsaicin treatment, the percentage of NEBs contacted by CGRP-positive nerve terminals was dramatically reduced (5.90%) compared to control lungs (51.80%), while the numbers of CGRP-IR NEBs revealed no significant changes (see above). To further strengthen the capsaicin data, immunocytochemistry with antibodies against the capsaicin receptor VR1 was applied. The results clearly showed that all CGRP-stained nerve ﬁbers in the vicinity of and contacting NEBs express VR1s (Fig. 12) and may therefore be considered capsaicin-sensitive. The VR1/CGRP-IR nerve ﬁber plexus mainly contacts the base of the NEBs, and again the NEBs themselves appeared to be VR1-negative. All available data on the spinal sensory component of the selective innervation of NEBs are summarized in the scheme of Figure 20 (nerve ﬁber population shown in green). Essentially, the sensory nerve ﬁber population involved forms a mainly basal plexus, most likely has its origin in the DRGs T1-T6, can be marked by its CGRP/SP IR, is capsaicin-sensitive, and expresses VR1s. It is now ﬁrmly established that rat pulmonary NEBs may receive at least two different sensory nerve ﬁber populations. Again, however, the question remains as to whether this double sensory innervation can explain the discrepancy between the good evidence that NEBs are hypoxia sensors, and the lack of physiological evidence for the vagal transmission of hypoxia-related stimuli. One possibility is that central hypoxic responses, originating from NEB “receptors” are mediated via a spinal afferent instead of a vagal pathway. However, it may be more complicated than that. Fig. 11. a and b: Immunocytochemical double-staining for substance P (SP; green FITC ﬂuorescence) and CGRP (red Cy3 ﬂuorescence) of an NEB in the intrapulmonary bronchus of a 10-day-old rat. MVP of 31 optical sections (0.8-m interval). a: Green channel showing SP-IR nerve ﬁbers (open arrows) contacting the base of the epithelium (open arrowheads). b: The combination of the green and red channels clearly shows an extensive network of SP/CGRP-IR (yellow ﬂuorescence being indicative of colocalization) nerve ﬁbers (open arrows) contacting the base (open arrowheads) of a CGRP-IR NEB. Fig. 12. Immunocytochemical double-staining for VR1 (green FITC ﬂuorescence) and CGRP (red Cy3 ﬂuorescence), as a marker for the spinal sensory subpopulation of nerve terminals contacting pulmonary NEBs. Bronchiolar CGRP-IR NEB in a 10-day-old rat contacted by extensively branching CGRP-containing nerve ﬁbers that also express VR1 (open arrows; the yellow ﬂuorescence is indicative of colocalization). CGRP IR NEB cells do not show VR1 IR. Reconstruction of seven optical sections (1-m interval). Fig. 13. Bronchial NEB in rat lung at PD10 double-stained for nNOS (red Cy3 ﬂuorescence) and CB (green FITC ﬂuorescence). Combined red and green channels showing nNOS-IR neuronal cell bodies (arrowheads) that give rise to a nitrergic nerve plexus (arrows) in the lamina propria and to intraepithelial nerve terminals (open arrowheads) selectively contacting the CB-IR NEB (asterisk). The image is suggestive of a direct relationship between the selective intraepithelial nitrergic innervation of the NEB, and the nearby nNOS-IR neurons. Note the absence of CB immunostaining from the nitrergic nerves (arrows). MVP of 27 confocal optical sections (1.5-m interval). Fig. 14. a– c: Detail of a CGRP-IR (green FITC-ﬂuorescence) NEB in a respiratory area, contacted by separate nNOS- (red Cy3 ﬂuorescence) and CGRP-IR nerve terminals. MVP of 30 confocal optical sections (0.9-m interval). a: Red channel showing an intraepithelial nNOS-expressing arborization (open arrowheads) originating from subepithelial nerve ﬁbers (arrows). b: Green channel showing CGRP-IR nerve ﬁbers (open arrows) contacting the base of a CGRP-IR NEB. c. Combination of both channels revealing that the intraepithelial nitrergic nerve terminals do not contain CGRP, although the subepithelial CGRP-IR nerve ﬁbers run in very close proximity to the nNOS-IR ﬁbers. Fig. 15. High-magniﬁcation detail of a CB-IR NEB in a rat bronchiole at postnatal day 2. nNOS (arrows) and CB (open arrows) IR are present in different nerve ﬁber populations. Clearly, high-resolution confocal imaging is necessary to differentiate the ﬁne intertwined terminals of both populations. MVPs of eight confocal optical sections (1.5-m interval). Fig. 16. a– c: Immunocytochemical double-staining for VIP (red Cy3ﬂuorescence) and nNOS (green FITC-ﬂuorescence). Detail of an intrapulmonary bronchus at PD10. MVP of 16 confocal optical sections (1.5-m interval). a: Red channel showing a VIP-IR intraepithelial arborization (open arrowheads), suggestive of the presence of an NEB. The lamina propria of the bronchus harbors a weakly VIP-IR neuronal cell body (open arrow). b: Green channel showing that the VIP-IR neuronal cell body is strongly stained for nNOS (arrow). In contrast, the VIP-IR intraepithelial arborization shows weak nNOS IR (arrowheads). c: Combination of both channels revealing the discrepancy in staining intensity between VIP (open arrowheads) and nNOS (arrow). Fig. 17. Confocal detail of nNOS-IR (green FITC-ﬂuorescence) neurons located in a small ganglion situated in the lamina propria of a bronchiole of a 10-day-old rat. Varicose CGRP-IR (red Cy3-ﬂuorescence) nerve ﬁbers intimately surround the two nNOS-IR neurons (asterisks) in this ganglion. Processes of the nitrergic neurons (arrows) follow a course conspicuously similar to that of the CGRP-IR nerve ﬁbers (open arrows). MVP of 17 optical sections (1-m interval). Fig. 18. a and b: Immunocytochemical double-staining for VIP (green FITC-ﬂuorescence) and CGRP (red Cy3-ﬂuorescence). MVPs of 14 confocal optical sections (1-m interval). a: VIP-IR nerve ﬁbers (open arrows) give rise to an extensive intraepithelial arborization (open arrowheads). b: Combination of green and red channels revealing that VIP-IR nerve terminals protrude between CGRP-IR NEB cells. Note that the VIP-IR nerve ﬁbers do not contain CGRP and can be seen to overlay apical NEB cells, close to the luminal surface. Fig. 19. a– c: Immunocytochemical double-staining for VAChT (red Cy3 ﬂuorescence) and CGRP (green FITC ﬂuorescence) in a bronchiole of a 10-day-old rat. MVPs of 15 confocal optical sections (0.9-m interval). a: Red channel showing strong VAChT-IR nerve ﬁbers that are abundant in the subepithelial region (SE). Epithelial cells (arrowheads) show a very faint VAChT IR, while stronger VAChT-IR “terminals” (open arrowheads) can be observed intraepithelially (E). VAChT-IR nerve ﬁbers (arrows) appear to contact the epithelium. b: Green channel demonstrating CGRP-IR spinal sensory nerve terminals (open arrows) contacting a CGRP-IR NEB. c. The combination of both channels shows that the epithelial VAChT staining is present in NEB cells. VAChT-IR nerve terminals appear to contact the NEBs. 34 ADRIAENSEN ET AL. Intrinsic motor component of the selective innervation of pulmonary NEBs. It is well known that nitric oxide (NO) plays an important role in lung physiology and pathophysiology. In relation to hypoxia, especially neuronal NO and endothelial NO appear to be important. Chronic hypoxia in rats induces an upregulation of neuronal NO synthase (nNOS) expression (Xue et al., 1994; Shaul et al., 1995; Gess et al., 1997). Therefore, we examined the possibility of a relationship between intrapulmonary nitrergic structures and pulmonary NEBs. Previous conventional immunocytochemical studies of nNOS, however, reported only very few nNOS-IR nerve ﬁbers in rat lungs (Kobzik et al., 1993). Observing in greater detail and using a much more sensitive detection method, we recently found that part of the NEBs in rat lungs are selectively innervated by nitrergic (nNOS-IR) nerve terminals that penetrate in the epithelium, between the NEB cells, and apparently originate from intrinsic pulmonary nitrergic neurons (Brouns et al., 2002a). From postnatal day 2 onward, nNOS-IR neurons, present mainly in small ganglia close to the mucosa at all levels of intrapulmonary airways, were seen to give rise to complex intraepithelial terminals that invariably colocalize with NEBs (Fig. 13). nNOS IR was absent from the spinal afferent CGRP-IR (Fig. 14) and from the vagal nodose afferent CB-IR (Fig. 15) nerve ﬁber populations that were previously shown to selectively contact NEBs. Quantitative analysis revealed that all NEBs receiving nNOS-IR terminals were also contacted by CGRP-positive nerve ﬁbers, while about 55% were additionally contacted by CB-IR nerves. nNOS-positive ﬁbers approaching NEBs are often very closely related to some of the CGRP-IR ﬁbers contacting the same NEB. The reported nitrergic neurons appeared to colocalize vasoactive intestinal polypeptide (VIP) (Fig. 16), did not express cholinergic markers, and were always surrounded by a basket of CGRP-IR nerve terminals. These nerve terminals originate from CGRP ﬁbers that spiral around the axons of the nitrergic neurons (Fig. 17), and presumably represent collaterals of the spinal CGRP-IR nerve ﬁbers that selectively innervate NEBs. The available data on the intrinsic nitrergic component of the selective innervation of NEBs are summarized in the scheme of Figure 20 (nerve ﬁber population shown in red). Essentially, the nitrergic nerve ﬁber population concerned provides intraepithelial terminals between the NEB cells, has its origin in neurons located in airway ganglia, and reveals a very strong interaction with the spinal afferent CGRP-IR component of the NEB innervation. The characterization of this rather extensive population of highly likely intrinsic intraepithelial motor terminals raises some serious questions about the validity of the mainly electron microscopic data contained in the initial scheme of Figure 1, and about the interpretation of the quantitative electron microscopic data of NEB innervation after vagotomy in rat lungs (Van Lommel and Lauweryns, 1993a). Simultaneous demonstration of vagal sensory, spinal sensory, and intrinsic nitrergic nerve terminals in pulmonary NEBs. The previous chapters revealed that pulmonary NEBs can be contacted by vagal (CB-IR), spinal (CGRP-IR), and nitrergic (nNOS-IR) nerve terminals. However, the evidence that NEBs receive all three of the fully characterized (to date) nerve ﬁber populations is indirect. Recently, we described a reliable multiple immunolabeling method (Brouns et al., 2002b) using unconjugated primary polyclonal antibodies raised in the same species. In this way, simultaneous detection in a single preparation of CB, CGRP, and nNOS in three different nerve ﬁber populations that selectively contact pulmonary NEBs can be achieved (Fig. 21). In the reported procedure, nNOS was visualized using TSA enhancement (PerkinElmer Life Sciences, Boston, MA), followed by detection of CGRP via a ﬂuorophore-coupled Fab secondary antibody, and the subsequent labeling of CB with a ﬂuorophore-coupled “conventional” secondary antibody. All possible remaining binding sites of the second primary antibody were blocked using unlabeled anti-rabbit Fab fragments between the second and third steps. Triple-labeling immunocytochemistry thus provided evidence that part of the NEBs in rat lungs selectively receive at least three different populations of nerve terminals, the characteristics of which are schematized in Figure 20. Other components of the selective innervation of pulmonary neuroepithelial bodies. The functional morphology of rat pulmonary NEBs is clearly much more complex than was predicted by electron microscopic studies (Van Lommel and Lauweryns, 1993a). Moreover, we have now good evidence that several other nerve ﬁber populations, which are not fully characterized yet, provide additional nervous connections of NEBs. A considerable number of NEBs appear to be contacted by profuse beaded VIP-IR intraepithelial nerve terminals (Fig. 18). Although VIP IR was seen to be localized in the intrinsic nitrergic neurons that give rise to nitrergic terminals contacting NEBs, we believe that an additional population of VIP-expressing nerve endings with an as yet unidentiﬁed origin may be involved in the selective innervation of NEBs. As mentioned above, electron microscopy showed that NEBs in rat lungs are contacted by nerve endings that contain small, clear cholinergic-like synaptic vesicles, and often reveal synaptic contacts with NEB cells. Therefore, antibodies against the vesicular acetylcholine transporter (VAChT; a marker for cholinergic nerves) were used to visualize these possible cholinergic nerve terminals in direct relation to NEBs. Weakly VAChT-IR intraepithelial cell groups, characterized as NEBs after multiple immunostaining (Fig. 19), appeared to be contacted by VAChT-IR cholinergic nerve ﬁbers. Although the latter nerve ﬁber population is not yet fully characterized, we have evidence suggesting that cholinergic motor ﬁbers, originating from preganglionic parasympathetic neurons (and hence an as yet uncharacterized population) may be involved. Preliminary data further revealed that tyrosine hydroxylase-IR nerve terminals selectively contact some rat pulmonary NEBs at their basal pole. These nerve ﬁbers likely have their origin in sympathetic ganglia. CONCLUDING REMARKS Selective Innervation of NEBs in the Rat Lung As touched on in the Introduction, it has been suggested that pulmonary NEBs are predominantly, if not exclusively, contacted by (vagal nodose) sensory nerve termi- NEBS: COMPLEX AIRWAY RECEPTORS Fig. 20. Schematic representation of the three fully characterized nerve ﬁber populations that selectively contact pulmonary NEBs. Fig. 21. a– c: Confocal images of an NEB present in the epithelium of an intrapulmonary bronchus of a 10-day-old rat, triple-stained for nNOS (red Cy3 ﬂuorescence), CGRP (green FITC ﬂuorescence), and CB (blue pseudocolor of Cy5 emission in far-red). The CGRP/CB-IR NEB cells are contacted by three different nerve ﬁber populations. In many places, the nerve ﬁbers of the different populations are so close together that they 35 could only be distinguished by confocal laser scanning microscopy. MVP of 33 confocal optical sections (1-m interval). a: Red nNOS-IR nerve ﬁbers (arrowheads) run in the lamina propria and penetrate between the epithelial cells, apparently almost reaching (open arrowheads) the airway lumen (L). b: Green channel showing thin varicose CGRP-IR nerve ﬁbers (open arrows) contacting the basal side of CGRP-IR NEB cells. c: Blue channel showing a bundle of CB-IR nerve ﬁbers (arrows) giving rise to a CB-IR nerve plexus contacting CB-IR NEB cells. 36 ADRIAENSEN ET AL. nals (Van Lommel et al., 1998, 1999; Cutz and Jackson, 1999; Widdicombe, 2001) in several mammalian species, and in particular in rats (Van Lommel and Lauweryns, 1993a). Most of the evidence referred to in these works was based on data obtained from electron microscopic studies, and consequently often from a very limited number of NEBs. In our investigations, neuronal tracing, chemical or mechanical denervation, and (immuno)cytochemistry, in combination with confocal microscopy, have proven to be valuable tools with which to study the overall pattern of NEBs innervation. The application of these techniques has resulted in extensive evidence that NEBs in rat lungs may be selectively contacted by at least ﬁve distinct nerve ﬁber populations that are both sensory and motor in nature, and have different origins. Multiple immunocytochemical staining with presently well-characterized markers revealed that at least part of the pulmonary NEBs are simultaneously contacted by several nerve ﬁber populations. Caution is recommended, however, when dividing NEBs into subpopulations on the basis of the light microscopic data reported in the present review only. It is clear that none of the nerve ﬁber populations contact all NEBs. Moreover, division into subgroups requires a thorough knowledge of the interrelationships between the various nerve ﬁber populations contacting pulmonary NEBs, and thus necessitates reliable, multiple staining procedures. Moreover, it should be taken into account that the use of other markers might reveal even more nerve ﬁber populations related to pulmonary NEBs. In a previous electron microscopic study of rat NEBs, Van Lommel and Lauweryns (1993a) reported “clusters” of nerve ﬁbers located between the neuroendocrine cells of rat NEBs, but stressed that all of these terminals belong to a single population of vagal nodose afferents presenting intraepithelial efferent-like collaterals. In our studies of rat lungs, however, vagal nodose sensory, intrinsic nitrergic, and VIP-IR motor terminals (and possibly extrinsic cholinergic motor terminals) all appeared to penetrate between the neuroendocrine cells of NEBs, thus providing evidence for the existence of more than just one intraepithelial nerve ﬁber population. Therefore, the nerve endings that were reported intact in the ispilateral lung after infranodosal vagotomy (Van Lommel and Lauweryns, 1993a) do not necessarily reﬂect a crossing-over of intact contralateral vagal ﬁbers (Lauweryns and Van Lommel, 1986; Van Lommel and Lauweryns, 1993a). Development of the Innervation of Pulmonary NEBs The migration of neuroblasts from the neural crest to the trachea in rats starts at around GD12 (Morikawa et al., 1978). Neuroblasts can be identiﬁed at the late bronchial bud stage (GD14) by their PGP9.5 IR, after which a primitive nerve network, associated with the airway walls and to a lesser degree with the vasculature, is seen to extend rapidly (Sorokin et al., 1997). It has been suggested that neuroendocrine cell groups are ﬁrst contacted by these postganglionic parasympathetic nerves, thereby becoming real NEBs, and only much later (just before birth) receive sensory nerve endings (Sorokin et al., 1997). These conclusions were based on observations that intrinsic PGP9.5-IR nerve terminals contact pulmonary NEBs around GD17 in in vitro fetal rat lung, while CGRP-IR nerve terminals contacting NEBs in vivo are not common until the end of postnatal week 1 (Sorokin et al., 1997). However, from our studies it is clear that CGRP IR labels spinal, and not vagal, sensory nerve terminals in contact with NEBs, and that the CGRP-negative vagal nodose sensory innervation most likely has been overlooked. It was also conﬁrmed that the ﬁrst CGRP-IR nerve ﬁbers contacting CGRP-IR NEBs could be detected later, at GD19. Our studies further showed that from GD15-16 onward, a large number of subepithelial nerve ﬁbers seen in rat airways express CB, a marker for the vagal nodose sensory nerve ﬁber population that selectively innervates NEBs from GD17 on. The latter observations were conﬁrmed by an ontogenetic study using P2X3 receptor immunolabeling as a marker for the vagal sensory component of NEB innervation (personal observations). On the other hand, pulmonary NEBs do receive part of their innervation from neurons intrinsic to the lungs (Sorokin et al., 1993, 1997). The present study showed that from GD17 onward, PGP9.5-IR NEB cells are contacted by PGP9.5-IR nerve ﬁbers, apparently originating from intrinsic PGP9.5-IR cell bodies that do not reveal nNOS IR at that time. It is likely that at least part of the initial intrapulmonary PGP9.5-IR neuroblasts or neurons, located along the pulmonary epithelial tubes, differentiate into nNOS-IR and/or VIP-IR neurons at a later stage of development and are at least partly responsible for the speciﬁc intraepithelial nitrergic nerve terminals in NEBs. An ontogenetic study (Brouns et al., 2002a) found that the nitrergic nerve terminals selectively contacting rat NEBs can only be visualized from postnatal day 2 onward, supporting the hypothesis that the innervation of rat NEBs undergoes further maturation after birth. Although previous studies proposed that ingrowth of autonomic and sensory ﬁbers converts clusters of neuroendocrine cells into bona ﬁde NEBs (Sorokin et al., 1997), that non-innervated PNEC clusters may be interpreted as developing NEBs, and that the entire pulmonary neuroendocrine system should be regarded as progressing toward complete capture by the nervous system (Sorokin and Hoyt, 1990), our data indicate that even in postnatal lungs the innervation pattern is far from identical for all NEBs, and it is likely that part of the NEBs do not receive a selective innervation. Nevertheless, all NEBs in postnatal rat lungs, regardless of whether they receive a demonstrable innervation or not, apparently produce a similar palette of amines, purines, and peptides, and do not degenerate after birth. It may be that the ingrowth of motor and sensory nerve ﬁbers into pulmonary NEBs does not determine their reaction to certain stimuli. The multicomponent innervation may regulate the sensitivity of NEB cells to stimuli, and serve as a tool for exerting additional local intrapulmonary and CNS reﬂexes. Functional Implications The data presented herein demonstrate that pulmonary NEBs represent an extensive population of very complex intraepithelial receptors that probably can accommodate various sensory modalities. Although the physiological signiﬁcance of the complex innervation pattern of NEBs is still a matter of speculation, the sensory nature of NEBs is beyond dispute. According to recent reviews, no physiological recordings have been obtained yet from single vagal afferent ﬁbers from NEBs, and in general all recordings of the effect of airway hypoxia have yielded negative results NEBS: COMPLEX AIRWAY RECEPTORS (Widdicombe, 2001). However, it seems very unlikely that lung physiologists, in performing many thousands of single ﬁber recordings in the vagus nerve to identify airway receptor populations in rat lungs, have never made registrations from the many hundreds of myelinated vagal nodose neurons that selectively contact pulmonary NEBs in each rat lung. More likely, the populations in question have not yet been recognized among the existing data. Undoubtedly, a considerable part of the already “characterized” airway receptors, a large part of which are mechanoreceptors, will eventually turn out to be related to NEBs. None of the nerve ﬁber populations characterized so far contacts all pulmonary NEBs. However, the observation that only half (or even 10%) of the NEBs receive a certain nerve ﬁber population does not mean they should be considered as less important. After all, the “different” NEB populations do account for at least a few hundred receptor points each. The potential importance of NEBs is supported by the fact that the total number of NEB cells and associated nerve ﬁbers in an animal easily outnumbers carotid body cells and their selective innervation (Sorokin and Hoyt, 1990). Receptosecretory NEB cells are excellent candidates for registering properties of the airway environment. Although there is accumulating evidence for a complete functional system for oxygen sensing in PNECs (Peers and Kemp, 2001), it should be stressed that the exact nature of the possible physiological stimulus modalities of NEB cells in healthy lungs is still unknown, and that it becomes increasingly likely that other stimuli, such as mechanical stimuli, may also be involved. Upon stimulation, NEB cells reveal changes in the exocytosis of DCVs, and presumably release the amines, peptides, ACh, and ATP stored in these secretory granules. In addition to acting on nerve endings in contact with NEBs, the released bioactive substances may exert local actions on, for instance, nearby airway or vascular smooth muscle (paracrine), or could be taken up by nearby blood vessels (endocrine). It is tempting to look at NEBs as local regulators of airway functions that do not necessarily require signaling to the CNS. The main sensor/effector action to hypoxia could be local, and a possible central transduction of hypoxic stimuli may be mediated by spinal rather than vagal afferents in rat lungs. Because they are located at strategic points along the intrapulmonary airways, NEBs appear to be ideally placed to effectuate and coordinate the ﬁne-tuning of local blood ﬂow to local aeration, a physiological function that cannot be performed by the carotid body or central hypoxia receptors. In this respect, pulmonary NEBs can be regarded as inexhaustible local pools of vasoactive transmitters, such as the potent pulmonary vasoconstrictor serotonin and vasodilator CGRP. Continuous release of endogenous CGRP from NEB cells may be responsible for at least part of the homeostatic control of blood vessel relaxation in normoxic lung areas. It has been established that intact primary sensory CGRP/SP-containing and capsaicin-sensitive nerve ﬁbers are required for endogenous CGRP to modulate pulmonary vascular tone in hypoxic pulmonary hypertension (Tjen-A-Looi et al., 1998). Because hypoxia apparently inhibits CGRP secretion from rat NEBs (Springall and Polak, 1993), local physiological hypoxia may result in local inhibition of vasodilation, implying local adjustment of pulmonary perfusion to ventilation. As demonstrated in the present 37 work, all NEBs with an intraepithelial nNOS-IR innervation also reveal basal contacts with CGRP-IR afferents, the presumable collaterals of which appear to follow the course of nitrergic axons and ﬁnally form baskets around the nitrergic neurons in the lamina propria. This nitrergic innervation may be a necessary component for the hypoxic inhibition of CGRP release from NEB cells, requiring CGRP-IR afferents for activation of the mechanism. On the other hand, a system that prevents NEB receptors from continuously transmitting hypoxic information to the CNS would be crucial for avoiding exaggerated central actions that may eventually lead to a general pulmonary vasoconstriction and hypertension. Therefore, a similar mode of action of NO on the transduction of hypoxic stimuli, as reported for carotid body cells (for review, see Prabhakar, 1999), may be proposed for NEBs. Release of NO from the terminals of intrinsic pulmonary nitrergic neurons in NEBs may result in an inhibition of the sensory discharge of NEBs in response to, e.g., local hypoxia. Such a mechanism may keep NEB receptors “quiet” as far as the CNS is concerned, creating time and space for local actions. In this way, afferent signaling to the CNS may be limited to powerful stimuli that necessitate central regulation of respiration. In addition to their receptor function(s), PNECs and NEBs may exert many other functions (independent of their innervation?) during prenatal, perinatal, and early neonatal life (for review, see Sorokin and Hoyt, 1989). Because the vagal nodose sensory component of the selective innervation of NEBs appears to be fully differentiated well before birth, it may be essential for neonatal respiratory adaptation. Future Prospects Although the innervation pattern of pulmonary NEBs in rats appears to be fairly complex, there is no conclusive evidence for assuming that the detailed functional morphological investigation is complete. We even have good (unreported) evidence that other markers for nerve ﬁber populations will provide additional features of the nerve ﬁber populations that have already been characterized, or will reveal additional nerve ﬁber populations contacting pulmonary NEBs. Further characterization of receptors for important neurotransmitters or neuromodulators, present in NEB cells (e.g., 5-HT and CGRP) and in nerve ﬁber endings associated with NEBs (e.g., VIP, CGRP, SP, and ACh), will be necessary to further elucidate the functional signiﬁcance of these substances. It is clear that PNECs show some marked species-speciﬁc differences in their palette of substances (Polak et al., 1993), and that the origin and chemical coding of nerve endings selectively contacting pulmonary NEBs may vary from species to species. Obviously, it will be necessary to validate the data obtained in rats (as discussed in the present review) in other species as well. As mentioned above, to date very little is known about the presence or origin of the different nerve ﬁber populations selectively innervating human pulmonary NEBs. During the last decade, various methods have been developed to study PNECs/NEBs in vitro (Speirs and Cutz, 1993). Because the close relationship between NEB cells themselves, and between NEBs and nerve ﬁbers, is completely lost in isolated cell suspensions of PNECs, more recent in vitro studies have focused on the use of lung slices (Fu et al., 1999). This technique offers a more real- 38 ADRIAENSEN ET AL. istic natural environment for NEBs, and in several cases probably does not even affect the relationship between intrinsic neuronal cell bodies and pulmonary NEBs. Physiological reactions of pulmonary NEBs and their selective innervation to administered neurotransmitters/modulators and (ant)agonists, or on environmental stimuli (e.g., hypoxia and nicotine), could be measured electrophysiologically or by microscopic visualization of physiologic parameters (e.g., calcium concentration and membrane potential) in these lung slices. In addition to investigations on healthy rat lungs, genetic models that reveal lung disorders, or even speciﬁc abnormalities of the pulmonary DNES, should be included in these studies. 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