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Electron microscopy of terminal buds on the barbels of the silurid fish Corydoras paleatus.

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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.
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taste buds induced by prolonged treatment with cycloheximide. Arch. Histol. Jap., 38:31-42.
141
Fujimoto, S., and R.G. Murray (19701 Fine structure of degeneration and regeneration in denervated rabbit vallate
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Hirata, Y. (1966) Fine structure of the terminal buds on the
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Luft, J.H. (1966) Ruthenium red staining of the striated
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Proceedings of the VIth International Congress of Electron Microscopy, vol. II:6566.
Murray, R.G., A. Murray, and S. Fujimoto (1969) Fine structure of gustatory cells in rabbit taste buds. J. Ultrastruct.
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Reutter, K. (1971) Die Geschmacksknospen des Zwergewelses, Arniurus nebulosus (Lesueur). Z. Zellforsch.,
120:28&308.
Trujillo-Cenoz, 0. (1961i Electron miscroscope observations
on chemo- a nd mechano-receptor cells of fishes. Z .
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Uga, S., and K. Hama (19671 Electron microscopic study on
the synaptic region of the taste organ of carps and frogs. J.
Electronmicr., 16:26%275.
Welsch, U., and I? Starch (1969) Die Feinstruktur der Geshmacksknospen von Welsen [Clarias batrachus (L.i und
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