Ultrastructural and Histochemical Changes in the Frog Taste Organ following Denervation

Transcription

1 Arch. histol. jap., Vol. 47, No.1(1984) p Ultrastructural and Histochemical Changes in the Frog Taste Organ following Denervation Kuniaki TOYOSHIMA,I Eiko HONDA,2 Satoshi NAKAHARA2 and Akitatsu SHIMAMURA1 Department of Oral Anatomy (Prof. A. SHIMAMURA)1 and Oral Physiology (Prof. S. NAKAHARA),2 Kyushu Dental College, Kitakyushu, Japan Received August 26, 1982; revised manuscript received July 6, 1983 Summary. The fine structure of the taste organ in the Rana catesbiana was observed by light and electron microscopy. The taste organ consists of three distinct cell types, the taste, supporting and basal cells as well as nerve elements. The present findings suggest that the taste cell might function not only as a chemoreceptor cell, but also as a paracrine cell. The basal cell also may have a dual function as both mechanoreceptor and paracrine cell. Furthermore, taste organs have been examined at intervals from 3 hrs to 120 days after sectioning of the glossopharyngeal nerves. The taste organs were almost intact throughout the experimental period after denervation and even after ceasing to produce impulses from chemical or mechanical stimuli. The dense-cored vesicles of the taste or basal cells, which may possess a paracrine action, still remain in the taste organ even 120 days after denervation. It can be concluded that the morphological integrity of the frog taste organ does not absolutely need the presence of the gustatory fibers in contrast to the nerve-dependent nature of the mammalian taste bud. The taste organs of frogs have been widely used in electrophysiological (KUSANo and SATO, 1957; RAPUZZI and CASELLA, 1965) and ultrastructural studies (GRAZIADEI and DEHAN, 1971; STENSAAS, 1971; SHIMAMURA and TOKUNAGA, 1970; DURING and ANDRES, 1976; TOYOSHIMA and SHIMAMURA, 1982) of taste sensation. It has long been known that the integrity of the taste bud is dependent on a trophic influence of the gustatory nerves. It is generally believed that the taste bud disappears rapidly after denervation and reappears following regeneration of the nerve fibers (GUTH, 1957; FARBMAN, 1969; IWAYAMA, 1970; FUJIMOTO and MURRAY, 1970). ROBBINS (1967) reported, however, that the taste organ of the frog never completely disappears even after a long time of denervation. The present study reports on the details of the changes in the frog taste organs after denervation as well as the fine structures of these sense organs.

2 32 K. ToYOSHIMA MATERIALS et al.: AND METHODS Frogs (Rana catesbiana) were anesthetized by intramuscular injection of tubocurarine chloride (20 mg/kg), and the glossopharyngeal nerves, mm long, were severed bilaterally in the oral cavities. The proximal and distal cut ends were turned backward along the course of the stumps. Impulses generated by chemical and mechanical stimuli were recorded in the peripherally cut ends of the glossopharyngeal nerves at intervals of 3, 6, 12 and 24 hrs and 2, 3, 10, 20, 30, 60, 90 and 120 days after surgery. Then the tongues were removed, minced and immediately fixed with 2.5 glutaraldehyde in a 0.1 M sodium cacodylate buffer (ph 7.3) at 4 C for 6 hrs. Postfixation was made in 1 osmium teroxide in the same buffer for 2 hrs. The tissues were then dehydrated in ethanol and embedded in Epon 812. Thin sections obtained with a Porter-Blum MT-2B ultramicrotome were stained with uranyl acetate followed by lead citrate, and then examined under a JEM-100C electron microscope. Thick sections were stained with toluidine blue for light microscopic examinations. Some thick sections were stained with PAS after the removal of epoxy resin. For detection of ATPase, frozen sections were cut in a cryostat to a 15 Pm thickness and incubated for 15 min at 37 C in the medium of WACHSTEINand MEtssEL (1957). Following incubation, the sections were washed in distilled water and then treated with an aqueous solution of yellow ammonium sulfide, and examined with a light microscope. RESULTS The taste organ of the frog is located in the dorsal epithelium of the fungiform papilla ( 1) and is recognized easily by the strong reaction for ATPase ( 2). Three type of cells: the taste, the supporting and the basal cells; and nerve elements were distinguished within the organ. Taste and supporting cells extended throughout the total thickness of the organ, while the basal cells were located only at its basal periphery ( 1) Vertical section of a taste organ in the fungiform with PAS-positive secretion granules are seen. x 230 papilla stained with PAS. Supporting cells Basal cells (arrows) are positive to PAS. 2. ATPase activity reaction of fungiform papillae. A strong is found in taste organs. x 110

3 Frog 3. Taste Organ following Denervation 33 Electron micrograph of a vertically sectioned taste organ. The taste organ contains three types of cells, taste (T), supporting (S) and basal (B) cells. Intragemmal nerve fibers (arrows) are seen in the basal part. x 1,650. Inset. A closer view of the basal part of a taste organ. x 6,200 The nuclei of the taste cells were located at the basal one third of the whole height of the taste organ, where the cells appeared thickest. The cytoplasm of the cell extended branches from the supranuclear region and terminated in a few short microvilli ( 3, 4). Characteristic of the taste cell was the presence of a number of densecored vesicles, nm in diameter, in the basal cytoplasm ( 5). The density of their contents was variable. They tended to accumulate towards the junction with the nerve fibers; synaptic contact between a taste cell and a nerve fiber was frequently observed in that region ( 6). In addition to coated invaginations, Q-shaped figures strongly suggesting exocytosis of the dense-cored vesicles were seen on the plasma membrane ( 5, 6). The site where these exocytotic structures occurred was not juxtaposed or approached by nerve fibers. A subsynaptic cistern located close to the cell surface contacting the nerve fibers was sometimes seen in the taste cell ( 7). Well-developed Golgi complexes, centrioles and an occasional single cilium were also components of this cell ( 8). The major part of the supporting cells, in which the nucleus and many secretory granules were located, was situated at the apical third of the taste organ ( 1, 4). They showed a positive reaction to PAS. A number of mitochondria and welldeveloped Golgi complexes were seen among them. The basal cells were located at the basal-lateral region of the taste organ ( 1,

4 34 K. ToYOSHIMA et al.: Electron micrograph of an apical region of a taste cell (T) ending in microvilli. ing cell. X6,500 S support- 5. Basal part of a taste organ. Basal processes of taste cells (7) contain many dense-cored vesicles. The arrow shows a coated invagination of the plasma membrane. N nerve ending, x 16,600 9) and numbered 8-10 in each taste organ. Their cytoplasm, positive in PAS reaction, extended along the basal lamina toward the center of the taste organ ( 1, 10). A number of dense-cored vesicles measuring nm in diameter were contained in the entire cytoplasm ( 9, 10). Coated and non-coated invaginations of the plasma membrane, which probably represented exocytosis of the vesicles, were occasionally seen ( 10). They did not necessarily occur in relation to nerves supplying the cell. No half -desmosomal attachment to the basal lamina was observed at the inferior border of the cell. Digital cytoplasmic processes protruding into the intercellular space (Fig. 9) and occasionally into the connective tissue in close association with the subgemmal nerve fibers ( 11) were observed. The first changes in some nerve terminals within the taste organ could be observed 6-12 hrs after denervation. These alterations were characterized by the appearance of electron-dense bodies and myelin-like membranous lamellar bodies ( 12). Intragemmal nerve terminals became totally degenerated within 24 hrs and completely disappeared by the 7th day. Myelin-like membranous lamellar bodies were found in the intercellular spaces at the basal region of the denervated taste organ from the 7th to the 120th day after surgery ( 13). As the nerve terminals within the taste organ degenerated, impulses to chemical and mechanical stimuli declined and ceased from the 7th day ( 14). The denervated taste organ was almost intact throughout the 120 days period after denervation ( 15, 16). The dense-cored vesicles of the taste cell still remained in the basal cytoplasm for this period, although considerably decreased in number ( 17). The size and density of individual vesicles were not remarkably altered. Occasionally,

5 Frog Taste Organ following Dencrvation i 6. A synaptic contact between a taste cell (T) and an afferent nerve ending (N). Abundant dense-cored vesicles accumulate near the latter. A coated invagination of the plasma membrane is seen (arrow). x 24,000. Inset. Exocytosis (arrow) of a dense-cored vesicles in a taste cell (T). x 45, Synaptic site between a taste cell (T) and an efferent nerve ending (N). A subsynaptic cistern is seen in the taste cell just beneath the synaptic membrane. x 33,200 S. Well-developed Golgi complex (G) and basal body in a taste cell (T). x 26,600. Inset. A single cilium in a taste cell. x 21,300

6 36 K. TovosHuIA et al.: 9 9. Basal cell (B) containing abundant dense-cored vesicles. Note the digital cytoplasmic processes (arrows). x 8,300 1(l A foot process of a basal cell (B) elongated along the basal lamina (arrows). x 11,400. Inset. A closer view showing a possible exocytosis of a dense-cored vesicles (B). N nerve fiber. x 29, Basal part of a taste organ. Note a digital process of a basal cell (B) penetrating lamina (arrows) into the connective tissue. N nerve fiber. x 14,100 the basal

7 Frog Taste Organ following denervation Basal region of a taste organ 6 hrs after denervation. A myelin-like membranous lamellar body is seen in the intragemmal nerve terminal. x 12, Basal region of a taste organ 7 days after denervation. Nerve fibers have completely disappeared. Myelin-like bodies (arrows) are seen in the intercellular spaces. x 12,000 A B 14. Response of the peripherally cut end of the glossopharyngeal nerve fibers to chemical and mechanical stimuli. Normal (A) and 120 days after denervation (B). Any electrophysiological response is completely absent (B).

8 38 K. TOYOSHIMA A vertically Taste its organ lacking et al.: sectioned taste 120 days after nerve elemens. organ 30 days denervation. x 1,300 after Its denervation. structure x 1,500 is intact except for

9 Frog 17. Taste Organ following Denervation 39 A highly magnified view of the basal part of a taste organ 120 days after denervation. The dense-cored vesicles are still seen in the basal cytoplasm of the taste cells (T). The basal cell (B) contains abundant dense-cored vesicles. x 16,000. Inset. 12-shaped membrane invaginations (arrows) found in the basal plasma membrane of a taste cell (T). x 21,600 fl-shaped membrane invaginations were found on the plasma membrane ( 17). The basal cells were recognized in the basal periphery of the taste organ throughout the experimental period ( 18). As were those in the taste cell, the vesicles of the basal cell were retained in the cytoplasm until the 120th day after denervation ( 17). Exocytotic figures of the vesicles were also observed at places where no nerve fiber seemed to approach ( 19). Mitotically dividing cells were encountered in the basal part of the denervated taste organ ( 20) as in the normal state. Strong ATPase activity persisted in the taste organ for 30 days after denervation ( 20); then the activity lessened in intensity. Trace amount of the reaction products, however, were still 18. Light micrograph of a taste organ stained with PAS, 120 days after denervation. PAS-positive basal cells (arrows) are seen in the basal periphery of a taste organ. X110

10 K. ToYOSHIMA 40 et al.: Electron micrograph of a part of the basal cell (B) 120 days after denervation. An exocytotic figure is seen (arrow). x 23,500. Inset. Another example of exocytosis seen on the basal plasma membrane of a basal cell (arrow). x 23, Light micrograph of a taste organ 120 days after denervation. the basal part of the denervated taste organ. x 970 A dividing cell is seen in observed in the taste organ even 120 days after denervation ( 22). No distinct change in ATPase activity was noticed in the intrapapillary connective tissue throughout the postoperative period. DISCUSSION The dependency of the taste buds upon the nerve supplying them has been long known in most vertebrates. It is generally believed that total denervation of the gustatory papilla results in a rapid degeneration followed by the complete disappearance of the taste buds as described before. Furthermore, when the gustatory papilla is removed, reappearance of the taste buds takes place only where the nerve fibers have been regenerated (ZALEWSKI,1970; ToYOSHIMA,1979). RoBBINs (1967), on the other hand, has demonstrated that the frog taste organ becomes gradually smaller over a period of months after section of the gustatory nerve fibers, but never completely disappears. The independency of the taste buds from the gustatory nerve fibers has also been reported in certain lower vertebrates (WRIGHT, 1968; PoRITSKYand SINGER,1977). In the present study, the frog taste organ maintained its structures without sensory innervation for 120 days. The morphological finding was also histochemically supported by the presence of ATPase activity. The ATPase activity observed in the connective tissue cores of denervated fungiform papillae was considered to be related

11 Frog Taste Organ following Denervation ATPase activity sists in the taste in a fungiform organ. x 230 papilla 30 days after denervation. 22. ATPase activity observed in the taste organ 120 days after also seen in the intrapapillary connective tissue. x 250 The activity denervation. The still peractivity is to Schwann cells surviving in their original sites for a long time as described previously by IWAYAMA and NASA (1969). The taste cell of the frog taste organ extended from the basal lamina to the apical surface of the organ and directly contacted the oral environment. The dense-cored vesicles observed in the taste cell tended to accumulate near the synaptic contact with afferent nerve endings. In both normal and denervated taste cells, exocytosis of these vesicles was observed at sites not associated with a nerve. These findings suggest that the taste cell may have a dual function as both chemoreceptor and paracrine cell. As for the basal cell, having characteristic digital cytoplasmic processes, its function is poorly understood. It seems reasonable to consider, however, that it may act as a mechanoreceptor when the frog preys on live bait with its tongue. A mechanoreceptive function of the frog taste organ was also confirmed in the present electrophysiological findings. The basal cell persisted in the denervated taste organ even after it ceased to produce an electrophysiological response to mechanical stimuli. Further, it seems likely, from the present findings, that the basal cell also may possess a paracrine action like the taste cell. It is of interest to speculate that the morphological and possibly functional relationships between the taste and the basal cells in the frog taste organ are reminiscent of those between the open and closed type of cells in the gastro-enteric basal-granulated cells (see review by FuJITA, 1977). Further studies into this problem seem worthy to be promoted. Acknowledgements. Heartfelt thanks are due to Prof. H. NAKAYAMAof Kyushu University for his critical reading of the manuscript and valuable advice.

12 42 K. TOYOSHIMA et al. REFERENCES During, M. v. and K. H. Andres : The ultrastructure of taste and touch receptors of the frog's taste organ. Cell Tissue Res. 165: (1976). Farbman, A. I.: Fine structure of degenerating taste buds after denervation. J. Embryol. exp. Morphol. 22: (1969). Fujimoto, S. and R. G. Murray : Fine structure of degeneration and regeneration in denervated rabbit vallate taste buds. Anat. Rec. 168: (1970). Fujita, T.: Concept of paraneurons. Arch. histol. jap. 40 (Suppl.):1-12 (1977). Graziadei, P. P. C. and R. S. DeHan : The ultrastructure of the frog's taste organ. Acta anat. 80: (1971). Guth, L.: The effects of glossopharyngeal nerve transection on the circumvallate papilla of the rat. Anat. Rec. 128: (1957). Iwayama, T.: Changes in the cell population of taste buds during degeneration and regeneration of their sensory innervation. Z. Zellf orsch. 110: (1970). Iwayama. T. and O. Nada : Histochemical observations on phosphatase activities of degenerating and regenerating taste buds. Anat. Rec. 163: (1969). Kusano, K. and M. Sato : Properties of fungiform papillae in frog's tongue. Jap. J. Physiol. 7: (1957). Poritsky, R. and M. Singer: Intraperitoneal transplants of taste buds in the newt. Anat. Rec. 188: (1977). Rapuzzi, G. and C. Casella : Innervation of fungiform papillae in the frog tongue. J. Neurophysiol. 28: (1965). Robbins, N.: The role of the nerve in maintenance of frog taste buds. Exp. Neurol. 17: (1967). Shimamura, A. and J. Tokunaga: Scanning electron microscopy of sensory (fungiform) papillae in the frog tongue. In: (ed. by) Om Johari: Scanning electron microscopy/1970. Chicago, IIT Research Institute, (p ). Stensaas, L. J.: The fine structure of fungiform papillae and epithelium of the tongue of a south toad, Calyptocephalella dayi. Amer. J. Anat. 131: (1971). Toyoshima, K.: Neurohistological study on the regeneration of taste organ after corrosion in rat. J. Kyushu Dent. Soc. 33:16-24 (1979). Toyoshima, K. and A. Shimamura : Comparative study of ultrastructures of the lateral-line organs and the palatal taste organs in the African clawed toad, Xenopus laevis. Anat. Rec. 204: (1982). Wachstein, M. and E. Meissel: Histochemistry of hepatic phosphatase at a physiological ph. Amer. J. clin. Pathol. 27: ). Wright, M.: Persistence of taste organs in tongue grafted to liver. Proc. Soc. Exptl. and Med. 97: (1968). Zalewski, A. A.: Regeneration of taste buds in the lingual epithelium after excision of the vallate papilla. Exp. Neurol. 26: (1970). Dr. Kuniaki TOYOSHIMA Department of Oral Anatomy Kyushu Dental College Manazuru, Kokurakita-ku Kitakyushu, 803 Japan