THE ANATOMICAL RECORD 234:419-431 (1992)
Ultrastructure and lmmunocytochemistry of the Neuroepithelial Bodies in the Lung of the Tiger Salamander, Ambystoma tigrinum (Urodela, Am phibia) L. GONIAKOWSKA-WITALINSKA, J.M. LAUWERYNS, G. ZACCONE, S. FASULO, AND G. TAGLIAFIERRO Department of comparative Anatomy, Jagielloonian University, Krakow, Poland (L.G.- W.); Department of Histopathology, Catholic University of Leuven, Belgium (J.M.L.); Department of Animal Biology and Marine Ecology, University of Messina, Italy (G.Z., S.F); and Institute of Comparative Anatomy, University of Genoa, Italy (G.T.)
ABSTRACT
Light and electron microscopy of the lungs of Ambystoma tigrinum (Urodela) revealed a relatively complex pattern of the neuroendocrine (NE) cells. In the apical parts of smaller septa single NE cells not associated with nerve fibres were covered and surrounded by pneumocytes. The larger septa possessed small areas of ciliated epithelium, in which the NE cells were grouped in a form of neuroepithelial bodies (NEB) consisting of 3-5 cells and covered by goblet cells. NE cells possessed a large nucleus with patches of condensed chromatin, clear cytoplasm, and membrane-bound vesicles of variable morphology and size, containing an electron dense interior surrounded by a lucent space. The size of these dense core vesicles (DCV) ranged from 70-140 nm, while rarely the larger ones exhibited a diameter of 300-600 nm. In some NEB a second type of NE cells was observed for the first time in an amphibian species: these cells communicated with the air space and exhibited on their surface microvilli and a single modified cilium with a 8 + 1 microtubule arrangement. Their cytoplasm contained two types of DCV: dense core granules with a diameter of 140-260 nm and vesicles 320-700 nm in diameter with a moderately electron dense interior. The NEB were associated with intracorpuscular, sensory nerve terminals morphologically afferent and efferent. By immunocytochemistry, the NE cells revealed the presence of serotonin, met-enkephalin, and leu-enkephalin. A paracrine and chemoreceptor role is proposed for NEB of Ambystoma tigrinum. Q 1992 Wiley-Liss, Inc. Key words: Neuroendocrine cells, Pulmonary epithelium, Salamander
The respiratory epithelium of many vertebrates reveals the presence of solitary neuroendocrine (NE) cells (Frohlich, 1949; Lauweryns and Peuskens, 1969; Cutz and Conen, 1972) and/or groups of these cells associated with nerve endings and termed neuroepithelial bodies (NEB) by Lauweryns and Peuskens (1972). The investigations of NE cells have been mostly focused on mammals (for review see Scheuermann, 1987; Sorokin and Hoyt, 19891,but data concerning other vertebrates such as birds (Cook and King, 1969; Wasano and Yamamoto, 1979; Walsch and MacLelland, 1974) and reptiles (Wasano and Yamamoto, 1976; Scheuermann et al., 1983; Pastor et al., 1987, 1989) are also available. The literature dealing with the morphology and immunohistochemistry of NE cells in the lungs of amphibians is rather scarce (Rogers and Haller, 1978, 1980; Wasano and Yamamoto, 1978; GoniakowskaWitalinska, 1981; Cutz et al., 1986; GoniakowskaWitalinska and Cutz, 1990; Goniakowska-Witalinska et al., 1990). As far as Urodela are concerned, solitary noninnervated NE cells were observed in the larval lung of Salamandra salamandra (Goniakowska0 1992 WILEY-LISS,
INC.
Witalinska, 1982) and simple NE cells associated with efferent nerve endings were found in the ciliated epithelium of the newt Triturus alpestris lung. These cells were serotonin-negative (Goniakowska-Witalinska, 1980), although further studies revealed the occurrence of bombesin in their cytoplasm (Cutz et al., 1986). Apart from solitary NE cells, Matsumura (1985) observed neuroepithelial bodies in the pulmonary epithelium of semiaquatic salamander Hynobius nebulosus, while Scheuermann et al. (1989) described single and grouped NE cells in the lungs of entirely aquatic Ambystoma mexicanum. The NE cells described so far in the lungs of tailed amphibians corresponded with the NE cell type common for all the vertebrates studied, Received May 1, 1991; accepted February 25, 1992. The ultrastructural data of this paper were presented at the Third International Congress of Vertebrate Morphology, Antwerp 1989 and published as a n abstract. Address reprint requests to L. Goniakowska-Witalinska, Department of Comparative Anatomy, Jagiellonian University, R. Ingardena 6, 30-060 Krakow, Poland.
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i.e., cells containing characteristic small, electrondense granules. In this paper we report the occurrence of NEB in the lung of the terrestrial salamander Ambystoma tigrinum; they have innervation atypical for amphibians and consist of two cell types: the usual cells with small dense core vesicles and a n unusual type of NE cell with very large dense core vesicles and a modified cilium protruding into the air space.
ined under the light microscope. Ultrathin serial sections were mounted on Formvar-coated grids, stained with uranyl acetate and lead citrate, and examined in a Phillips CM 10 electron microscope at 60 kV. RESULTS General Remarks
The lungs of Ambystoma tigrinum possess several septa of the first, second, and third order which divide MATERIALS AND METHODS the internal lung space into several irregular chambers. The apical part of the 1st and IInd order septa are lmmunohistochemistry thickened by the occurrence of smooth muscle cells and Pieces of the lung tissue were fixed with 4% paraforblood vessels. Both sides of the septa and their apical maldehyde buffered to pH 7.2 with 0.05 M phosphate for 12 h and rinsed in 0.1 M phosphate buffer a t + 4°C. parts are covered by one type of pneumocytes characThe tissues were embedded in Paraplast. Sections of 5 teristic for the lungs of amphibia. Only in the apical p.m were placed on clear glass slides. These sections part of the first order septa small dispersed patches of were subjected to the peroxidase-antiperoxidase ciliated cells are observed (Fig. la). Single neuroendomethod of Sternberger (1979) and the indirect fluores- crine (NE) cells occur intercalated and covered by cence technique of Coons et al. (1955). In order to re- pneumocytes in the apical part of the second order duce the nonspecific binding of proteins, the sections septa, while the first order septa reveal the presence of were incubated with normal goat serum (NGS 1:30,30 neuroepithelial bodies (NEB) surrounded and covered min) diluted in phosphate-buffered saline (PBS) 0.1 M by goblet cells. In some semithin sections, two NEB can at pH 7.4. The PAP procedure was carried out in PBS be found on the top and side of the apical part of the containing 1%NGS. Dewaxed sections were rinsed in septum. The distance between the NEB base and the PBS, pretreated with 2% hydrogen peroxide in PBS, large vessel inside the septum ranges from 14 to 66 p.m. lmmunohistochemistry and incubated in a moist chamber at room temperature for 24 h with the antisera raised in rabbit against seEndocrine cells occur as single or in groups of two to rotonin (INCSTAR, USA, diluted 1 5 0 0 in PBS), syn- five cells. Immunostaining with antibody against serothetic met-benkephalin (Cambridge Research Bio- tonin showed a positive reaction (Fig. lb) in both single chemical Limited, CRB, England, diluted 1:200 in and clusters cells. Met-5-enkephalin immunostaining PBS), synthetic leu-5-enkephalin (CRB, England di- was observed in grouped cells only (Fig. lc), while sinluted 1:200 in PBS), and amphibian bombesin (CRB, gle cells sometimes communicating with the air space England diluted 1:400). Following a rinse in PBS (30 were leu-5-enkephalin-positive(Fig. Id). Immunomin), the sections were incubated in moist chamber for staining with bombesin antiserum was negative in all 1h at room temperature with goat anti-rabbit gamma lung specimens, although control sections from mamglobulin conjugated with fluorescein isothiocyanate malian lungs (adult rats) showed a strong immunore(FITC, Dakopatts, Denmark, diluted 1 : l O O in PBS) or activity. with anti-rabbit IgG (H + L) peroxidase (Chemical CreElectron Microscopy dential ICN, Immunobiologicals, England, diluted 1: All neuroepithelial bodies (NEB) consist of 3-5 100 in PBS) and rinsed in PBS. Peroxidase activity was detected by fresh solution of 3,3’ diaminobenzidine, 4 round or oval neuroendocrine (NE) cells, surrounded HC1 (DAB), 30 mg/100 ml, and H,02 (0.01%) in PBS (5 and covered exclusively by goblet cells (Fig. 2). Solimin); then the sections were dehydrated, and mounted tary NE cells, ultrastructurally identical with those in DPX. The specificity of the antisera were tested by of NEB, are intercalated between pneumocytes on absorption overnight a t + 4°C with different quanti- smaller septa (Fig. 3). The size of NE cells ranges from ties (10-50 p.g/ml of diluted antiserum) of their corre- 13 to 16 p.m. Both NEB and single NE cells are localsponding haptens. Additional control procedures in- ized in the simple epithelium, being separated from the cluded omission of the specific antiserum, or its air space by a cytoplasmic layer (0.1 to 4.0 p.m thick) substitution by a non-immune serum and the use of belonging to the surrounding cells (Figs. 2, 3, 10). In known positive tissue, e.g., mammalian nervous tissue some cases this layer is discontinuous, allowing a direct communication of the NE cells with the air space. and mammalian lung tissue. Some images even suggest that NE cells can be exElectron Microscopy pulsed towards the lung lumen (Fig. 10). The goblet Seven adult males and females (length 22 cm, weight cells surrounding NEB exhibit typical structure, al60 g) of Ambystoma tigrinum were purchased from a though their basal regions may contain dense bodies commercial supplier (Dr. W. De Rover, Herpetologisch and lamellar bodies characteristic for pneumocytes. The NE cell possess a large nucleus with deep inInstituut and Dr. Ce Den Dolder, The Netherlands). The animals were kept for 3 weeks and fed with earth- vaginations of the nuclear envelope and characteristic worms. Before being sacrified they were anaesthetized patches of condensed chromatin (Figs. 2, 3). The cytowith 0.01% MS 222. Lung tissue was immediately fixed plasm contains numerous elongated mitochondria, in a cold mixture of two parts of 1%osmium tetroxide multivesicular bodies, lysosomes, a centrosome located to one part 2.5% glutaraldehyde, cacodylate-buffered to in the supranuclear region, RER and free ribosomes, pH 7.4, rinsed in cacodylate buffer, and quickly dehy- numerous actin filaments, and Golgi complexes, mostly drated in ethanol to be embedded in Epon. Semithin grouped in the basal part of the cell (Fig. 4a-c). Nusections were stained with toluidine blue and exam- merous dense core vesicles (DCV) occur throughout in
NEUROEPITHELIAL BODIES IN THE LUNG OF AMBYSTOMA
Fig. 1, a: Cross section through the first order septa covered in apical part by ciliated epithelium (black arrow) while the other parts of the septa are lined by respiratory epithelium (open arrow). A, air space; V, blood vessel, haematoxylin, eosin. X 370. b: Serotonin immunostaining in two NE cells covered by a thin cytoplasmic layer of
42 1
surrounding cells. Immunoperoxidase staining. A, air space; ct, connective tissue. x 1,100, c: A cluster of met-5-enkephalin immunoreactive NE cells close to the air space (A). Immunoperoxidase staining. X 1,100. d Leu-5-enkephalin immunoreactivity in a solitary NE cells. A, air space. Immunoperoxidase staining. x 1,100.
Fig. 2. A neuroepithelial body in the lung of Ambystoma tigrinum consisting of three neuroendocrine cells surrounded by goblet cells (GI and separated from the air space (A) by a thin cytoplasmic layer. Note condensed chromatin and invaginations of the nuclear envelope. Afferent and efferent nerve endings (arrows) can be seen between NE cells in the upper part of NEB. CT, connective tissue. ~ 5 , 0 0 0 .
Fig. 3. Solitary NE cells resting on basal lamina and covered by neighbouring pneumocytes (P) with characteristic numerous lamellar bodies (asterisk). The elongated mitochondria and small DCV are accumulated in the basal part of the cell. A, air space; C, capillary; CT, connective tissue. x 4,300.
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the entire cytoplasm, though their majority may be eter, glycogen particles, and rare concentric lamellated observed in the basal cell region, adjacent to the un- bodies. The efferent nerve endings (Fig. 8b) are charderlying connective tissue (Figs. 4a-q 5, 6). acterized by the presence of numerous small lucent vesNEB and solitary NE cells usually remain in a direct icles, 40-90 nm in diameter, occasional larger vesicles contact with the basal lamina, although they may be and relatively scarce dense core vesicles. The synaptic separated in some cases from this lamina by a thin junctions between NE cells and nerve terminals reveal cytoplasmic layer belonging to the surrounding cells electron dense layers associated with both synaptic (Figs. 2,4b,c). NE cells are connected to the neighbour- membranes, separated by a 20 nm wide cleft, and a ing cells and between each other by desmosomes (Fig. synaptic grid on the NE cell side (Fig. 9). Vesicles can 4a) in which long tonofilaments are anchored. be observed on both sides of the junction: electron luSome NEB contain single cells of another type: cent in the nerve endings and with dense core in the larger and approximately 20 pm in length. They are NE cell. Serial sections of the intraepithelial nerves located adjacent to type 1 cells on the one hand and to reveal that both morphologically afferent and efferent goblet or ciliated cells on the other, but never inbe- types of endings can be formed by the same nerve fiber tween type 1 cells (Figs. 4b, 5, 6, 11).Some of type 2 which branches a t some distance from the terminals cells (probably the mature ones) are pyramidal in (Fig. 9). The structure of the NEB and their innervashape and extend from the basal lamina to the air tion is summarized on Figure 12. space. Their apical surface is small (approx. 2.0 km in DISCUSSION diameter) and equipped with short microvilli and a single modified cilium a 8 + 1 arrangement of the axoneThe NEB located in bronchopulmonary epithelium of ma1 microtubules. The apical and subapical cytoplasm mammals usually communicate with the air space via contains several small (50-70 nm in diameter) electron NE cells extending to the lumen and possessing short lucent vesicles and very rare dense core granules (Figs. microvilli on their surface (Cutz and Connen, 1972; 11, 12). The main ultrastructural difference between Ericsson et al., 1972; Lauweryns et al., 1972; Hung et type 1 and type 2 cells also concerns the morphology of al., 1973; Hage e t al., 1974; Cutz et al., 1975; Cutz, the dense core vesicles (Fig. 4a,c, 5, 6). In type 1 cells, 1982; Hoyt et al., 1982; Hung, 1982; Sorokin e t al., both solitary and corpuscular, vesicles can be divided 1982, 1983; Lauweryns and Van Lommel, 1987). The into several morphological forms, probably represent- “open” NEB type was also observed in the chicken ing different stages of maturation (Fig. 6): a) “open” (Walsh and McLelland, 1974) and turtle (Scheuermann vesicles 180 nm in diameter, with moderately dense e t al., 1983). content and incomplete limiting membrane, b) vesicles As appears from previous studies, most amphibians 150 nm in diameter with moderately dense core sur- possess “closed” NEB surrounded and covered by a rounded by a clear space 15-30 nm wide, c) electron thick layer of ciliated cells, e.g., in Rana nigromadense core vesicles 70-140 nm in diameter, with a lu- culata (Wasano and Yamamoto, 1978) or goblet cells, cent halo 15-30 nm wide, and d) exceptionally rare e.g., in Hynobius nebulosus (Matsumura, 1985). They large vesicles, 300-600 nm in diameter, with a mod- may also be coated by a thin layer of pneumocytes erately electron dense core surrounded by a lucent and/or ciliated cells, a s in Hyla arborea, Bornbina space 20 nm in width. uariegata, Bufo bufo, Bufo uiridis, Bombina orientalis Type 2 cells possess only two vesicle forms (Figs. 4b, (Goniakowska-Witalinska, 1981; Goniakowska-Wita5, 6), i.e., a) dense core vesicles, 140-260 nm in diam- linska and Cutz, 1990; Goniakowska-Witalinska et al., eter, with a clear and 20 nm wide space and b) large 1990) and in Ambystoma mexicanum (Scheuermann et vesicles, 320-700 nm in diameter, with a moderately al., 1989). The “open” type of amphibian NEB was reelectron dense core surrounded by a lucent halo 20-70 ported exclusively in Bufo rnarinus (Rogers and Haller, nm wide. 1978, 1980). In all NE cells, mature DCV can be observed in close There are both “open and closed” NEB in the lung of vicinity to the basal cell membrane (Figs. 4b, 5, 6, 7). Ambystoma tigrinum. The predominant morphological The solitary NE cells are in a direct contact with the type consist of type 1 NE cells covered and separated basal lamina only in a relatively small area (Fig. 31, from the air space by a thin cytoplasmic layer of goblet whereas the cell membrane forms several invagina- cells. Some NEB however contain type 2 NE cells tions (up to 0.5 pm long) containing DCV in different which often communicate with the lung lumen, thus secretory phases. Coated vesicles can also be observed accounting for the “open” form of the entire body (Fig. in this region of the cell (Fig. 7). The solitary NE cells 11). are devoid of any detectable innervation, while the In all amphibian species investigated so far, the NE NEB are associated with sensory nerves containing cells of NEB tightly adhere to each other. Only in Bornmorphologically afferent and efferent intraepithelial bina uariegata they are separated by the cytoplasmic terminals (Figs. 2, 8a,b) located between both types of processes of the surrounding ciliated cells (GoniaNE cells (Fig. 6), sometimes in the vicinity of the air kowska-Witalinska and Cutz, 19901, as also observed space (Figs. 2, 12) as well as between NE cells and in some birds (Cook and King, 1969) and mammals goblet cells. The main part of the innervation occurs in (Sorokin and Hoyt, 1989). Apart from the NEB, solitary NE cells were dethe basal region of NEB (Figs. 9,121, where apart from nerve endings some nonmyelinated axons can be ob- scribed in the respiratory epithelium of mammals, served in the connective tissue close to the basal lam- birds, reptiles, and amphibians (for review see ina (Fig. 5). The afferent nerve endings (Fig. 8a) are Scheuermann, 1987; Sorokin and Hoyt, 1989). In more numerous and contain abundant mitochondria Urodela, such NE cells are mostly found in the ciliated with long cristae, small vesicles, 40-100 nm in diam- epithelium, displaying different levels of specialization
Fig. 4. a: Part of type 1 NE cell with two multivesicular bodies (black arrows), elongated mitochondrium, several actin filaments (open arrow), free ribosomes scattering over the cytoplasm, small dense core vesicles, and a part of rough endoplasmic reticulum (arrowhead). Note the desmosome between NE cells and pneumocyte in left lower corner. x 49,000. b: Type 2 NE cells with both types of dense
core vesicles: large with moderately electron dense interior (asterisk) and several smaller ones with electron dense core. The goblet cell (GI, contain lamellar body. L, lysosome. x 31,800.c: Type 1 NE cell with Golgi complex (asterisk), and dense core vesicles in their vicinity. P, pneumocyte. x 48,000.
Fig, 5. Type 2 NE cell (11) located between goblet (G)and type 1 NE cells (I).Two types of DCV are seen. Larger DCV (arrows) with moderately electron dense interior are scattered between the smaller DCV with electron dense core. Note an afferent nerve (N) in the connective tissue near the basement membrane (BM). X 11,500.
Fig. 6. Two types of NE cells (I and 11) with afferent nerve endings (N) between them. Note the differences between the DCV in the two types of cells. Type 1 cell (I) contains open vesicles (black arrow), vesicles with moderately electron interior (arrowhead), and dense core vesicles (open arrow). Some DCV in type 2 cells are located near the cell membrane. CT, connective tissue; BM, basal membrane. x 38,000.
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Fig.7. Basal part of solitary NE cells with DCV a t different secretory stages. Coated vesicles (arrow) are also observed. BM, basement membrane. x 71,500.
Fig.8.Nerve endings between type 1 NE cells. x 42,000.a: Morphologically afferent type with small lucent vesicles and mitochondria containing long cristae. b: Morphologically efferent type with numerous small lucent vesicles and some larger ones.
Fig. 9. Basal part of NEB with afferent nerve plexus and several intraepithelial nerve endings of morphologically afferent and efferent type. The cytoplasmic continuity between the upper efferent and lower afferent ending is evident (asterisk). The synapse (arrow) between the cell membrane of type 1 cell and the afferent nerve ending reveals the presence of an electron dense grid on the NE cell side.
Fig. 10. The second order septum. Solitary NE cells separated from the air space (A) by a thin layer of pneumocyte (P) cytoplasm. CT, connective tissue. x 4,200.
x 41,000.
Fig. l l . Apical part of type 2 NE cell with microvilli and atypical cilium containing 8 + 1pattern of axonemal microtubules. Note small electron lucent vesicles (asterisk) and large dense core vesicle (arrow) of 230 nm in diameter. The NE cell is flanked by ciliated (CL) and goblet (G) cell. A, air space. x 38,300.
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Figs. 9-1 1.
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Fig. 12. Schematic representation of open type of neuroepithelial bodies in the lung of Ambystoma tigrinum. The NEB consists of NE cells of type 1 (I) possessing small dense core vesicles, NE cell type 2 (11) with large DCV, and intracorpuscular nerve endings of afferent and efferent type (arrows). The main innervation of NEB is localized in the basal part, where cytoplasmic continuity between afferent and efferent nerve endings is seen (star). NEB is intercalated between goblet (G)and ciliated (CL) cells. A, air space; CT, connective tissue with nerve profile of afferent type.
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TABLE 1. Diameter ranges of dense core vesicles from different types of NE cells in the lungs of tailed amvhibians
Species Hynobius nebulosus Ambystoma mexicanum Salamandra salamandra
Triturus a fpestris Ambystoma tigrinum
Diameter ranges (nm) I. 46-69 11. 80-110 I. 58 11. 73 I. 90-150 and 150-270 11. 150-450 I. 69-189 I. 70-140 and 300-600 11. 140-260 and 320-700
including cells without innervation in Hynobius nebulosus (Matsumura, 1985), Ambystoma mexicanum (Scheuermann e t al., 1989) and A. tigrinum; two types of NE cells in Salamandra salamandra and single cells with efferent innervation in Triturus alpestris (Goniakowska-Witalinska, 1980, 1982). Solitary NE cells devoid of innervation were also described in the lungs of a n ancient fish Polypterus (Scheuermann and De Groodt-Lasseel, 1982), further studies having shown that these cells communicate with the air space (Zaccone e t al., 1989). The secretory vesicles (DCV) found in the NE cells of tailed amphibians exhibit a considerable diversity in size (Table 1) and morphology. The occurrence in the same NE cells of open vesicles, a s well a s vesicles with moderately electron dense interior and dense core vesicles suggest that these forms may represent different maturation stages of DCV. The DCV size (Table 1) ranges from the smallest observed in H . nebulosus and A. mexicanum to the largest occurring in A. tigrinum. Type 1 NE cells of A. tigrinum contain apart from small DCV, also occasionally large ones, similar to those described in NE cells of such anurans as Hyla, Bufo, and Bombina (Goniakowska-Witalinska, 1981, Goniakowska-Witalinska and Cutz, 1990). Type 2 NE cells of A. tigrinum are quite unusual and hitherto have not been described in the lungs of either amphibians or other vertebrates. They contain smaller DCV, comparable to those occurring in type 2 NE cells of S. salamandra, although revealing a more electron dense interior and wider lucent halo. Apart from lungs, type 2 cells in A . tigrinum were also observed in the gill epithelium of neotenic animals and were serotonin, met&enkephalin, and leu-5-enkephalin-positive(Goniakowska-Witalinska et al., in preparation). In the basal part of both solitary and NEB-associated NE cells of A. tigrinum, mature DCV are often seen in contact with the cell membrane, suggesting the release of their content to the basement membrane. In the solitary NE cells, the basal cell membrane forms invaginations which increase the secretory surface and DCV a t different stages of secretion were observed in this region. A similar configuration of the basal part of NE cells was described in Polypterus (Zaccone e t al., 1989). In three species, a toad, Bufo marinus (Rogers and Haller, 1980), a turtle, Pseudemis scripta elegans (Scheuermann et al., 1983; Pastor et al., 19871, and Ambystoma tigrinum, some NE cells communicate with the air space via the apical surface equipped with
References Matsumura, 1985
Scheuermann et al., 1989 Goniakowska-Witalinska, 1982
Goniakowska-Witalinska,1980
short microvilli and a single modified cilium. In the turtle, normal NE cell bear on their surface a cilium with 9 + 0 or 8 + 1 microtubules, while in A. tigrinum only type 2 cell communicating with the air space display the presence of a cilium with 8 + 1 axonemal configuration. The apical cells in the lung of Bufo marinus are highly specialized and possess in their apical part numerous coated vesicles and a single 8 + 1-type cilium. The ultrastructure of these modified cilia suggests a sensory function for the cells bearing them (Sorokin, 1968; Afzelius, 1975). Rogers and Haller (1980) hypothesized that the apical cells in B. marinus serve as receptors transducing stimuli to the basal NE cells. A recepto-sensory role was also postulated for NEB in the turtle (Scheuermann et al., 1983). Our results seem to agree with this supposition, although it should be noted that type 2 cells in Ambystoma are situated between goblet and ciliated cells, their modified cilia being embedded in the surface lining layer. Therefore it seems more plausible that these cells function a s receptors of local chemical changes as well a s sensors monitoring the oxygen and carbon dioxide levels. The investigations carried out so far point out that in amphibians the nerve endings are located in the basal part of NEB (Rogers and Haller, 1978, 1980; Wasano and Yamamoto, 1978; Goniakowska-Witalinska, 1981; Matsumura, 1985; Scheuermann et al., 1989; Goniakowska-Witalinska and Cutz, 1990; GoniakowskaWitalinska et al., 1990). The present study revealed a morphologically atypical innervation of NEB in A . tigrinum: apart from the plexus of morphologically afferent and efferent nerve endings located in the basal portion, some endings of both types occur in the apical region of NEB, a s well between NE cells alone as between NE cells and the surrounding goblet cells (Fig. 11). A similar type of afferent innervation was found in rabbit NEB (Lauweryns and Van Lommel, 1986). Further analysis of serial sections revealed that both morphologically afferent and efferent nerve endings form very often a cytoplasmic continuity as previously reported by Lauweryns and Van Lommel(1987). The ultrastructural features of the synaptic junctions associated with NE cells suggest a transmission of stimuli from NE cell to the nerve ending, thus supporting the hypothetical neuroreceptor function of NEB. Some attention should be also paid to the observations concerning the life cycle of the NE cells in A. tigrinum. Both solitary and NEB-associated NE cells are initially separated from the air space by a cytoplas-
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mic layer of pneumocytes and/or goblet cells. This layer becomes gradually thinner (Fig. 10) up to the point of disruption, whereupon the NE cells are extruding from the epithelium to the lung lumen. This phenomenon has not been so far observed in NE cells of other vertebrates. The comparison of NE cells in the lungs of five species of tailed amphibia reveals some correlation between the mode of life of animal and the structure of NEB. In entirely aquatic A. mexicanum exist single or grouped NE cells, but typical NEB are absent (Scheuermann et al., 1989). The semi-aquatic H . nebulosus possesses closed NEB and solitary NE cells (Matsumura, 1985). Terrestrial tiger salamander has relatively large oxygen requirement during its activity (Full et al., 1988) and the uptake of oxygen proceeds mainly via the lung (Whitford and Hutchison, 1965).In these animals the structure of open NEB is most complicated, with innervation similar to that of the mammalian NEB. The occurrence of serotonin was identified in many vertebrates (see Scheuerman, 1987; Sorokin and Hoyt, 1989), including several amphibian species (Rogers and Haller, 1978; Wasano and Yamamoto, 1978; Cutz et al., 1986; Scheuermann et al., 1989; GoniakowskaWitalinska et al., 1990). On the other hand, the presence of enkephalins in NE cells ofAmbystoma tigrinum is also of interest. Met-5-enkephalin was found in mammals (Cutz et al., 1981; Lauweryns and Van Ranst, 1987) and the present study reports the first observation of this peptide in NE cells of amphibia. Among the investigated species of amphibia only single leu-enkephalin cells were observed in the lungs of Bombina uariegata (Cutz et al., 19861, as well as in the gill NE cells of A. tigrinum (Goniakowska-Witalinska et al., in preparation) and lamprey Lampetra japonica (Zaccone, personal communication). Co-occurrence of met-enkephalin and leu-enkephalin was also found in the NE cells of the gills of some teleost fish species (Zaccone et al., 1992). The present results reveal that single leu-enkephalin positive cells in the lung of A. tigrinum may correspond to type 2 NE cells, although it requires further immunocytochemical studies in EM. In the course of ontogeny the air tract originates from the alimentary canal and the neuroendocrine cells occurring in the lungs are a part of gastroenteric paraneurons (Fujita et al., 1988). The role of NEB in the respiratory epithelium of vertebrates has not been fully elucidated so far, but the recent observations, especially the occurrence of type 2 NE cell, suggest that in Ambystoma tigrinum NEB may serve as receptors sensitive to local chemical changes and are modulated by the central nervous system. They probably also have endocrine andlor paracrine functions. ACKNOWLEDGMENTS
We are grateful to R. Renward, K. Armee for technical and A. Van Dormael for photographical assistance. This investigation was supported by a grant number 93/90 UJ, Poland and a grant from M.U.R.S.T. Rome. LITERATURE CITED Afzelius, B.A. 1975 Ultrastructure of cilia and flagella. In: Handbook of Molecular Cytology. A. Lima-de-Faria, ed. North Holland Publ., Amsterdam, pp. 1219-1242.
Cook, R.D., and AS. King 1969 A neurite-receptor complex in the avian lung: Electron microscopical observations. Experientia, 25: 1162-1164. Coons, A.H., E.H. Leduc, and J.M. Conelly 1955 Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of hiperimmune rabbit. J . Exp. Med., 102:49-59. Cutz, E., and P.E. Conen 1972 Endocrine-like cells in human fetal lungs: An electron microscopic study. Anat. Rec., 173:115-122. Cutz, E., W. Chan, V. Wong, and P.E. Conen 1975 Ultrastructure and fluorescence histochemistry of endocrine (APUD-type) cells in tracheal mucosa of human and various animal species. Cell Tissue Res., 158:425-437. Cutz, E., W. Chan, and N.S. Track 1981 Bombesin, calcitonin and leu-enkephalin immunoreactivity in endocrine cells of human lung. Experientia, 37:765-767. Cutz, E. 1982 Neuroendocrine cells of the lung: An overview of morphologic characteristics and development. Exp. Lung Res., 3: 185-208. Cutz, E., L. Goniakowska-Witalinska, and W. Chan 1986 An immunohistochemical study of regulatory peptides in lungs of amphibians. Cell Tissue Res., 244:227-233. Ericsson, L.E., R. HakAnson, B. Larcon, C. Owman, and E. Sundler 1972 Fluorescence and electron microscopy of amine-storing enterochromaffn-like cells in tracheal epithelium of mouse. Z. Zellforsch., 124532-545, Frohlich, F. 1949 Die “Helle Zelle” der Bronchialschleimhaut und ihre Beziehungen zum Problem der Chemoreceptoren. Frankf. 2. Pathol., 60517-559. Fujita, T., T. Kanno, and S. Kobayashi 1988 Bronchopulmonary paraneuron. In: The Paraneuron. Springer Verlag, Tokyo, pp. 190198. Full, R.J., B.D. Anderson, C.M. Finnerty, and M.E. Feder 1988 Exercising with and without lungs. I. The effects of metabolic cost maximal oxygen transport and body size on terrestrial locomotion in salamander species. J . Exp. Biol., 138:471-485. Goniakowska-Witalinska, L. 1980 Endocrine-like cells in the lungs of the newt Triturus alpestris Laur. Cell Tissue Res., 210521-524. Goniakowska-Witalinska, L. 1981 Neuroepithelial bodies in the lung of the tree frog Hyla arborea L. Cell Tissue Res., 21 7:435-441. Goniakowska-Witalinska, L. 1982 Development of the larval lung of Salamandra salamandra L., A Scanning and transmission electron microscopic study. Anat. Embryol., 164:113-137. Goniakowska-Witalinska, L., and E. Cutz 1990 Ultrastructure of neuroendocrine cells in the lungs of three anuran species. J . Morphol., 203:l-9. Goniakowska-Witalinska, L., J.M. Lauweryns, and L. Van Ranst 1990 Neuroepithelial bodies in the lungs of Bombina orientalis (Boul.). In: Chemoreceptors and Chemoreceptor Reflexes. H. Acker, ed. Plenum Press, New York, pp. 111-117. Goniakowska-Witalihska, L., G. Zaccone, and S. Fasulo 1992 Neuroepithelial cells in the gills of the neotenic tiger salamander Ambystoma tigrinum (in preparation). Hage, E. 1974 Histochemistry and fine structure of endocrine cells in foetal lungs of the rabbit, mouse and guinea pig. Cell Tissue Res., 149513-524. Hoyt, R.F., S.P. Sorokin, and H. Feldman 1982 Small-granule (Neuro) endocrine cells in the infracardial lobe of hamster lung. Number, subtypes and distribution. Exp. Lung Res., 3~273-298. Hung, K-S. 1982 Development of neuroepithelial bodies in pre- and postnatal mouse lung: Scanning electron microscopic study. Anat. Rec., 203:285-291. Hung, K-S., M.S. Hertweck, J.D. Hardy, and C.G. Loosli 1973 Ultrastructure of nerves and associated cells in bronchiolar epithelium of mouse lung. J . Ultrastruct. Res., 43:426-437. Lauweryns, J.M., M. Cokelaere, and P. Theunynck 1972 Neuroepithelial bodies in the respiratory mucosa of various mammals. 2. Zellforsch., 135569-592. Lauweryns, J.M., and J.C. Peuskens 1969 Argyrophil (storing and amine producing?) cells in human infant airway epithelium. Life Sci., 8577-585. Lauweryns, J.M., and J.C. Peuskens 1972 Neuroepithelial bodies (neuroreceptor or secretory organs?) in human infant bronchial and bronchiolar epithelium. Anat. Rec., 172:471-482. Lauweryns, J.M., and A. Van Lommel 1986 Effect of various vagotomy procedures in the reaction to hypoxia of rabbit neuroepithelial bodies: Modulation by intrapulmonary axon reflexes? Exp. Lung Res., llr319-339. Lauweryns, J.M., and A. Van Lommel 1987 Ultrastructure of nerve endings and synaptic junctions in rabbit intrapulmonary neu-
NEUROEPITHELIAL BODIES IN THE LUNG OF AMBYSTOMA roepithelial bodies: A single and serial sections analysis. J. Anat., 151:65-83. Lauweryns, J.M., and L. Van Ranst 1987 Leu-7 immunoreactivity in human, monkey and pig bronchopulmonary neuroepithelial bodies and neuroendocrine cells. J . Histochem. Cytochem., 35t687690. Matsumura, H. 1985 Electron microscopic studies of the lung of the salamander, Hynobius nebulosus. 111. A scanning and transmission electron microscopic observations on neuroepithelial bodies. Okajimas Folia Anat. Jpn., 62:187-204. Pastor, L.M., J . Ballesta, M.T. Castells, R. Perez-Tomas, J.A. Marin, and J.F. Madrid 1989 A microscopic study of the lung of Testudo graeca (Chelonia).J . Anat., 164:19-39. Pastor, L.M., J. Ballesta, F. Hernandez, R. Perez-Tomas, A. Zuasti, and C. Ferrer 1987 A microscopic study of the tracheal epithelium of Testudo graeca and Pseudemis scripta elegans. J. Anat., 153:171-183. Rogers, D.C., and C.J. Haller 1978 Innervation and cytochemistry of neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus). Cell Tissue Res., 195:395-410. Rogers, D.C., and C.J. Haller 1980 The ultrastructural characteristics of the apical cell in the neuroepithelial bodies of the toad lung (Bufo marinus). Cell Tissue Res., 209:485-498. Scheuermann, D.W., M.H.A. De Groodt-Lasseel, C. Stilman, and M.L. Meisters 1983 A correlative light-, fluorescence and electron microscopic study of neuroepithelial bodies in the lung of the redeared turtle, Pseudemis scripta elegans. Cell Tissue Res., 234: 249-269. Scheuermann, D.W. 1987 Morphology and cytochemistry of the endocrine epithelial system in the lung. Internat. Rev. Cytol., 106: 35-88. Scheuermann, D.W., D. Andriaensen, J.P. Timmermans, and M.H.A. De Groodt-Lasseel 1989 Neuroepithelial endocrine cells in the lung of Ambystoma mexicanum. Anat. Rec., 225:139-149. Scheuermann, D.W., and M.H.A. De Groodt-Lasseel 1982 Monoam-
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ine-containing granulated cells in the Polypterus lung. Verh. Anat. Ges., 76:301-302. Sorokin, S.P. 1968 Reconstruction of centriole-formation on ciliogenesis in mammalian lungs. J. Cell Sci., 3:207-230. Sorokin, S.P., and R.F. Hoyt 1989 Neuroepithelial bodies and solitary small-granule cells. In: Lung Cell Biology. D. Massaro ed. M. Dekker Inc., New York, Basel, pp. 191-344. Sorokin, S.P., R.F. Hoyt, and M. Grant 1982 Development of neuroepithelial bodies in fetal rabbit lungs. I. Appearance and functional maturation as demonstrated by high-resolution light microscopy and formaldehyde induced fluorescence. Exp. Lung Res., 3237259. Sorokin, S.P., R.F. Hoyt, and A.D. Persall 1983 Comparative biology of small granule cells and neuroepithelial bodies in the respiratory system: Short review. Am. Rev. Respir. Dis., 128:S26S31. Sternberger, L.A. 1979 Immunocytochemistry. 2nd ED., J . Wiley and Sons, New York. Walsh, C., and J. McLelland 1974 Granular “endocrine”cells in avian respiratory epithelia. Cell Tissue Res., 153269-276. Wasano, K., and T. Yamamoto 1976 Granule-containing cells in the snake respiratory mucosa. Acta Anat. Nippon, 51:299. Wasano, K., and T. Yamamoto 1978 Monoamine-containing granulated cells in the frog lung. Cell Tissue Res., 193:201-209. Wasano, K., and T. Yamamoto 1979 APUD-type recepto-secretory cells in the chicken lung. Cell Tissue Res., 201:197-205. Whitford, W.G., and V.H. Hutchison 1965 Gas exchange in salamanders. Physiol. Zool., 38228-242. Zaccone, G., L. Goniakowska-Witalinska, J.M. Lauweryns, M. Fasulo, and G. Tagliafierro 1989 Fine structure and serotonin immunohistochemistry of the neuroendocrine cells in the lungs of the bichirs Polypterus delhezi and P. ornatipinnis. Bas. Appl. Histochem., 33:277-287. Zaccone, G., L.M. Lauweryns, S. Fasulo, G. Tagliafierro, L. Ainis, and L. Licata 1992 Immunocytochemical localization of serotonin and neuropeptides in the neuroepithelial endocrine cells of teleost and lungfish gills. Acta Zool. Stockh., in press.
Ultrastructure and lmmunocytochemistry of the Neuroepithelial Bodies in the Lung of the Tiger Salamander, Ambystoma tigrinum (Urodela, Am phibia) L. GONIAKOWSKA-WITALINSKA, J.M. LAUWERYNS, G. ZACCONE, S. FASULO, AND G. TAGLIAFIERRO Department of comparative Anatomy, Jagielloonian University, Krakow, Poland (L.G.- W.); Department of Histopathology, Catholic University of Leuven, Belgium (J.M.L.); Department of Animal Biology and Marine Ecology, University of Messina, Italy (G.Z., S.F); and Institute of Comparative Anatomy, University of Genoa, Italy (G.T.)
ABSTRACT
Light and electron microscopy of the lungs of Ambystoma tigrinum (Urodela) revealed a relatively complex pattern of the neuroendocrine (NE) cells. In the apical parts of smaller septa single NE cells not associated with nerve fibres were covered and surrounded by pneumocytes. The larger septa possessed small areas of ciliated epithelium, in which the NE cells were grouped in a form of neuroepithelial bodies (NEB) consisting of 3-5 cells and covered by goblet cells. NE cells possessed a large nucleus with patches of condensed chromatin, clear cytoplasm, and membrane-bound vesicles of variable morphology and size, containing an electron dense interior surrounded by a lucent space. The size of these dense core vesicles (DCV) ranged from 70-140 nm, while rarely the larger ones exhibited a diameter of 300-600 nm. In some NEB a second type of NE cells was observed for the first time in an amphibian species: these cells communicated with the air space and exhibited on their surface microvilli and a single modified cilium with a 8 + 1 microtubule arrangement. Their cytoplasm contained two types of DCV: dense core granules with a diameter of 140-260 nm and vesicles 320-700 nm in diameter with a moderately electron dense interior. The NEB were associated with intracorpuscular, sensory nerve terminals morphologically afferent and efferent. By immunocytochemistry, the NE cells revealed the presence of serotonin, met-enkephalin, and leu-enkephalin. A paracrine and chemoreceptor role is proposed for NEB of Ambystoma tigrinum. Q 1992 Wiley-Liss, Inc. Key words: Neuroendocrine cells, Pulmonary epithelium, Salamander
The respiratory epithelium of many vertebrates reveals the presence of solitary neuroendocrine (NE) cells (Frohlich, 1949; Lauweryns and Peuskens, 1969; Cutz and Conen, 1972) and/or groups of these cells associated with nerve endings and termed neuroepithelial bodies (NEB) by Lauweryns and Peuskens (1972). The investigations of NE cells have been mostly focused on mammals (for review see Scheuermann, 1987; Sorokin and Hoyt, 19891,but data concerning other vertebrates such as birds (Cook and King, 1969; Wasano and Yamamoto, 1979; Walsch and MacLelland, 1974) and reptiles (Wasano and Yamamoto, 1976; Scheuermann et al., 1983; Pastor et al., 1987, 1989) are also available. The literature dealing with the morphology and immunohistochemistry of NE cells in the lungs of amphibians is rather scarce (Rogers and Haller, 1978, 1980; Wasano and Yamamoto, 1978; GoniakowskaWitalinska, 1981; Cutz et al., 1986; GoniakowskaWitalinska and Cutz, 1990; Goniakowska-Witalinska et al., 1990). As far as Urodela are concerned, solitary noninnervated NE cells were observed in the larval lung of Salamandra salamandra (Goniakowska0 1992 WILEY-LISS,
INC.
Witalinska, 1982) and simple NE cells associated with efferent nerve endings were found in the ciliated epithelium of the newt Triturus alpestris lung. These cells were serotonin-negative (Goniakowska-Witalinska, 1980), although further studies revealed the occurrence of bombesin in their cytoplasm (Cutz et al., 1986). Apart from solitary NE cells, Matsumura (1985) observed neuroepithelial bodies in the pulmonary epithelium of semiaquatic salamander Hynobius nebulosus, while Scheuermann et al. (1989) described single and grouped NE cells in the lungs of entirely aquatic Ambystoma mexicanum. The NE cells described so far in the lungs of tailed amphibians corresponded with the NE cell type common for all the vertebrates studied, Received May 1, 1991; accepted February 25, 1992. The ultrastructural data of this paper were presented at the Third International Congress of Vertebrate Morphology, Antwerp 1989 and published as a n abstract. Address reprint requests to L. Goniakowska-Witalinska, Department of Comparative Anatomy, Jagiellonian University, R. Ingardena 6, 30-060 Krakow, Poland.
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i.e., cells containing characteristic small, electrondense granules. In this paper we report the occurrence of NEB in the lung of the terrestrial salamander Ambystoma tigrinum; they have innervation atypical for amphibians and consist of two cell types: the usual cells with small dense core vesicles and a n unusual type of NE cell with very large dense core vesicles and a modified cilium protruding into the air space.
ined under the light microscope. Ultrathin serial sections were mounted on Formvar-coated grids, stained with uranyl acetate and lead citrate, and examined in a Phillips CM 10 electron microscope at 60 kV. RESULTS General Remarks
The lungs of Ambystoma tigrinum possess several septa of the first, second, and third order which divide MATERIALS AND METHODS the internal lung space into several irregular chambers. The apical part of the 1st and IInd order septa are lmmunohistochemistry thickened by the occurrence of smooth muscle cells and Pieces of the lung tissue were fixed with 4% paraforblood vessels. Both sides of the septa and their apical maldehyde buffered to pH 7.2 with 0.05 M phosphate for 12 h and rinsed in 0.1 M phosphate buffer a t + 4°C. parts are covered by one type of pneumocytes characThe tissues were embedded in Paraplast. Sections of 5 teristic for the lungs of amphibia. Only in the apical p.m were placed on clear glass slides. These sections part of the first order septa small dispersed patches of were subjected to the peroxidase-antiperoxidase ciliated cells are observed (Fig. la). Single neuroendomethod of Sternberger (1979) and the indirect fluores- crine (NE) cells occur intercalated and covered by cence technique of Coons et al. (1955). In order to re- pneumocytes in the apical part of the second order duce the nonspecific binding of proteins, the sections septa, while the first order septa reveal the presence of were incubated with normal goat serum (NGS 1:30,30 neuroepithelial bodies (NEB) surrounded and covered min) diluted in phosphate-buffered saline (PBS) 0.1 M by goblet cells. In some semithin sections, two NEB can at pH 7.4. The PAP procedure was carried out in PBS be found on the top and side of the apical part of the containing 1%NGS. Dewaxed sections were rinsed in septum. The distance between the NEB base and the PBS, pretreated with 2% hydrogen peroxide in PBS, large vessel inside the septum ranges from 14 to 66 p.m. lmmunohistochemistry and incubated in a moist chamber at room temperature for 24 h with the antisera raised in rabbit against seEndocrine cells occur as single or in groups of two to rotonin (INCSTAR, USA, diluted 1 5 0 0 in PBS), syn- five cells. Immunostaining with antibody against serothetic met-benkephalin (Cambridge Research Bio- tonin showed a positive reaction (Fig. lb) in both single chemical Limited, CRB, England, diluted 1:200 in and clusters cells. Met-5-enkephalin immunostaining PBS), synthetic leu-5-enkephalin (CRB, England di- was observed in grouped cells only (Fig. lc), while sinluted 1:200 in PBS), and amphibian bombesin (CRB, gle cells sometimes communicating with the air space England diluted 1:400). Following a rinse in PBS (30 were leu-5-enkephalin-positive(Fig. Id). Immunomin), the sections were incubated in moist chamber for staining with bombesin antiserum was negative in all 1h at room temperature with goat anti-rabbit gamma lung specimens, although control sections from mamglobulin conjugated with fluorescein isothiocyanate malian lungs (adult rats) showed a strong immunore(FITC, Dakopatts, Denmark, diluted 1 : l O O in PBS) or activity. with anti-rabbit IgG (H + L) peroxidase (Chemical CreElectron Microscopy dential ICN, Immunobiologicals, England, diluted 1: All neuroepithelial bodies (NEB) consist of 3-5 100 in PBS) and rinsed in PBS. Peroxidase activity was detected by fresh solution of 3,3’ diaminobenzidine, 4 round or oval neuroendocrine (NE) cells, surrounded HC1 (DAB), 30 mg/100 ml, and H,02 (0.01%) in PBS (5 and covered exclusively by goblet cells (Fig. 2). Solimin); then the sections were dehydrated, and mounted tary NE cells, ultrastructurally identical with those in DPX. The specificity of the antisera were tested by of NEB, are intercalated between pneumocytes on absorption overnight a t + 4°C with different quanti- smaller septa (Fig. 3). The size of NE cells ranges from ties (10-50 p.g/ml of diluted antiserum) of their corre- 13 to 16 p.m. Both NEB and single NE cells are localsponding haptens. Additional control procedures in- ized in the simple epithelium, being separated from the cluded omission of the specific antiserum, or its air space by a cytoplasmic layer (0.1 to 4.0 p.m thick) substitution by a non-immune serum and the use of belonging to the surrounding cells (Figs. 2, 3, 10). In known positive tissue, e.g., mammalian nervous tissue some cases this layer is discontinuous, allowing a direct communication of the NE cells with the air space. and mammalian lung tissue. Some images even suggest that NE cells can be exElectron Microscopy pulsed towards the lung lumen (Fig. 10). The goblet Seven adult males and females (length 22 cm, weight cells surrounding NEB exhibit typical structure, al60 g) of Ambystoma tigrinum were purchased from a though their basal regions may contain dense bodies commercial supplier (Dr. W. De Rover, Herpetologisch and lamellar bodies characteristic for pneumocytes. The NE cell possess a large nucleus with deep inInstituut and Dr. Ce Den Dolder, The Netherlands). The animals were kept for 3 weeks and fed with earth- vaginations of the nuclear envelope and characteristic worms. Before being sacrified they were anaesthetized patches of condensed chromatin (Figs. 2, 3). The cytowith 0.01% MS 222. Lung tissue was immediately fixed plasm contains numerous elongated mitochondria, in a cold mixture of two parts of 1%osmium tetroxide multivesicular bodies, lysosomes, a centrosome located to one part 2.5% glutaraldehyde, cacodylate-buffered to in the supranuclear region, RER and free ribosomes, pH 7.4, rinsed in cacodylate buffer, and quickly dehy- numerous actin filaments, and Golgi complexes, mostly drated in ethanol to be embedded in Epon. Semithin grouped in the basal part of the cell (Fig. 4a-c). Nusections were stained with toluidine blue and exam- merous dense core vesicles (DCV) occur throughout in
NEUROEPITHELIAL BODIES IN THE LUNG OF AMBYSTOMA
Fig. 1, a: Cross section through the first order septa covered in apical part by ciliated epithelium (black arrow) while the other parts of the septa are lined by respiratory epithelium (open arrow). A, air space; V, blood vessel, haematoxylin, eosin. X 370. b: Serotonin immunostaining in two NE cells covered by a thin cytoplasmic layer of
42 1
surrounding cells. Immunoperoxidase staining. A, air space; ct, connective tissue. x 1,100, c: A cluster of met-5-enkephalin immunoreactive NE cells close to the air space (A). Immunoperoxidase staining. X 1,100. d Leu-5-enkephalin immunoreactivity in a solitary NE cells. A, air space. Immunoperoxidase staining. x 1,100.
Fig. 2. A neuroepithelial body in the lung of Ambystoma tigrinum consisting of three neuroendocrine cells surrounded by goblet cells (GI and separated from the air space (A) by a thin cytoplasmic layer. Note condensed chromatin and invaginations of the nuclear envelope. Afferent and efferent nerve endings (arrows) can be seen between NE cells in the upper part of NEB. CT, connective tissue. ~ 5 , 0 0 0 .
Fig. 3. Solitary NE cells resting on basal lamina and covered by neighbouring pneumocytes (P) with characteristic numerous lamellar bodies (asterisk). The elongated mitochondria and small DCV are accumulated in the basal part of the cell. A, air space; C, capillary; CT, connective tissue. x 4,300.
NEUROEPITHELIAL BODIES IN T H E LUNG O F AMBYSTOMA
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the entire cytoplasm, though their majority may be eter, glycogen particles, and rare concentric lamellated observed in the basal cell region, adjacent to the un- bodies. The efferent nerve endings (Fig. 8b) are charderlying connective tissue (Figs. 4a-q 5, 6). acterized by the presence of numerous small lucent vesNEB and solitary NE cells usually remain in a direct icles, 40-90 nm in diameter, occasional larger vesicles contact with the basal lamina, although they may be and relatively scarce dense core vesicles. The synaptic separated in some cases from this lamina by a thin junctions between NE cells and nerve terminals reveal cytoplasmic layer belonging to the surrounding cells electron dense layers associated with both synaptic (Figs. 2,4b,c). NE cells are connected to the neighbour- membranes, separated by a 20 nm wide cleft, and a ing cells and between each other by desmosomes (Fig. synaptic grid on the NE cell side (Fig. 9). Vesicles can 4a) in which long tonofilaments are anchored. be observed on both sides of the junction: electron luSome NEB contain single cells of another type: cent in the nerve endings and with dense core in the larger and approximately 20 pm in length. They are NE cell. Serial sections of the intraepithelial nerves located adjacent to type 1 cells on the one hand and to reveal that both morphologically afferent and efferent goblet or ciliated cells on the other, but never inbe- types of endings can be formed by the same nerve fiber tween type 1 cells (Figs. 4b, 5, 6, 11).Some of type 2 which branches a t some distance from the terminals cells (probably the mature ones) are pyramidal in (Fig. 9). The structure of the NEB and their innervashape and extend from the basal lamina to the air tion is summarized on Figure 12. space. Their apical surface is small (approx. 2.0 km in DISCUSSION diameter) and equipped with short microvilli and a single modified cilium a 8 + 1 arrangement of the axoneThe NEB located in bronchopulmonary epithelium of ma1 microtubules. The apical and subapical cytoplasm mammals usually communicate with the air space via contains several small (50-70 nm in diameter) electron NE cells extending to the lumen and possessing short lucent vesicles and very rare dense core granules (Figs. microvilli on their surface (Cutz and Connen, 1972; 11, 12). The main ultrastructural difference between Ericsson et al., 1972; Lauweryns et al., 1972; Hung et type 1 and type 2 cells also concerns the morphology of al., 1973; Hage e t al., 1974; Cutz et al., 1975; Cutz, the dense core vesicles (Fig. 4a,c, 5, 6). In type 1 cells, 1982; Hoyt et al., 1982; Hung, 1982; Sorokin e t al., both solitary and corpuscular, vesicles can be divided 1982, 1983; Lauweryns and Van Lommel, 1987). The into several morphological forms, probably represent- “open” NEB type was also observed in the chicken ing different stages of maturation (Fig. 6): a) “open” (Walsh and McLelland, 1974) and turtle (Scheuermann vesicles 180 nm in diameter, with moderately dense e t al., 1983). content and incomplete limiting membrane, b) vesicles As appears from previous studies, most amphibians 150 nm in diameter with moderately dense core sur- possess “closed” NEB surrounded and covered by a rounded by a clear space 15-30 nm wide, c) electron thick layer of ciliated cells, e.g., in Rana nigromadense core vesicles 70-140 nm in diameter, with a lu- culata (Wasano and Yamamoto, 1978) or goblet cells, cent halo 15-30 nm wide, and d) exceptionally rare e.g., in Hynobius nebulosus (Matsumura, 1985). They large vesicles, 300-600 nm in diameter, with a mod- may also be coated by a thin layer of pneumocytes erately electron dense core surrounded by a lucent and/or ciliated cells, a s in Hyla arborea, Bornbina space 20 nm in width. uariegata, Bufo bufo, Bufo uiridis, Bombina orientalis Type 2 cells possess only two vesicle forms (Figs. 4b, (Goniakowska-Witalinska, 1981; Goniakowska-Wita5, 6), i.e., a) dense core vesicles, 140-260 nm in diam- linska and Cutz, 1990; Goniakowska-Witalinska et al., eter, with a clear and 20 nm wide space and b) large 1990) and in Ambystoma mexicanum (Scheuermann et vesicles, 320-700 nm in diameter, with a moderately al., 1989). The “open” type of amphibian NEB was reelectron dense core surrounded by a lucent halo 20-70 ported exclusively in Bufo rnarinus (Rogers and Haller, nm wide. 1978, 1980). In all NE cells, mature DCV can be observed in close There are both “open and closed” NEB in the lung of vicinity to the basal cell membrane (Figs. 4b, 5, 6, 7). Ambystoma tigrinum. The predominant morphological The solitary NE cells are in a direct contact with the type consist of type 1 NE cells covered and separated basal lamina only in a relatively small area (Fig. 31, from the air space by a thin cytoplasmic layer of goblet whereas the cell membrane forms several invagina- cells. Some NEB however contain type 2 NE cells tions (up to 0.5 pm long) containing DCV in different which often communicate with the lung lumen, thus secretory phases. Coated vesicles can also be observed accounting for the “open” form of the entire body (Fig. in this region of the cell (Fig. 7). The solitary NE cells 11). are devoid of any detectable innervation, while the In all amphibian species investigated so far, the NE NEB are associated with sensory nerves containing cells of NEB tightly adhere to each other. Only in Bornmorphologically afferent and efferent intraepithelial bina uariegata they are separated by the cytoplasmic terminals (Figs. 2, 8a,b) located between both types of processes of the surrounding ciliated cells (GoniaNE cells (Fig. 6), sometimes in the vicinity of the air kowska-Witalinska and Cutz, 19901, as also observed space (Figs. 2, 12) as well as between NE cells and in some birds (Cook and King, 1969) and mammals goblet cells. The main part of the innervation occurs in (Sorokin and Hoyt, 1989). Apart from the NEB, solitary NE cells were dethe basal region of NEB (Figs. 9,121, where apart from nerve endings some nonmyelinated axons can be ob- scribed in the respiratory epithelium of mammals, served in the connective tissue close to the basal lam- birds, reptiles, and amphibians (for review see ina (Fig. 5). The afferent nerve endings (Fig. 8a) are Scheuermann, 1987; Sorokin and Hoyt, 1989). In more numerous and contain abundant mitochondria Urodela, such NE cells are mostly found in the ciliated with long cristae, small vesicles, 40-100 nm in diam- epithelium, displaying different levels of specialization
Fig. 4. a: Part of type 1 NE cell with two multivesicular bodies (black arrows), elongated mitochondrium, several actin filaments (open arrow), free ribosomes scattering over the cytoplasm, small dense core vesicles, and a part of rough endoplasmic reticulum (arrowhead). Note the desmosome between NE cells and pneumocyte in left lower corner. x 49,000. b: Type 2 NE cells with both types of dense
core vesicles: large with moderately electron dense interior (asterisk) and several smaller ones with electron dense core. The goblet cell (GI, contain lamellar body. L, lysosome. x 31,800.c: Type 1 NE cell with Golgi complex (asterisk), and dense core vesicles in their vicinity. P, pneumocyte. x 48,000.
Fig, 5. Type 2 NE cell (11) located between goblet (G)and type 1 NE cells (I).Two types of DCV are seen. Larger DCV (arrows) with moderately electron dense interior are scattered between the smaller DCV with electron dense core. Note an afferent nerve (N) in the connective tissue near the basement membrane (BM). X 11,500.
Fig. 6. Two types of NE cells (I and 11) with afferent nerve endings (N) between them. Note the differences between the DCV in the two types of cells. Type 1 cell (I) contains open vesicles (black arrow), vesicles with moderately electron interior (arrowhead), and dense core vesicles (open arrow). Some DCV in type 2 cells are located near the cell membrane. CT, connective tissue; BM, basal membrane. x 38,000.
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Fig.7. Basal part of solitary NE cells with DCV a t different secretory stages. Coated vesicles (arrow) are also observed. BM, basement membrane. x 71,500.
Fig.8.Nerve endings between type 1 NE cells. x 42,000.a: Morphologically afferent type with small lucent vesicles and mitochondria containing long cristae. b: Morphologically efferent type with numerous small lucent vesicles and some larger ones.
Fig. 9. Basal part of NEB with afferent nerve plexus and several intraepithelial nerve endings of morphologically afferent and efferent type. The cytoplasmic continuity between the upper efferent and lower afferent ending is evident (asterisk). The synapse (arrow) between the cell membrane of type 1 cell and the afferent nerve ending reveals the presence of an electron dense grid on the NE cell side.
Fig. 10. The second order septum. Solitary NE cells separated from the air space (A) by a thin layer of pneumocyte (P) cytoplasm. CT, connective tissue. x 4,200.
x 41,000.
Fig. l l . Apical part of type 2 NE cell with microvilli and atypical cilium containing 8 + 1pattern of axonemal microtubules. Note small electron lucent vesicles (asterisk) and large dense core vesicle (arrow) of 230 nm in diameter. The NE cell is flanked by ciliated (CL) and goblet (G) cell. A, air space. x 38,300.
NEUROEPITHELIAL BODIES IN T H E LUNG OF AMBYSTOMA
Figs. 9-1 1.
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Fig. 12. Schematic representation of open type of neuroepithelial bodies in the lung of Ambystoma tigrinum. The NEB consists of NE cells of type 1 (I) possessing small dense core vesicles, NE cell type 2 (11) with large DCV, and intracorpuscular nerve endings of afferent and efferent type (arrows). The main innervation of NEB is localized in the basal part, where cytoplasmic continuity between afferent and efferent nerve endings is seen (star). NEB is intercalated between goblet (G)and ciliated (CL) cells. A, air space; CT, connective tissue with nerve profile of afferent type.
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TABLE 1. Diameter ranges of dense core vesicles from different types of NE cells in the lungs of tailed amvhibians
Species Hynobius nebulosus Ambystoma mexicanum Salamandra salamandra
Triturus a fpestris Ambystoma tigrinum
Diameter ranges (nm) I. 46-69 11. 80-110 I. 58 11. 73 I. 90-150 and 150-270 11. 150-450 I. 69-189 I. 70-140 and 300-600 11. 140-260 and 320-700
including cells without innervation in Hynobius nebulosus (Matsumura, 1985), Ambystoma mexicanum (Scheuermann e t al., 1989) and A. tigrinum; two types of NE cells in Salamandra salamandra and single cells with efferent innervation in Triturus alpestris (Goniakowska-Witalinska, 1980, 1982). Solitary NE cells devoid of innervation were also described in the lungs of a n ancient fish Polypterus (Scheuermann and De Groodt-Lasseel, 1982), further studies having shown that these cells communicate with the air space (Zaccone e t al., 1989). The secretory vesicles (DCV) found in the NE cells of tailed amphibians exhibit a considerable diversity in size (Table 1) and morphology. The occurrence in the same NE cells of open vesicles, a s well a s vesicles with moderately electron dense interior and dense core vesicles suggest that these forms may represent different maturation stages of DCV. The DCV size (Table 1) ranges from the smallest observed in H . nebulosus and A. mexicanum to the largest occurring in A. tigrinum. Type 1 NE cells of A. tigrinum contain apart from small DCV, also occasionally large ones, similar to those described in NE cells of such anurans as Hyla, Bufo, and Bombina (Goniakowska-Witalinska, 1981, Goniakowska-Witalinska and Cutz, 1990). Type 2 NE cells of A. tigrinum are quite unusual and hitherto have not been described in the lungs of either amphibians or other vertebrates. They contain smaller DCV, comparable to those occurring in type 2 NE cells of S. salamandra, although revealing a more electron dense interior and wider lucent halo. Apart from lungs, type 2 cells in A . tigrinum were also observed in the gill epithelium of neotenic animals and were serotonin, met&enkephalin, and leu-5-enkephalin-positive(Goniakowska-Witalinska et al., in preparation). In the basal part of both solitary and NEB-associated NE cells of A. tigrinum, mature DCV are often seen in contact with the cell membrane, suggesting the release of their content to the basement membrane. In the solitary NE cells, the basal cell membrane forms invaginations which increase the secretory surface and DCV a t different stages of secretion were observed in this region. A similar configuration of the basal part of NE cells was described in Polypterus (Zaccone e t al., 1989). In three species, a toad, Bufo marinus (Rogers and Haller, 1980), a turtle, Pseudemis scripta elegans (Scheuermann et al., 1983; Pastor et al., 19871, and Ambystoma tigrinum, some NE cells communicate with the air space via the apical surface equipped with
References Matsumura, 1985
Scheuermann et al., 1989 Goniakowska-Witalinska, 1982
Goniakowska-Witalinska,1980
short microvilli and a single modified cilium. In the turtle, normal NE cell bear on their surface a cilium with 9 + 0 or 8 + 1 microtubules, while in A. tigrinum only type 2 cell communicating with the air space display the presence of a cilium with 8 + 1 axonemal configuration. The apical cells in the lung of Bufo marinus are highly specialized and possess in their apical part numerous coated vesicles and a single 8 + 1-type cilium. The ultrastructure of these modified cilia suggests a sensory function for the cells bearing them (Sorokin, 1968; Afzelius, 1975). Rogers and Haller (1980) hypothesized that the apical cells in B. marinus serve as receptors transducing stimuli to the basal NE cells. A recepto-sensory role was also postulated for NEB in the turtle (Scheuermann et al., 1983). Our results seem to agree with this supposition, although it should be noted that type 2 cells in Ambystoma are situated between goblet and ciliated cells, their modified cilia being embedded in the surface lining layer. Therefore it seems more plausible that these cells function a s receptors of local chemical changes as well a s sensors monitoring the oxygen and carbon dioxide levels. The investigations carried out so far point out that in amphibians the nerve endings are located in the basal part of NEB (Rogers and Haller, 1978, 1980; Wasano and Yamamoto, 1978; Goniakowska-Witalinska, 1981; Matsumura, 1985; Scheuermann et al., 1989; Goniakowska-Witalinska and Cutz, 1990; GoniakowskaWitalinska et al., 1990). The present study revealed a morphologically atypical innervation of NEB in A . tigrinum: apart from the plexus of morphologically afferent and efferent nerve endings located in the basal portion, some endings of both types occur in the apical region of NEB, a s well between NE cells alone as between NE cells and the surrounding goblet cells (Fig. 11). A similar type of afferent innervation was found in rabbit NEB (Lauweryns and Van Lommel, 1986). Further analysis of serial sections revealed that both morphologically afferent and efferent nerve endings form very often a cytoplasmic continuity as previously reported by Lauweryns and Van Lommel(1987). The ultrastructural features of the synaptic junctions associated with NE cells suggest a transmission of stimuli from NE cell to the nerve ending, thus supporting the hypothetical neuroreceptor function of NEB. Some attention should be also paid to the observations concerning the life cycle of the NE cells in A. tigrinum. Both solitary and NEB-associated NE cells are initially separated from the air space by a cytoplas-
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mic layer of pneumocytes and/or goblet cells. This layer becomes gradually thinner (Fig. 10) up to the point of disruption, whereupon the NE cells are extruding from the epithelium to the lung lumen. This phenomenon has not been so far observed in NE cells of other vertebrates. The comparison of NE cells in the lungs of five species of tailed amphibia reveals some correlation between the mode of life of animal and the structure of NEB. In entirely aquatic A. mexicanum exist single or grouped NE cells, but typical NEB are absent (Scheuermann et al., 1989). The semi-aquatic H . nebulosus possesses closed NEB and solitary NE cells (Matsumura, 1985). Terrestrial tiger salamander has relatively large oxygen requirement during its activity (Full et al., 1988) and the uptake of oxygen proceeds mainly via the lung (Whitford and Hutchison, 1965).In these animals the structure of open NEB is most complicated, with innervation similar to that of the mammalian NEB. The occurrence of serotonin was identified in many vertebrates (see Scheuerman, 1987; Sorokin and Hoyt, 1989), including several amphibian species (Rogers and Haller, 1978; Wasano and Yamamoto, 1978; Cutz et al., 1986; Scheuermann et al., 1989; GoniakowskaWitalinska et al., 1990). On the other hand, the presence of enkephalins in NE cells ofAmbystoma tigrinum is also of interest. Met-5-enkephalin was found in mammals (Cutz et al., 1981; Lauweryns and Van Ranst, 1987) and the present study reports the first observation of this peptide in NE cells of amphibia. Among the investigated species of amphibia only single leu-enkephalin cells were observed in the lungs of Bombina uariegata (Cutz et al., 19861, as well as in the gill NE cells of A. tigrinum (Goniakowska-Witalinska et al., in preparation) and lamprey Lampetra japonica (Zaccone, personal communication). Co-occurrence of met-enkephalin and leu-enkephalin was also found in the NE cells of the gills of some teleost fish species (Zaccone et al., 1992). The present results reveal that single leu-enkephalin positive cells in the lung of A. tigrinum may correspond to type 2 NE cells, although it requires further immunocytochemical studies in EM. In the course of ontogeny the air tract originates from the alimentary canal and the neuroendocrine cells occurring in the lungs are a part of gastroenteric paraneurons (Fujita et al., 1988). The role of NEB in the respiratory epithelium of vertebrates has not been fully elucidated so far, but the recent observations, especially the occurrence of type 2 NE cell, suggest that in Ambystoma tigrinum NEB may serve as receptors sensitive to local chemical changes and are modulated by the central nervous system. They probably also have endocrine andlor paracrine functions. ACKNOWLEDGMENTS
We are grateful to R. Renward, K. Armee for technical and A. Van Dormael for photographical assistance. This investigation was supported by a grant number 93/90 UJ, Poland and a grant from M.U.R.S.T. Rome. LITERATURE CITED Afzelius, B.A. 1975 Ultrastructure of cilia and flagella. In: Handbook of Molecular Cytology. A. Lima-de-Faria, ed. North Holland Publ., Amsterdam, pp. 1219-1242.
Cook, R.D., and AS. King 1969 A neurite-receptor complex in the avian lung: Electron microscopical observations. Experientia, 25: 1162-1164. Coons, A.H., E.H. Leduc, and J.M. Conelly 1955 Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of hiperimmune rabbit. J . Exp. Med., 102:49-59. Cutz, E., and P.E. Conen 1972 Endocrine-like cells in human fetal lungs: An electron microscopic study. Anat. Rec., 173:115-122. Cutz, E., W. Chan, V. Wong, and P.E. Conen 1975 Ultrastructure and fluorescence histochemistry of endocrine (APUD-type) cells in tracheal mucosa of human and various animal species. Cell Tissue Res., 158:425-437. Cutz, E., W. Chan, and N.S. Track 1981 Bombesin, calcitonin and leu-enkephalin immunoreactivity in endocrine cells of human lung. Experientia, 37:765-767. Cutz, E. 1982 Neuroendocrine cells of the lung: An overview of morphologic characteristics and development. Exp. Lung Res., 3: 185-208. Cutz, E., L. Goniakowska-Witalinska, and W. Chan 1986 An immunohistochemical study of regulatory peptides in lungs of amphibians. Cell Tissue Res., 244:227-233. Ericsson, L.E., R. HakAnson, B. Larcon, C. Owman, and E. Sundler 1972 Fluorescence and electron microscopy of amine-storing enterochromaffn-like cells in tracheal epithelium of mouse. Z. Zellforsch., 124532-545, Frohlich, F. 1949 Die “Helle Zelle” der Bronchialschleimhaut und ihre Beziehungen zum Problem der Chemoreceptoren. Frankf. 2. Pathol., 60517-559. Fujita, T., T. Kanno, and S. Kobayashi 1988 Bronchopulmonary paraneuron. In: The Paraneuron. Springer Verlag, Tokyo, pp. 190198. Full, R.J., B.D. Anderson, C.M. Finnerty, and M.E. Feder 1988 Exercising with and without lungs. I. The effects of metabolic cost maximal oxygen transport and body size on terrestrial locomotion in salamander species. J . Exp. Biol., 138:471-485. Goniakowska-Witalinska, L. 1980 Endocrine-like cells in the lungs of the newt Triturus alpestris Laur. Cell Tissue Res., 210521-524. Goniakowska-Witalinska, L. 1981 Neuroepithelial bodies in the lung of the tree frog Hyla arborea L. Cell Tissue Res., 21 7:435-441. Goniakowska-Witalinska, L. 1982 Development of the larval lung of Salamandra salamandra L., A Scanning and transmission electron microscopic study. Anat. Embryol., 164:113-137. Goniakowska-Witalinska, L., and E. Cutz 1990 Ultrastructure of neuroendocrine cells in the lungs of three anuran species. J . Morphol., 203:l-9. Goniakowska-Witalinska, L., J.M. Lauweryns, and L. Van Ranst 1990 Neuroepithelial bodies in the lungs of Bombina orientalis (Boul.). In: Chemoreceptors and Chemoreceptor Reflexes. H. Acker, ed. Plenum Press, New York, pp. 111-117. Goniakowska-Witalihska, L., G. Zaccone, and S. Fasulo 1992 Neuroepithelial cells in the gills of the neotenic tiger salamander Ambystoma tigrinum (in preparation). Hage, E. 1974 Histochemistry and fine structure of endocrine cells in foetal lungs of the rabbit, mouse and guinea pig. Cell Tissue Res., 149513-524. Hoyt, R.F., S.P. Sorokin, and H. Feldman 1982 Small-granule (Neuro) endocrine cells in the infracardial lobe of hamster lung. Number, subtypes and distribution. Exp. Lung Res., 3~273-298. Hung, K-S. 1982 Development of neuroepithelial bodies in pre- and postnatal mouse lung: Scanning electron microscopic study. Anat. Rec., 203:285-291. Hung, K-S., M.S. Hertweck, J.D. Hardy, and C.G. Loosli 1973 Ultrastructure of nerves and associated cells in bronchiolar epithelium of mouse lung. J . Ultrastruct. Res., 43:426-437. Lauweryns, J.M., M. Cokelaere, and P. Theunynck 1972 Neuroepithelial bodies in the respiratory mucosa of various mammals. 2. Zellforsch., 135569-592. Lauweryns, J.M., and J.C. Peuskens 1969 Argyrophil (storing and amine producing?) cells in human infant airway epithelium. Life Sci., 8577-585. Lauweryns, J.M., and J.C. Peuskens 1972 Neuroepithelial bodies (neuroreceptor or secretory organs?) in human infant bronchial and bronchiolar epithelium. Anat. Rec., 172:471-482. Lauweryns, J.M., and A. Van Lommel 1986 Effect of various vagotomy procedures in the reaction to hypoxia of rabbit neuroepithelial bodies: Modulation by intrapulmonary axon reflexes? Exp. Lung Res., llr319-339. Lauweryns, J.M., and A. Van Lommel 1987 Ultrastructure of nerve endings and synaptic junctions in rabbit intrapulmonary neu-
NEUROEPITHELIAL BODIES IN THE LUNG OF AMBYSTOMA roepithelial bodies: A single and serial sections analysis. J. Anat., 151:65-83. Lauweryns, J.M., and L. Van Ranst 1987 Leu-7 immunoreactivity in human, monkey and pig bronchopulmonary neuroepithelial bodies and neuroendocrine cells. J . Histochem. Cytochem., 35t687690. Matsumura, H. 1985 Electron microscopic studies of the lung of the salamander, Hynobius nebulosus. 111. A scanning and transmission electron microscopic observations on neuroepithelial bodies. Okajimas Folia Anat. Jpn., 62:187-204. Pastor, L.M., J . Ballesta, M.T. Castells, R. Perez-Tomas, J.A. Marin, and J.F. Madrid 1989 A microscopic study of the lung of Testudo graeca (Chelonia).J . Anat., 164:19-39. Pastor, L.M., J. Ballesta, F. Hernandez, R. Perez-Tomas, A. Zuasti, and C. Ferrer 1987 A microscopic study of the tracheal epithelium of Testudo graeca and Pseudemis scripta elegans. J. Anat., 153:171-183. Rogers, D.C., and C.J. Haller 1978 Innervation and cytochemistry of neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus). Cell Tissue Res., 195:395-410. Rogers, D.C., and C.J. Haller 1980 The ultrastructural characteristics of the apical cell in the neuroepithelial bodies of the toad lung (Bufo marinus). Cell Tissue Res., 209:485-498. Scheuermann, D.W., M.H.A. De Groodt-Lasseel, C. Stilman, and M.L. Meisters 1983 A correlative light-, fluorescence and electron microscopic study of neuroepithelial bodies in the lung of the redeared turtle, Pseudemis scripta elegans. Cell Tissue Res., 234: 249-269. Scheuermann, D.W. 1987 Morphology and cytochemistry of the endocrine epithelial system in the lung. Internat. Rev. Cytol., 106: 35-88. Scheuermann, D.W., D. Andriaensen, J.P. Timmermans, and M.H.A. De Groodt-Lasseel 1989 Neuroepithelial endocrine cells in the lung of Ambystoma mexicanum. Anat. Rec., 225:139-149. Scheuermann, D.W., and M.H.A. De Groodt-Lasseel 1982 Monoam-
431
ine-containing granulated cells in the Polypterus lung. Verh. Anat. Ges., 76:301-302. Sorokin, S.P. 1968 Reconstruction of centriole-formation on ciliogenesis in mammalian lungs. J. Cell Sci., 3:207-230. Sorokin, S.P., and R.F. Hoyt 1989 Neuroepithelial bodies and solitary small-granule cells. In: Lung Cell Biology. D. Massaro ed. M. Dekker Inc., New York, Basel, pp. 191-344. Sorokin, S.P., R.F. Hoyt, and M. Grant 1982 Development of neuroepithelial bodies in fetal rabbit lungs. I. Appearance and functional maturation as demonstrated by high-resolution light microscopy and formaldehyde induced fluorescence. Exp. Lung Res., 3237259. Sorokin, S.P., R.F. Hoyt, and A.D. Persall 1983 Comparative biology of small granule cells and neuroepithelial bodies in the respiratory system: Short review. Am. Rev. Respir. Dis., 128:S26S31. Sternberger, L.A. 1979 Immunocytochemistry. 2nd ED., J . Wiley and Sons, New York. Walsh, C., and J. McLelland 1974 Granular “endocrine”cells in avian respiratory epithelia. Cell Tissue Res., 153269-276. Wasano, K., and T. Yamamoto 1976 Granule-containing cells in the snake respiratory mucosa. Acta Anat. Nippon, 51:299. Wasano, K., and T. Yamamoto 1978 Monoamine-containing granulated cells in the frog lung. Cell Tissue Res., 193:201-209. Wasano, K., and T. Yamamoto 1979 APUD-type recepto-secretory cells in the chicken lung. Cell Tissue Res., 201:197-205. Whitford, W.G., and V.H. Hutchison 1965 Gas exchange in salamanders. Physiol. Zool., 38228-242. Zaccone, G., L. Goniakowska-Witalinska, J.M. Lauweryns, M. Fasulo, and G. Tagliafierro 1989 Fine structure and serotonin immunohistochemistry of the neuroendocrine cells in the lungs of the bichirs Polypterus delhezi and P. ornatipinnis. Bas. Appl. Histochem., 33:277-287. Zaccone, G., L.M. Lauweryns, S. Fasulo, G. Tagliafierro, L. Ainis, and L. Licata 1992 Immunocytochemical localization of serotonin and neuropeptides in the neuroepithelial endocrine cells of teleost and lungfish gills. Acta Zool. Stockh., in press.
