Occurrence of calcitonin-positive C cells within the distal vagal ganglion and the recurrent laryngeal nerve of the chicken.код для вставкиСкачать
THE ANATOMICAL RECORD 224:43-54 (1989) Occurrence of Calcitonin-Positive C Cells Within the Distal Vagal Ganglion and the Recurrent Laryngeal Nerve of the Chicken YOKO KAMEDA Department of Anatomy, Fukuoka University School of Medicine, Fukuoka 814-01, Japan ABSTRACT The chicken ultimobranchial glands are richly supplied with nerve fibers originating from both the main trunk of the vagus nerve and its branch-the recurrent laryngeal nerve. C cells immunoreactive for calcitonin were invariably found in the large nerve bundles distributed throughout the ultimobranchial glands. In addition, these cells were often present within the distal vagal ganglia and the recurrent laryngeal nerves. The frequency of occurrence and the pattern of distribution of the C cells in the distal vagal ganglia and the recurrent laryngeal nerves were determined in chickens of various ages by means of an immunoperoxidase method with anticalcitonin and antineurofilament antisera. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland was in close contact with the vagus nerve trunk, especially with the distal vagal ganglion, but it was separated from the recurrent laryngeal nerve. The right gland contacted the recurrent laryngeal nerves, its medial edge being frequently penetrated by the nerve, but the gland was separated from the distal vagal ganglion. On the left side, C cells were found in 25 out of 39 distal vagal ganglia but they were not distributed in the recurrent laryngeal nerve. On the right side, the cells were present in 28 out of 43 recurrent laryngeal nerves but absent in the distal vagal ganglia. The results indicate that the C cells secreting a hormone calcitonin can enter into nerves, but their occurrence is restricted to the nerves in close proximity to the ultimobranchial glands. Electron microscopic studies revealed that C cells in the nerves received numerous axon clusters enveloped with Schwann cell cytoplasm. Naked axons regarded as axon terminals were found in direct contact with the surface of C cells. They were mainly composed of efferenttype nerve endings showing the accumulation of numerous small clear vesicles and a few large dense-cored vesicles. In addition, C cells were partly covered with the long cytoplasmic processes of Schwann cells and were also in contact with the Schwann cell perikarya. The C cells in nerves appear to be controlled by neural stimulation. In lower vertebrates including birds, C cells are not distributed in thyroid glands, but form separate organs called ultimobranchial glands. In contrast to mammalian thyroid C cells which have no relation to neural elements, ultimobranchial glands of lower vertebrates receive numerous nerve fibers (Watzka, 1933) and some C cells of frog and chick ultimobranchial glands are in close contact with axon clusters (Robertson, 1967; Hodges and Gould, 1969; Stoeckel and Porte, 1969). The innervation of ultimobranchial glands in the chicken has been studied in detail by the use of an immunoperoxidase staining with antiserum to chick neurofilaments (Kameda et al., 1988). The chick ultimobranchial glands are supplied with a large number of branches derived from both the main trunk of the vagus nerve, especially the distal vagal ganglion, and one of its branches-the recurrent laryngeal nerve. In the course of study of the innervation of chick ultimobranchial glands, I noticed that C cells are distributed in large nerve bundles projecting to the ultimobran0 1989 ALAN R. LISS, INC. chial glands, and furthermore that the cells can migrate into the distal vagal ganglia and the recurrent laryngeal nerves. The present study was undertaken t o show the frequency of occurrence, distribution pattern, and electron microscopic features of the C cells in the distal vagal ganglia and the recurrent laryngeal nerves. The results indicate that C cells, functioning its endocrine cells, can be dispersed in the vagus nerve trunk and its branches and fit into the category of “vagal paraganglionic cells.” MATERIALS AND METHODS Chickens (white leghorn) of both sexes and various ages, newly hatched to 12 months old, were used. Received March 1, 1988; accepted November 28, 1988. 44 Y. KAMEDA C CELLS IN THE VAGUS NERVE OF CHICKENS 45 TABLE 1. The occurrence of C cells in the left distal vagal ganglia from chickens of various ages ment antisera. The preparation and characterization of each antiserum have been described previously (Kameda and Ikeda, 1979; Kameda et al., 1988). The porNo. of cine and salmon calcitonin antisera were used at No. of animals showing 1:3,000 to 1:5,000 and the neurofilaments antisemm a t C cells in the Age animals 1:1,000 to 1:3,000 dilutions. The rabbit primary anti(days) examined distal vagal ganglion bodies (IgGs) were localized using goat antirabbit IgG 1 9 6 (Cappel Labs.) at a dilution of 1:20 for 30 min, rinsed in 5 8 4 phosphate-buffered saline (PBS),pH 7.3, and incubated 10 5 2 with rabbit horseradish peroxidase antiperoxidase com7 6 30 plex (PAP, Miles-Yeda LTD) a t a dilution of 1:lOO for Adult 10 7 (6-12 months) 30 min. The peroxidase reaction was developed with 5 Total 39 Incidence 25/39 (64.1%) mg diaminobenzidine tetrahydrochloride per 100 ml 0.05 M Tris, pH 7.6, plus 0.01%H202. Control reactions included replacing the primary antisera with normal (nonimmune) rabbit serum and absorbing the primary TABLE 2. The occurrence of C cells in the right antisera with an excess of the respective antigens. recurrent laryngeal nerves from chickens of various Electron Microscopy ages Fifteen chickens, 1 day, 10 days, and 6 months old, No. of were used for electron microscopy. Anesthetized aniNo. of animals showing mals were perfused through the heart for 15 min with Age animals C cells in the 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, (davs) examined recurrent nerve with 0.02 mM CaClz and 3% sucrose; then the ultimo1 6 2 branchial glands were excised and fixed for an addi6 6 5 tional 2 hr in the same fixative. The tissues were post10 10 4 fixed for 2 hr in 1%osmium tetroxide in the cacodylate 20 4 3 buffer. After dehydration in ethanol and propylene ox11 8 30 ide, the specimens were embedded in Epon epoxy resin Adult 6 5 (6-12 months) by the standard method. Thin sections were made with Total 43 Incidence 28/43 (65.1%) a diamond knife and were then doubly stained with uranyl acetate and lead citrate. RESULTS Histological and lmmunoperoxidase Staining Ultimobranchial glands, together with adjacent tissues from chickens aged 1day, 5 days, 10 days, 20 days, 1month, 6 months, and 12 months, were removed and processed. Forty-three right glands and 39 left glands were examined (see Tables 1,2). Specimens were fixed in Bouin's solution for 24-48 hr, embedded in paraffin, and then cut into 5 pm-thick serial sections. Some sections were stained with hematoxylin-eosin or periodic acid-Schiff (PAS) reaction. For immunoperoxidase staining, the unlabeled antibody peroxidase-antiperoxidase technique of Sternberger (1979) was used. Prior to incubation of primary antisera, deparaffinized slides were placed in methanolic peroxide solution (one part 3%H202 to five parts methanol, v/v) for 30 min at room temperature to suppress endogenous peroxidase activity. Sections were incubated with the primary antisera at different dilutions for 18 hr at 4°C. The following primary antisera were employed: antiporcine calcitonin, antisalmon calcitonin, and antichick neurofila- Figs. 1-3. Three serial sections of the left ultimobranchial gland from a 6-month-old chicken, stained by three different methods. The left distal vagal ganglion (DVG) is located adjacent to the ultimobranchial gland. x 90. Fig. 1 . Hematoxylin-eosin staining. T w o large branches (arrows) originating from the distal vagal ganglion enter the ultimobranchial gland. Fig. 2. Immunoperoxidase staining with antichick neurofilament The ultimobranchial glands of chickens consist of C cells arranged in large and small clusters in the connective tissue stroma. In addition, there were cysts of various sizes and shapes. They were mostly lined with a single layer of squamous to cuboidal cells, and partially included a multilayered epithelium. The cyst lumina contained various amounts of colloid-like and flocculent materials. The C cells distributed in connective tissue stroma and in cyst epithelia were intensely immunoreactive to anticalcitonin antisera. Numerous nerve fibers immunoreactive to the antineurofilaments antiserum were observed in chick ultimobranchial glands (Figs. 1, 2). They varied in size. Some nerves were large and thick with only a few branches, while others divided into delicate branches inside the glands and exhibited complex ramifications. Innervation of chick ultimobranchial glands has been described in detail in a previous study (Kameda et al., 1988). The ultimobranchial gland was located between the main trunk of the vagus nerve, especially the distal vagal ganglion, and its branch-the recurrent laryn- antiserum. Numerous nerve fibers immunoreactive for neurofilaments are distributed within the ultimobranchial gland. Fig. 3. Immunoperoxidase staining with antiporcine calcitonin antiserum. C cells immunoreactive for calcitonin are observed within the distal vagal ganglion. Fig. 4. Higher magnification of the distal vagal ganglion in Figure 3. C cells gathered in small clusters are scattered among nerve fibers and surround the ganglion cells. x 230. 46 Y.KAMEDA C CELLS IN THE VAGUS NERVE OF CHICKENS Figs. 9, 10. Consecutive sections of the right ultimobranchial gland from a 1-month-old chicken, stained by two different methods. The right recurrent laryngeal nerve (RN) is in close contact with the ultimobranchial gland. C, cyst. X 140. 47 rent nerve enters the ultimobranchial gland. Fig. 10. Immunoperoxidase staining with the calcitonin antiserum. Numerous C cells (arrows) immunoreactive for calcitonin are distributed within the branch. Fig. 9. Hematoxylin-eosin staining. A large branch of the recur- geal nerve-at the beginning of the common carotid artery. The vagus nerve trunk was situated lateral to the ultimobranchial gland and the recurrent laryngeal nerve medial to it. The topographical locations of the right and left ultimobranchial glands were asymmetrical. On the left side, the ultimobranchial gland was attached to the distal vagal ganglion, but it was separated from the recurrent laryngeal nerve. The left ultimobranchial gland received a large number of branches originating from the distal vagal ganglion (Figs. 1, 2). The left carotid body was located cranially in contact with the ultimobranchial gland (Fig. 5).The prominent nerve bundles and several ganglion cells derived from the distal vagal ganglion were observed in the lateral cranial portion of the ultimobranchial gland, i.e., just inferior to the carotid body (Fig. 6). At the lateral cranial portion of the gland, numerous C cells were invariably scattered in the large nerve bundles and surrounded the ganglion cells (Figs. 7,8).The large nerve bundles in the ultimobranchial gland ran toward the carotid body and contributed to the carotid body innervation. C cells were also distributed in the nerves surrounding the carotid body. Furthermore, the cells were found within the distal vagal ganglion (Fig. 3). They were concentrated near the origins of the large Figs. 5-7. Three serial sections of the left ultimobranchial gland from a 1-month-old chicken, stained by three different methods. The left carotid body (CB) is situated cranially in contact with the ultimobranchial gland. C, cyst. x 115. ultimobranchial gland. Fig. 5. Hematoxylin-eosin staining. Arrowheads indicate nerve cells derived from the distal vagal ganglion. Fig. 6. Immunoperoxidase staining with the neurofilament antiserum.The carotid body is enclosed with numerous nerve fibers. Many large bundles are observed in the lateral cranial portion of Fig. 7. Immunoperoxidase staining with the calcitonin antiserum. C cells intermingle with nerve bundles and ganglion cells in the lateral cranial portion of ultimobranchial gland. Fig. 8. Higher magnification of the lateral cranial portion of ultimobranchial gland in Figure 7. C cells (arrows) are dispersed within the large nerve bundles. They (arrowheads) also surround the ganglion cells. x 230. 48 Y. KAMEDA C CELLS IN THE VAGUS NERVE OF CHICKENS 49 B A VN VN RN I DvIfi I RN DVG Fig. 15. A,B: Schematic drawings showing the distribution of C cells within the distal vagal ganglion, the recurrent laryngeal nerve, and their branches. Each dot represents C cell clusters in the nerve. C cells always enter the large nerve bundles distributed in the ultimobranchial gland. In addition, the cells follow the reverse course of the large nerve bundles and migrate into the right recurrent nerve or the left distal vagal ganglion which is located adjacent to the ultimobranchial gland. UB, ultimobranchial gland; CB, carotid body; VN, vagus nerve trunk; DVG, distal vagal ganglion; RN, recurrent laryngeal nerve. A: Ventral view of the right side. The right ultimobranchial gland contacts the recurrent nerve; the medial edge of the gland is penetrated by the nerve. Within the recurrent nerve, C cells are concentrated near the origin of the branches projecting to the ultimobranchial gland. B: Ventral view of the left side. The left ultimobranchial gland is located adjacent to the distal vagal ganglion. Within the distal vagal ganglion, C cells are concentrated near the origin of the branches projecting to the ultimobranchial gland. branches projecting into the ultimobranchial gland. The C cells in the distal vagal ganglia were similar in appearance and immunohistochemical reaction for calcitonin to the cells in ultimobranchial glands (Fig. 3). They were intensely immunoreactive to the calcitonin antisera and usually oval to round in shape. The C cells were dispersed singly or arranged in small groups among the nerve fibers, and intermingled with or surrounded the neural cell bodies (Fig. 4). The number of C cells distributed in the distal vagal ganglia was different from individual to individual. In some ganglia only 5-6 cells per section were observed, while other ganglia contained as many as 50-100 cells per section plane. The occurrence of C cells in the distal vagal ganglia from chickens at various ages ranging from 1 day to 12 months is shown in Table 1. Twenty-five out of 39 distal vagal ganglia contained variable numbers of C cells; the frequency of occurrence was 64.1%.The incidence of C cells in the ganglia did not differ with age. At 1 day after hatching, C cells were already apparent in the distal vagal ganglia, although only a few were present. The branches originating from the recurrent laryngeal nerve often entered the medial caudal portion of the left ultimobranchial gland. C cells were also distributed in the branches of the recarrent laryngeal nerve inside the ultimobranchial glands. However, they were never detected in the left recurrent laryngeal nerve itself which was separated from the gland. Thus, in the ultimobranchial gland, C cells were always dispersed in the large nerve bundles which arose from both the distal vagal ganglia and the recurrent laryngeal nerves. Figure 15B shows the schematic distribution of C cells in the branches originating from the distal vagal ganglion and the recurrent laryngeal nerve, and also within the distal vagal ganglion. On the right side, the ultimobranchial gland contacted the recurrent laryngeal nerve; the medial edge of the gland was frequently penetrated by the nerve. However, it was separated from the distal vagal ganglion. Two prominent branches originating from the recurrent laryngeal nerve entered the medial cranial and caudal poles of the right ultimobranchial gland (Fig. 9). C cells were always detected in these large branches (Fig. 10). In addition, the cells occurred within the recurrent laryngeal nerve; they were concentrated near the origins of these large branches (Figs. 11-13). The C cells were arranged in various Figs. 11-1 3. Three serial sections of the recurrent laryngeal nerve Fig. 13. Immunoperoxidase staining with the calcitonin antiserum. C cells immunoreactive for calcitonin are distributed within the recurrent nerve. (RN) adjacent to the right ultimobranchial gland from 6-month-old chicken, stained by three different methods. X 115. Fig. 11. Hematoxylin-eosin staining. Fig. 12. Immunoperoxidase staining with the neurofilament antiserum. Fig. 14. Higher magnification of the recurrent nerve in Figure 13. The C cells are dispersed singly or arranged in small clusters within the recurrent nerve. x 280. 50 Y. KAMEDA 51 C CELLS IN THE VAGUS NERVE OF CHICKENS sizes of clusters or dispersed singly within the recurrent laryngeal nerve (Fig. 14).The occurrence of C cells in the recurrent nerves from chickens a t various ages ranging from 1 day to 12 months is shown in Table 2. Twenty-eight out of 43 recurrent nerves contained variable numbers of C cells; the frequency of occurrence was 65.1%. The right carotid body was located cranially, somewhat apart from the ultimobranchial gland. The large branches which arose from the recurrent nerve and entered the ultimobranchial gland, ran toward and terminated in the carotid body. One or two small branches originating from the distal vagal ganglion penetrated the lateral edge of the ultimobranchial gland. They intermingled with the branches from the recurrent nerve a t the lateral cranial part of the gland, and then reached the carotid body. C cells were observed in and along the nerves surrounding the carotid body. Figure 15A shows the schematic distribution of C cells in the branches originating from the recurrent laryngeal nerve and the distal vagal ganglion, and also within the recurrent laryngeal nerve. In contrast to the left side, no C cells were present in the distal vagal ganglion which was separated from the ultimobranchial gland. ked axons were in contact with the C cells. The axons contained microtubules, neurofilaments, and small round or spherical mitochondria. In addition, numerous axon terminals forming synaptic contacts with the surface of C cells were detected (Figs. 17, 18). They showed the accumulation of synaptic vesicles and a special differentiation in the membranes, desmosomelike thickening. The synaptic vesicles were commonly composed of numerous small, clear (30-50 nm in diameter) and occasional large, dense-cored (60-100 nm in diameter) vesicles (Fig. 18). Fig. 16. Electron micrograph of a C cell distributed within the nerve. The C cell contains numerous characteristic secretory granules. The perikaryon of Schwann cell (S) and axons (arrows) are in close contact with the C cell. N, nucleus of C cell. x 7,100. axons (arrowheads) showing the accumulation of synaptic vesicles come into direct contact with the C cell. Large electron-pale granules intermingle with typical electron-opaque secretory granules in the cell. N, nucleus of C cell. x 8,500. Fig. 17. A C cell distributed within the nerve. The cell is partially enclosed with the long cytoplasmic process of Schwann cell. Besides the axon clusters ensheathed with the Schwann cell cytoplasm, naked Fig. 18. An axon terminal (arrowhead) apposed the surface of C cell within the nerve. It contains numerous small clear vesicles and a few large dense-cored vesicles. X 15,000. DISCUSSION The ultimobranchial glands of chickens are richly innervated by the branches originating from the vagus nerve trunk, especially from the distal vagal ganglion, and the recurrent laryngeal nerve (Kameda et al., 1988). The present study showed that C cells immunoreactive for calcitonin were invariably distributed in the large branches from the distal vagal ganglion and the recurrent laryngeal nerve inside the ultimobranchial glands. Furthermore, the cells were found within the distal vagal ganglion and the recurrent nerve. Since the topographical locations of ultimobranchial glands are asymmetrical, the pattern of distribution of Electron Microscopy C cells within the nerves was completely different on The ultrastructure of C cells in chick ultimobran- the right side and the left side. The left ultimobranchial gland has been described in a previous study (It0 chial gland had intimate contact with the distal vagal et al., 1986). The C cells distributed in the nerves were ganglion, but it was separated from the recurrent similar in ultrastructural features to the cells in ulti- nerve, whereas the right ultimobranchial gland conmobranchial glands, except that they were iqtimately tacted the recurrent nerve, the medial edge being often associated with neural elements. C cells were filled penetrated by the nerve, but it was separated from the with characteristic secretory granules, ranging from distal vagal ganglion. The invasion of C cells was re100 to 300 nm in diameter (Figs. 16-18). The granules stricted to the left distal vagal ganglion and the right were electron-opaque and usually round t o oval, al- recurrent nerve adjacent to the ultimobranchial though they sometimes revealed pleomorphic shapes. glands. There were no C cells in the right distal vagal In addition, large granules (400 nm in mean diameter) ganglion, and the left recurrent nerve which were sepwith paler content were occasionally observed among arated from the gland. The C cells were concentrated the typical electron-dense secretory granules (Fig. 17). around the origins of the large branches projecting to Each granule was surrounded by a limiting membrane. the ultimobranchial glands within the left distal vagal The C cells were easily discernible by these conspicu- ganglia and the right recurrent nerves. The regions ous secretory granules from other cellular elements in remote from these branches were devoid of C cells. the nerves. The C-cell nuclei were round or oval with a Thus, C cells appear to be able to migrate into the smooth outline. Spheroid and elongated mitochondria distal vagal ganglion and the recurrent nerve along the were dispersed in the cytoplasm. Golgi complexes were large branches, but their migration distance in the seen around the nucleus. Free ribosomes arranged in nerves seems to be short. The number of C cells distributed in the left distal rosettes and cisternae of the rough endoplasmic reticulum were numerous throughout the cytoplasm. In vagal ganglia and the right recurrent nerves differed most cases, the C cells were in close contact with the from individual to individual. In the cases showing nuperikarya of Schwann cells (Fig. 16) and were also merous C cells, 50-100 cells per section plane were partially enveloped by long cytoplasmic processes of detected, while in some cases only a few C cells were Schwann cells (Fig. 17). Schwann cells were agranular present in the nerves. The frequency of occurrence of C and had irregular nuclear profiles. They contained nu- cells in the left distal vagal ganglia was 64.1% and that merous filaments and microtubules and relatively few in the right recurrent nerves was 65.1%; the incidence cytoplasmic organelles. A large number of axon clus- of C cells in the nerves was almost the same on the left ters ensheathed with Schwann cell cytoplasm and na- and right sides. 52 Y. KAMEDA The occurrence of C cells in the nerves did not differ with age. In chickens at various ages ranging from 1 day to 12 months, C cells were present in the nerve almost at the same rate. The calcitonin production of ultimobranchial C cells starts at around 16 days of incubation age; weak immunoreactivity for calcitonin begins to appear in C cells (Kameda, 1984). Thereafter, calcitonin immunoreactivity of C cells rapidly increases during late embryonic periods. At 1 day after hatching, almost all C cells in the ultimobranchial glands exhibit intense immunoreactivity for calcitonin. At this stage, C cells were already observed in the nerves and showed the same distribution patterns as the adults, although the number of C cells in the nerves was still small in proportion to the volume of ultimobranchial glands. It seems that C cells migrate into nerves during early embryonic periods. The ultimobranchial anlage arises from the last pair of pharyngeal pouches during early embryonic development. By the use of the techniques of heterospecific grafting (Le Douarin and Le Lievre, 1971) and fluorogenic amine tracer (Pearse and Polak, 1971), it has been reported that the ultimobranchial bodies of avian and mammalian embryos are colonized by cells derived from the neural crest and that the bodies are only the penultimate origin of the C cells. Although there are conflicting data concerning the origin of the carotid body (see Bock, 1982 for reviews), Pearse et al. (1973) also reported that chief cells (type I cells) of the carotid body are derived from the neural crest. The expression of the various phenotypes characterizing neural crest derivatives has been considered t o depend largely on environmental cues encountered by crest cells during their migration and in the site where they settle (see Le Douarin, 1982 for reviews). Furthermore, exogenous fibronectin and high cell density seem to be necessary for effective directional migration of the crest cells (Rovasio et al., 1983). In the chickens, the ultimobranchial gland is located in contact with the carotid body. C cells often surround the carotid body. The carotid body as well as the ultimobranchial gland are supplied by the branches from the vagus nerve trunk and the recurrent laryngeal nerve, although the carotid body receives more numerous nerve fibers than the ultimobranchial gland (Kameda et al., 1988). The hypothesis of neural crest origin of ultimobranchial C cells and carotid body chief cells cannot be supported or refuted by the results of the present study. Even if both C cells and chief cells are derived from the neural crest, environmental cues for the expression of each phenotype may be each anlage of the ultimobranchial gland and the carotid body. Migratory crest cells expressing the same phenotype could not invade different tissues, i.e., the ultimobranchial anlage and the nerves. It is more likely that after the ultimobranchial gland has received the innervation, C cells proliferate rapidly and migrate into the distal vagal ganglion and the recurrent nerve. In mammals, C cells are distributed in thyroid glands. During early fetal development, mammalian ultimobranchial bodies are incorporated into and then dispersed throughout thyroid parenchyma as C cells. In some animal species, including rabbits, cats, goats, and dogs, the C cells are also observed in the parathyroid gland IV and thymus IV, which have an intimate to- pographical connection with the ultimobranchial anlage during embryonic development (Kameda, 1971, 1981). However, no C cells are dispersed in the parathyroid I11 and thymus 111, which are separated from the ultimobranchial anlage. Thus, C cells of many animal species seem to have an ability to migrate into adjacent organs or tissues from their residential places; in mammals they can enter parathyroid gland and thymus, and in chickens they can enter nervous tissue. In the chick ultimobranchial gland, C cells distributed around large bundles and complex ramifications of nerves are in close contact with numerous axon clusters enveloped with Schwann cell cytoplasm (Hodges and Gould, 1969; Stoeckel and Porte, 1969; Kameda et al., 1988). In addition, naked axons are observed to form synaptic contacts with the surface of C cells; they show an accumulation of synaptic vesicles and a special differentiation in the membranes, desmosome-like thickening. Stoeckel and Porte (1969) reported that the axon terminals in contact with C cells contained small dense-cored vesicles, which were considered to be sympathetic adrenergic nerve terminals. However, our previous study (Kameda et al., 1988) indicated that the nerve endings containing small dense-cored vesicles are only rarely observed and a majority of axon terminals on the C cells show the accumulation of numerous small clear vesicles and a few large dense-cored vesicles. In addition, very few fluorescent fibers are detected around C-cell groups after treatment with Falck-Hillarp procedure, in contrast to the presence of numerous neurofilaments-positive nerve fibers around C-cell groups. It is considered that C cells of chick ultimobranchial glands are mostly innervated by cholinergic parasympathetic fibers. The C cells distributed in the distal vagal ganglion and the recurrent laryngeal nerve also had intimate relation t o neural elements. Many axon clusters ensheathed with Schwann cell cytoplasm and naked axons regarded as axon terminals apposed the C cells. The axon terminals showed the accumulation of numerous small clear vesicles and a few large dense-cored vesicles; the C cells in nerves also receive mainly cholinergic efferent type fibers. Furthermore, they were in close association with Schwann cell perikarya and partially enveloped by long cytoplasmic processes of Schwann cells. It appears that the secretory activity of chicken C cells is controlled by nerve stimulation. The epithelial cell clusters which are distributed in and around nerves and show high contents of catecholamines, are often called “paraganglion.”The occurrence of paraganglia in the vagus nerve trunk and their branches has been described in various mammalian species; the vagal paraganglia are very numerous and widely distributed (Deane et al., 1975; McDonald and Blewett, 1981). They are frequently observed in or near the nodose ganglion, which corresponds to the distal vagal ganglion of the bird (Watzka and Scharf, 1951; Grill0 et al., 1974; Kondo, 1977), and within the recurrent laryngeal nerves (Dahlqvist et al., 1984). It has been reported that the vagal paraganglionic cells are morphologically similar to chief cells of the carotid body and small intensely fluorescent (SIF) cells of the superior cervical ganglion; they contain numerous membrane-bound, electron-opaque granules (60-140 nm in diameter) and are in close contact with numer- C CELLS IN THE VAGUS NERVE OF CHICKENS 53 ous axon clusters (Grillo et al., 1974; Morgan et al., Dahlqvist, A,, B. Carlsoo, S. Domeij, and S. Hellstrom 1984 Morphometric analysis of glomus cells within the recurrent laryngeal 1976; Kondo, 1977). In addition, the cells show intense nerve of the rat. J. Neurocytol., 13r407-416. fluorescence for catecholamines after the treatment of Deane, B.M., A. Howe, and M. Morgan 1975 Abdominal vagal Falck-Hillarp procedure (Gorgas and Bock, 1976). Raparaganglia: Distribution and comparison with carotid body, in the rat. Acta Anat. (Basel), 93:19-28. dioactive labelling is concentrated over the secretory granules of the vagal paraganglionic cells after the in- Grillo, M.A., L. Jacobs, and J.H. Comroe, Jr. 1974 A combined fluorescence histochemical and electron microscopic method for jection of 3H-dopa (Chen and Yates, 1970). The nerve studying special monoamine-containing cells (SIF cells). J. Comp. terminals apposed the vagal paraganglionic cells are Neurol., 153:l-14. Gorgas, K., and P. Bock 1976 Formaldehyde induced catecholamine mainly provided with numerous small clear vesiclesfluorescence in the mouse inferior laryngeal paraganglion. Cell efferent-type terminals-although afferent nerve Tissue Res., 173r139-142. endings also make synaptic contacts with the cells Hodges, R.D., and R.P. Gould 1969 Partial nervous control of the (Grillo et al., 1974; Morgan et al., 1976; Kondo, 1977). avian ultimobranchial body. Experientia, 25~1317-1319. These synapses have been considered to correspond Hodges, R.D., A.S. King, D.Z. King, and E.I. French 1975 The general ultrastructure of the carotid body of the domestic fowl. Cell Tisfully to those seen in the carotid body. Although the sue Res., 162:483-497. functional significance of the vagal paraganglia is not Ito, M., Y. Kameda, and T. Tagawa 1986 An ultrastructural study of known, in view of their close morphological resemthe cysts in chicken ultimobranchial glands, with special reference to C-cells. Cell Tissue Res., 246:39-44. blance to the carotid body, chemoreceptive properties Kameda, Y. 1971 The occurrence and distribution of the parafollicuhave been suggested. lar cells in the thyroid, parathyroid IV and thymus IV in some In contrast to mammalian species, there is little remammals. Arch. Histol. Jpn., 33:283-299. search into the vagal paraganglia in lower vertebrates, Kameda, Y. 1981 Distribution of C-cells in parathyroid gland IV and thymus IV of different mammals studied by immunoperoxidase including birds. In chickens, however, chief cells of the method using anti-calcitonin and anti-C-thyroglobulin antisera. carotid body and granule-containing cells of the aortic Kawasaki Med. J., 7:97-111. wall, which belong to “paraganglionic cells,” have been Kameda, Y. 1984 Ontogeny of chicken ultimobranchial glands studfrequently investigated by electron microscopy (Hodges ied by a n immunoperoxidase method using calcitonin, somatostatin and 19s-thyroglobulin antisera. Anat. Embryol. (Berl.), et al., 1975; Taha and King, 1983; Ookawara et al., 170:139-144. 1974; Kondo, 1974; Abdel-Magied and King, 1984). Kameda, Y., and A. Ikeda 1979 C cell (parafollicular cellf-immunoBased on the definition of “paraganglion,” chicken C reactive thyroglobulin: Purification, identification and immunocells distributed within the distal vagal ganglia and logical characterization, Histochemistry, 60:155-168. the recurrent laryngeal nerves may be included in the Kameda, Y., K. Okamoto, M. Ito, and T. Tagawa 1988 Innervation of the C cells of chicken ultimobranchial glands studied by immucategory of “vagal paraganglionic cells.” In fact, C cells nohistochemistry, fluorescence microscopy and electron microsof chick ultimobranchial glands show intense green copy. Am. J. Anat. 182:353-368. fluorescence for dopamine after freeze-drying and fix- Kine. AS.. D.Z. Kine. R.D. Hodees. and J. Henrv 1975 Svnautic morpholob of the cirotid body Gf the domestic 6wl. C e l f T i k e Res., ation in paraformaldehyde vapor (Almqvist et al., 162:459-473. 1971; Kameda et al., 1988). In addition, avian C cells Kondo, H. 1974 On the granule-containing cells in the aortic wall of exhibit immunoreactivity for tyrosine hydroxylase, inthe young chick. Anat. Rec., 178t253-266. dicatingdopamine-containing cells (Takagi et al., 1984). Kondo, H. 1977 Innervation of SIF cells in the superior cervical and nodose ganglia: An ultrastructural study with serial sections. However, C cells in the nerves do not resemble the Biol. Cell, 30:253-264. paraganglionic cells of the carotid and aortic bodies in Le Douarin, N. 1982 The Neural Crest. Cambridge Univ. Press, Camtheir ultrastructural features. The major difference is bridge. the size of secretory granules; the secretory granules of Le Douarin, N., and C. Le Lievre 1971 Sur l’origine des cellules a calcitonine du corps ultimobranchial de l’embryon d’oiseau. Bull. C cells are larger than those of chief cells and granuleAssoc. Anat. (Nancy), 56:558-568. containing cells. Furthermore, axon clusters or axon McDonald, D.M., and R.W. Blewett 1981 Location and size of carotid terminals in close contact with the C cells are less nubody-like organs (paraganglia) revealed in rats by the permeabilmerous than those on chief cells of the carotid body. ity of blood vessels to Evans blue dye. J. Neurocytol., 10:607-643. The nerve terminals apposing the C cells are provided Morgan, M., R.J. Pack, and A. Howe 1976 Structure of cells and nerve endings in abdominal vagal paraganglia of the rat. Cell Tissue with numerous small clear vesicles-cholinergic efferRes., 169:467-484. ent-type endings. No afferent-type endings are found in Ookawara, S., K. Suzuki, Y. Yoshida, and G. Ooneda 1974 Monoamine-storing cells in the media of the thoracic aorta of Gallus relation to the C cells. Afferent and reciprocal synapses domesticus. Cell Tissue Res., 151~309-316. are demonstrated in the chief cells of chick carotid bodA.G.E., and J.M. Polak 1971 Cytochemical evidence for the ies (King et al., 1975; Taha and King, 1983). In con- Pearse, neural crest origin of mammalian ultimobranchial C cells. Histrast to other paraganglionic cells, C cells synthesize tochemie, 27:96-102. and secrete a hormone, calcitonin, with an endocrine Pearse, A.G.E., J.M. Polak, F.W.D. Rost, J. Fontaine, C. Le Lievre, and N. Le Douarin 1973 Demonstration of the neural crest origin function, and do not show chemoreceptive properties. Yl LITERATURE CITED Aldel-Magied, E.M., and A S . King 1984 Intramural granular cells in the arteries of the carotid body region of the domestic fowl. J. Anat., 139:483 -490. Almqvist, S., E. Malmqvist, C. Owman, M. Ritzen, F. Sundler, and G. Swedin 1971 Dopamine synthesis and storage, calcium-lowering activity, and thyroidal properties of chicken ultimobranchial cells. Gen. Comp. Endocrinol., 17:512-525. Bock, P. 1982 The paraganglia. In: Handbuch der Mikroskopischen Anatomie des Menschen. A. Oksche and L. Vollrath, eds. Springer, Berlin, Vol. VU8. Chen, I.L., and R.D. 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