Electron microscopy of terminal buds on the barbels of the silurid fish Corydoras paleatus.код для вставкиСкачать
THE ANATOMICAL RECORD 197:133-141 (1980) Electron Microscopy of Terminal Buds on the Barbels of the Silurid Fish, Corydoras paleatus SUNAO FTJJIMOTO AND KOJI YAMAMOTO Department o f d n a t o m y , Uniuersity of Occupational and Environmental Health, School of Medicine, Kitakyushu 807, Japan ABSTRACT The terminal buds of the Corydoras paleatus were observed with the electron microscope. Almost all the cells constituting the buds can be classified into two distinct cell types, supporting and receptor cells. In addition, a few cells designated as basal cells exist in the bottom of the buds and appear to be an immature form of each distinct cell type in the course of cell renewal. The receptor cells are characterized by the presence of tubules extending from the apical process. By the application of lanthanum nitrate as an extracellular marker, we demonstrated that the tubular system is in continuity with the extracellular space. The data suggest that the tubular system represents an amplification of the apical cell surface as a particular site of chemoreceptive activities, although we do not rule out a role for active absorptions of ions in a very hypotonic environment. In chemoreceptors of freshwater fish, spe- observed in other freshwater fish (Hirata, cific properties developed as an adaptation for '66). Our marker experiments using lanvery hypotonic environments might be ex- thanum nitrate clearly demonstrated t h a t pected. Since the first description of the fish these tubules communicate directly with the taste organs by Leydig (1851), the fine struc- extracellular space through the apical cell ture of the terminal buds has been studied in process. It is quite reasonable to assume that several species of freshwater fish (Trujillo- such a vast amplification of t h e receptor Cenoz, '61; Cordier, '64; Hirata, '66; Uga and membrane may have a specific role for the Hama, '67; Welsch and Storch, '69; Reutter, chemoreceptions in a dilute ionic environ'71). In these previous observations it was rec- ment. ognized that terminal buds are basically hoMATERIALS AND METHODS mologous t o those in other vertebrates, and the variation in appearance of the cells conBarbels of the silurid fish Corydoras pastituting the buds was interpreted as two or leatus were fixed in 2.5% glutaraldehyde in three distinct cell types. Furthermore, the 0.1 M cacodylate buffer for two hours. Some synaptic morphology has no fundamental in- were immersed in 1%lanthanum nitrate disconsistencies between fish and mammalian solved in the buffer for one hour before the taste buds. However, these works revealed a fixation. few morphological characteristics that have After a brief rinse in the buffer, the specinot been pointed out in mammalian taste mens were post-fixed in 2% osmium tetroxide buds. and dehydrated in graded concentrations of This paper deals with the fine structure of acetone. Sections were made by a Porterterminal buds of the freshwater fish Cor- Blum MT-1 Ultramicrotome, s t a i n e d by ydoras paleatus. Although they were briefly uranyl acetate and lead acetate, and exobserved by Trujillo-Cenoz ('61) and Cordier amined in a Hitachi HU-12A electron micro('64), further investigations are necessary t o scope. compare their morphology with that of other Barbels for scanning electron microscopy fishes and to provide more detailed informa- (SEM) were fixed i n t h e same way a s tion t o electrophysiologists. described above, dehydrated i n ethanol, The present observation revealed that the soaked in amyl acetate, dried by the critical receptor cells are characterized by the presReceived September 11,1979, accepted November 21,1979 ence of tubules, similar in structure to those 0003-276X/80/1972-0133$01.70 c 1980 ALAN R. LISS. INC. 133 134 SUNAO FUJIMOTO AND KOJI YAMAMOTO point method using COz, coated with gold- With higher magnified SEM pictures, t h e palladium, and observed with a Hitachi field number of these apical processes ranges between 40 a n d 80 per bud: These n u m b e r s emission electron microscope. might correspond to those of the receptor cells RESULTS AND INTERPRETATIONS per bud. The t e r m i n a l buds a r e s e t i n somewhat ’ h o kinds of tubules a s intracytoplasmic orelevated regions of t h e epidermis of the bar- ganelles can be found in t h e receptor cells. bel (fig. I). They a r e ovoid in shape and their One kind a r e microtubules, approximately of a p e x u s u a l l y p r o t r u d e s o u t w a r d from a n 20 A in diameter, running in parallel in the opening in the surface of the epidermis (taste center of the main portion of the cell (fig. 4). pore). By SEM pictures, the mean diameter of The other kind a r e smooth surface tubular t h e taste pore can be measured as approxi- membranes with a rather constant diameter mately 20 p m (fig. 2). approximately of 0.06 p m , which extend from Almost all the cells constituting t h e buds t h e apical process to the Golgi region. These a r e elongated or cylindrical i n form (3C50 t u b u l e s a r e c l e a r l y d i s t i n g u i s h a b l e f r o m p m i n l e n g t h ) a n d e x t e n d from t h e b a s a l s m o o t h e n d o p l a s m i c r e t i c u l u m by t h e i r lamina to t h e pore region of t h e epidermis. thicker limiting membrane of approximately Under the electron microscope, they a r e clas- 90 A and their electron dense amorphous consified into supporting and receptor cells by tents (figs. 4,5 , and 7). Long mitochondria are each general cytoplasmic feature and the way often associated with t h e tubules. As they they contact nerves. I n addition to t h e two m a y sometimes t a k e a t o r t u o u s o r s p i r a l distinct cell types, a few cells, usually oval in course i n t h e apical process, their sectioned shape, exist in the basal part of t h e buds (fig. profiles often appear as vesicles fused to each 11).They are similar in general features to other (fig. 5 ) . Furthermore, some of these prothose described a s “basal cells” by e a r l i e r files are often apposed closely with the lateral workers (Hirata, ’66; Reutter, ’71). surface of t h e apical process, although the diThe supporting cells a r e more slender in rect continuity is difficult to visualize (fig. 5 ) . outline and more compact in both cytoplasm By the application of lanthanum nitrate as and nucleus. Their apical cell surface is pro- a n extracellular space marker, every tubule vided with a few short microvilli, approxi- contains electron opaque l a n t h a n u m commately 0.6 p m in length and 0.12 p m in diam- pound (fig. 8 ) . However, t h e m a r k e r is not eter (figs. 3 and 4). Each microvillus is en- present i n t h e endoplasmic reticulum a n d closed with a n extension of t h e plasma mem- Golgi system. This indicates that the interior brane, of which the outer leaflet bears long of the tubular system is in continuity with t h e delicate filaments. These filaments anasto- exterior of t h e apical process. The experiment mose with each other and form extensive net- also shows t h a t each tubule t h a t extends sepworks on the surface of the buds (fig. 4).Their arately from the apical process anastomoses intense staining by Ruthenium red, according with another in the main portion of the cell to Luft’s method (’661, suggests t h a t they a r e body (fig. 8). Another prominent feature of a n acid mucopolysaccharide surface coat in t h e s e t u b u l e s i s t h a t t h e i r i n t e r i o r i s i n n a t u r e . T h e cytoplasmic organelles of t h e tensely stained by Ruthenium red (fig. 6). supporting cells a r e relatively few in number Golgi complexes a r e well-developed in the except for the existence of abundant cytoplas- receptor cells. The limiting membrane of the mic filaments of 75-100A diameter, which cisterns in their distal face (matured face) is give t h e cell a more electron-dense appear- slightly increased in density. Many profiles of ance. Golgi vesicles possibly originating from t h e The receptor cells differ consistently from distal face (for convenience i n descriptions, the supporting cells i n several respects. They “thick-walled vesicles”) a r e seen near the tuare always isolated by thin cytoplasm of t h e bles (fig. 9). supporting cells except i n t h e basal zone of A myelinated nerve plexus, branches of t h e t h e buds, where t h e receptor cells a r e fre- facial nerve, exists in the core of the barbel, quently in contact with one another. The apex and many profiles of unmyelinated nerve fiof t h e cells does not possess microvilli b u t bers which penetrate into t h e buds are in a ends bluntly a s a single t a p e r i n g process, close contact with both cell types a t t h e i r 1.5-3 p m in length and O . S O . 5 p m in diame- basal zone. “Classic” synaptic morphology is t e r at t h e thickest portion, which protrudes lacking i n t h e supporting cells. Where nerve outward from the taste pore (figs. 2 , 3 , and 5 ) . endings show a small protrusion toward the ELECTRON MICROSCOPY O F TERMINAL BUDS OF CORYDORAS PALEATUS 135 Fig. 1. SEM picture showing a barbel of Corydoras paleatus. Terminal buds a r e set in elevated regions of the epidermis (arrows). x 140. Fig. 2. SEM picture showing a taste pore. Many long slender projections proturde from the pore (arrows~.x 5,700 Fig. 3. The apical portion of a terminal bud is shown. Supporting cells (SC) possess a few short microvilli, but receptor cells (RC) end as a single apical process (AP)without rnicrovilli. x 12,000. 136 SUNAO FUJIMOTO AND KOJI YAMAMOTO Fig. 4. Microvilli of supporting cells (SC) bear long dehcate filaments (arrows). Receptor cells (RC) are characterized by the presence of microtubules (MT] and smooth-surfaced tubules (ST]. x 24,700. Fig. 5. The tubules (ST) take a tortuous or spiral course in the apical process (AP,, and one end o f a tubule associated with the lateral surface of the apical process (arrow). x 67,500. Fig. 6. The internal aspect of the tubules is intensely stained by Ruthenium red. x 35,600 IS closely ELECTRON MICROSCOPY OF TERMINAL BUDS OF CORYDORAS PALEATUS 137 Fig. 7.The tubules (ST)of receptor cells (RC) contain dense fibrillar materials in their interior. Long mitochondrion (MI) is closely associated with the tubule. Supporting cells (SCI appear darker by their abundant cytoplasmic filaments. x 69,400. Fig. 8. By the application of' lanthanum nitrate a s a n extracellular space marker, the tubular system (ST)of a receptor cell contains electron-dense lanthanum compound. Each tubule branches and anastomoses to another (arrows). x 38,000. SUNAO FUJIMOTO AND KOJI YAMAMOTO 138 Fig. 9. Thick-walled vesicles ( G V ) possibly derived from the mature face of the Golgi (GO) a r e seen near the tubules 40,000. (ST).x Fig. 10. Synaptic relation of a receptor cell (RCi with a nerve ending (NE) is shown. The apposed membranes display patches of increased density. Abundant smaller vesicles crowd against these specialized areas of the receptor membrane (presynaptic side). x 50,000. ELECTRON MICROSCOPY OF TERMINAL BUDS OF CORYDORAS PALEATUS receptor cells, however, features which characterize chemically transmitting synapses, similar to those in mammalian taste buds (Murray, et al, ’69), are clearly recognizable: The increase i n density and thickness is present along the plasma membrane of the cell a d j a c e n t to t h e n e r v e e n d i n g s , a n d smaller vesicles (electron-lucent type of synaptic vesicles) crowd against these specialized areas of the plasma membrane (fig. 10). Many nerve fibers are also in contact with the basal cells, and large numbers of smaller vesicles and a few cored vesicles occur in their cytoplasm near the contact areas (figs. 11 and 12). However, our electron micrographs do not show typical synaptic specializations between the cell and nerve as observed in the receptoneural contacts. DISCUSSION The receptor cells are characterized by the presence of two kinds of tubules. The microtubules running in parallel in the center of the cytoplasm might be considered as supportive elements i n n a t u r e . However, t h e smooth membranous tubules extending from the apical process are similar in structure to those described in other freshwater fishes (Hirata, ’66).They seem to occur uniquely in terminal buds of freshwater fishes since such structures have never been pointed out in other vertebrates. These tubules can clearly be distinguished from ordinary smooth endoplasmic reticulum by their rather constant diameter and their thicker limiting membrane. Using lanthanum nitrate a s a n extracellular space marker, we demonstrated that the interior of these tubules is in continuity with the exterior of the apical process. Furthermore, the intense staining by Ruthenium red indicates t h a t their internal surface is covered w i t h a b u n d a n t acid mucopolysaccharide coats. We have little physiological information about chemoreceptive activities in freshwater fishes. Since their taste receptors are constantly exposed to a dilute ionic environment, specific properties in t h e chemoreceptions might be expected. Konishi and Niwa (’64),by t h e i r electrophysiological s t u d y of t h e freshwater fish, have suggested the presence of a specific mechanism involved in the perception of sapid substances in extremely low concentrations. If we could assume that the tubular system, the vast amplification of the apical cell surface, is involved in the chemoreceptive activi- 139 ties, the cellular response to ionic influx into this expanded membrane system would also be responsible for changes in electrical potentials. Abundant mucopolysaccharide surface coats at the internal surface may have a role for “adsorption” of various ions a s postulated by Beidler (’541, possibly functioning a s a n ion-trap. Chloride cells of saltwater and freshwater fishes characterized by a n amplification of the surface cell membrane have been generally considered to participate in osmoregulatory function. In some freshwater fish in a dilute ionic environment, a role for a n absorption of ions has been proposed (Komuro and Yamamoto, ’75). Thus, one might speculate that active transport of ions through the tubular system in the receptor cells reported here might take place, but we cannot explain how ions pass into t h e extracellular tissue space. More detailed observations including enzyme histochemistry will be necessary t o elucidate whether the tubular system is only a n amplification of the receptor membrane or may represent a n ion-transporting mechanism. The present marker experiments do not show direct continuity between the tubules and the Golgi system in the receptor cells. However, our electron micrographs strongly suggest that “thick-walled vesicles” originating from the Golgi cisterns are incorporated into the tubular system. Thus, as previously described by one of the authors (Fujimoto, ’73), these Golgi-derived vesicles would represent preformed units of the tubular system, providing membrane itself and t h e surface coat materials. A few basal cells exist in the bottom of the buds lying directly upon the basal lamina. Although their general features, both in nucleus and cytoplasm, have no fundamental inconsistencies with those in mammalian taste buds, they are uniquely in contact with abund a n t nerve fibers. By occassional aggregations of smaller vesicles in the basal cells of other fishes, Hirata (’66) has postulated that these cells may represent “accessory receptor cell,” which would be activated by impulse from the ordinary receptor cells. The occurrence of vesicles in various sizes near t h e nerve contact areas is also true in our basal cells. However, our electron micrographs do not show the “classic” synaptic morphology a s the case in the receptor cells: Densities of the apposed plasma membranes and aggregations of vesicles against these specialized areas are not apparent. 140 SUNAO FUJIMOTO AND KOJI YAMAMOTO Fig. 11. Aggregations of smaller vesicles (arrows)are seen in a basal cell (BC). The cell contains abundant mitochondria BL, basal lamina. x 14,000. Fig. 12. Abundant smaller vesicles (SVI and a few granulated ones appear in the cytoplasm of a basal cell IBC). However, synaptic specializations as shown in fig. 10 are not apparent in the cell-to-nerve contacts. x 27,500. ELECTRON MICROSCOPY OF TERMINAL BUDS OF CORYDORAS PALEATUS In mammalian taste buds it is widely accepted t h a t basal cells represent a lessdifferentiated form of taste bud cells in the course of cell renewal. In their regenerative and degenerative processes, it was observed that each cell type originates separately from a single less-differentiated form (Fujimoto and Murray, ’70; Fujimoto, ’75).Thus, the appearance of smaller vesicles in the cytoplasm of a basal cell in our specimens may imply that this cell is differentiating to a receptor cell. Although we have no direct evidence to deny a specific transductive function of basal cells in the fish taste buds, the present observation favors a consideration that they represent precursor cells for the independent origins of each distinct cell type. LITERATURE CITED Beidler, L.M. (19541 A theory of taste stimulation. J. Gen. Physiol., 38:133-139. Cordier, R. (1964) Sensory cells. In J. Brachet and A.E. Mirsky, eds.: “The Cell,” Vol. VI. New York: Academic Press, pp. 311386. Fujimoto, S. (1973) On the Golgi-derived vesicles in the rabbit taste bud cells: An electron microscopy and related cytochemistry. Kurume Med. J., 20:133-148. Fujimoto, S. 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