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Histochemical observation on phosphatase activities of degenerating and regenerating taste buds.

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Histochemical Observation on Phosphatase
Activities of Degenerating and
Regenerating Taste Buds
TAKASHI IWAYAMA AND OSAMI NADA
Department of Anatomy, Faculty of Medicine, Kyushu University,
Fukuoka, Japan
ABSTRACT
Using rat's circumvallate papillae, ATPase, alk. Pase and acid Pase of
taste buds were observed after the transection of the glossopharyngeal nerve.
The taste buds began to disappear after the nerve war cut and were completely lost
after ten days. Following the regeneration of the glossopharyngeal nerve, taste buds
reappeared from the bottom of the gutters of circumvallate papillae about 25 days after
the operation.
ATPase was strongly present on the cell membrane of taste bud cells as far as they
existed during degeneration and regeneration. Alk. Pase, which is normally localized
on the superficial layers of the epithelium overlying the gutters of circumvallate papilla,
gradually diminished as the taste buds degenerated and reappeared as the taste buds
regenerated; that is, the activity began to diminish three days after the operation, became feeble after ten days and reappeared after 25 days. It is concluded that taste
bud cells secrete alk. Pase in the gutters of circumvallate papillae. Acid Pase activity,
usually found i n the supranuclear portion of taste bud cells, was intensely reactive
during degeneration but did not reappear a t the early stage of regeneration of taste
bud cells.
Histochemical observations have reIt is of particular interest to examine
vealed that three kinds of phosphatase the changes of phosphatase activities after
activities are localized in the gustatory re- the operaion on the nerve to a taste bud.
gions of the tongue (Iwayama and Nada, The present work, a short account of which
'67a,b). Adenosine triphosphatase (ATPase) has already been reported (Iwayama et al.,
and acid phosphatase (acid Pase) are found '67), was mainly undertaken to clarify
to be specific in taste bud cells, while alka- the relation of alk. Pase to taste bud cells.
line phosphatase (alk. Pase) is present on
MATERIAL AND METHODS
the superficial layers of the epithelium
The
circumvallate
papilla of albino rat
lining the gutters of circumvallate and foliate papillae. In these papillae, alk. Pase was chosen in this experiment because this
activity, which is confined only to the papilla is solely innervated by the glossogutters with taste buds, is probably not pro- pharyngeal nerve, while the foliate papilla
duced by such inactive cells as cornified of rat is supplied mostly by this nerve and
squamous epithelia but seems to have close partly by the chorda tympani (Kitamura,
relationship to taste bud cells. This en- '65).
Under anesthesia with an intramuscular
zyme, however, is not present on the superficial epithelium of the fungiform papilla administr,ition of pentobarbital sodium
where a taste bud is located thereon. Judg- (200 mg/ kg), the glossopharyngeal nerve
ing from this point of view, the direct was exposed by a median incision on the
correlation of alk. Pase to taste buds is un- neck and separation of the stylohyoid and
certain. On the other hand, a number of digastric muscles. The nerve was bilaterexperiments showed that taste buds dis- ally transected at the point after it passes
appear and reappear following the dener- over the greater horn of the hyoid bone and
vation and regeneration of the innervating just before it enters the tongue muscles.
nerve (Meyer, 1897; Olmsted, '20, '21, '22; In the deiiervation experiments, the proxof the glossopharyngeal nerve
May, '25; Whiteside, '26; Torrey, '34, '40; imal stump
Guth, '57, '58).
Received Aug. 13, '68. Accepted Oct. 10, '68.
ANAT. REC., 163: 31-38.
31
32
TAKASHI I W A Y A M A A N D O S A M I N A D A
Four to seven days after the operation,
taste buds, as well as taste bud cells, apparently diminished in number. ATPase
still persisted on the membrane of taste
bud cells although it might be less reactive
(fig. 6 ) . Acid Pase was intensely present
in the cytoplasm of a few surviving cells
(fig. 10). Alk. Pase at this stage indicated
apparent changes; the activity became less
intense and decreased in the upper part of
the gutters (fig. 2). The upper limits of the
reactive epithelia were close to the surviving taste buds.
On the 20th postoperative day, taste bud
cells had already completely disappeared
and were replaced by squamous epithelium. No ATPase activity was observable
in the epithelial layer, which became thinner than normal, but subgemmal nerve
fibers running along the gutters still gave
the definite positive reaction (fig. 7). No
discernible difference of ATPase activity
was found in the subgemmal nerve plexus
of both normal and operated circumvallate
RESULTS
papillae. Some reaction products of acid
Alk. Pase, ATPase and acid Pase were Pase were found scattered in the epithelocalized in specific relationship to the lium covering the bottom of the gutter
taste bud as already reported (Iwayama
Each group of figures 1, 5 and 9, figures 2, 6
and Nada, '67b) ; alk. Pase was present not
in the taste bud but on the superficial layer and 10, figures 3, 7 and 11, and figures 4, 8 and
12 contains photomicrographs of three serial secof the epithelium lining the gutter with tions
obtained from the different circumvallate
taste buds in the side of circumvallate papillae. All the figures were photographed a t
papilla (fig. 1). ATPase and acid Pase, on x 80.
Fig. 1 Alk. Pase i n control. The superficial
the other hand, were present in the taste
layers of the epithelium covering the gutters indibud. The former was associated with the cate a n intense reaction, which is confined to the
membrane of taste bud cells (fig. 5) and epithelium with taste buds. The capillary walls in
the latter was found in the supranuclear the lamina propria are also responsible for the
reaction.
cytoplasm (fig. 9).
Fig. 2 Alk. Pase seven days after the operaAs shown by previous investigators, in tion. Up to second postoperative day, the activity
general, taste buds gradually disappeared is still as strong as in the controls. But the refirst from the upper level in the gutter and action becomes gradually less intense and recedes
along the gutters downward. The upper limit of
finally were completely lost seven to ten the
activity corresponds to the location of the
days after the operation. This fact is uppermost surviving taste bud (compare with
clearly shown in figures 5, 6 , and 7, which fig. 6).
Fig. 3 Alk. Pase 20 days after the operation.
illustrate the ATPase reaction.
activity becomes very feeble except as a
Up to the second day after the operation, The
strongly reactive fleck near a n opening of a linthe number of taste bud cells contained in gual gland. The reaction o n the capillary walls
a taste bud seemed to decrease but they in the lamina propria is unaltered. The lingual
were not counted. In this point, ATPase gland with the activity is shown at the lower left.
Fig. 4 Alk. Pase 37 days after the operation.
activity was still intense in association with The
intense reaction is present along three walls
the membranes of taste bud cells, while of two gutters. In the left gutter, the upper limit
acid Pase activity was strongly positive in of the activity is higher on the outer wall than
all the taste bud cells, but some lacking in o n the inner wall, while the activity on the right
wall is negligible. The distribution of the
the controls. Alk. Pase was also as strongly inner
activity is well correlated with the regenerated
reactive as in the normal state.
t-ste buds which are shown in figure 8.
was ligated to prevent regeneration, while
in the animals in which regeneration was
anticipated, much care was paid not to impair the proximal portion of the nerve and
both stumps were left in situ without any
devices. The animals were sacrificed between 1 and 60 days after the operation.
The tongue containing the circumvallate
papilla was removed from an anesthetized
animal with ether. A small piece of specimen was trimmed in order to get suitable
sections of the papilla and was directly
frozen in isopentane cooled with solid carbon dioxide. The cryostat sections were
serially cut in 15 u thickness, dried with an
electric fan at room temperature and incubated to demonstrate alk. Pase, ATPase
and acid Pase after the method of Burstone
('61), Wachstein and Meisel ('57) and
Goldberg and Barka ( ' 6 2 ) respectively,
using every third section for the demonstration of each phosphatase activity.
HISTOCHEMISTRY OF DE- AND REGENERATING TASTE BUD
Figures 1-4
33
34
TAKASHI IWAYAMA AND OSAMI NADA
(fig. 11) and might indicate an infiltration
of leukocytes (Guth, '57) or reaction artifacts. Alk. Pase became feeble with the exception of intensely reactive speckles near
the openings of posterior lingual gland
(fig. 3). Further examination up to the
60th day showed no additional loss of alk.
Pase activity from the gutters.
About 25 days after the operation for
regeneration, following the reinnervation
of the glossopharyngeal nerve, taste buds
began to reappear from the lower portion
of the gutter upwards. ATPase activity
was strongly positive on the membrane of
newly formed taste bud cells in the epithelial layer which had regrown to an original
thickness. Following the regeneration of
taste buds, alk. Pase again appeared on the
gutter wall. Acid Pase, on the other hand,
was not distinct yet in the cytoplasm of
newly formed taste bud cells. In this experiment, the renewal of the taste buds
was rather poor as a result of the poor regeneration of the amputated nerve fibers.
Figures 4, 8 and 12 were taken from an
animal 37 days after the operation. Several
regenerated taste buds were clearly indicated by the staining of ATF'ase in figure 8
where taste buds reappeared higher in the
outer wall of the left gutter than in the
inner wall, while in the right gutter, the
inner wall indicated complete absence of
taste buds. These relations were well correlated with the alk. Pase activity in figure
4, that is, the upper limit of the activity
was higher on the left outer wall than on
the left inner wall and the activity on the
right inner wall was negligible. Acid Pase
were moderately present in some of taste
bud cells of figure 12 and this result indicated that acid Pase of taste bud cells developed somewhat later than the reappearance of taste bud cells.
DISCUSSION
Alk. Pase of the gustatory epithelia has
been histochemically indicated by several
authors (Bourne, '48; Baradi and Bourne,
'51, '53, '59; Rakhawy, '62; Baradi and
Brandes, '63). This enzyme is endowed
with important but different roles in taste
perception by Baradi and Bourne ('51) and
Rakhawy ('62), but the absence of this enzyme on fungiform papilla is unfavorable
for their hypothesis (Iwayama and Nada,
'67a). Moreover, the superficial layers of
the gutter, whcih are going to slough off,
have a strong enzymatic reaction. Such
strong enzymatic activity is, of course, not
produced by inanimate cells like superficial
squamous cells. Where can one look out
for the source of the alk. Pase of the gutter
then? Rakhawy ('62) assumed it might be
carried from the lingual toncil by lymphocytes. In the present experiment, alk. Pase
activity showed a consistent parallel alteration in accordance with the vicissitudes
of taste buds. Therefore, it seems reasonable to assume that almost all alk. Pase
found on the desquamating epithelia must
originate from taste bud cells. The secretory function of taste buds was already
demonstrated by the existence of the dense
substance which filled taste pores and the
secretory droplets of the same nature in
taste bud cells (de Lorenzo, '58, '63; Murray and Murray, '67), although the dense
substance bears probably no direct relationship to alk. Pase. The circumvallate and
foliate papillae have a considerable number of taste buds and their grooves may
prevent the enzyme from being easily removed by saliva. The fungiform papilla,
on the other hand, has a single taste bud
on its top, and therefore the secretion
would be scanty and easily dissolved in
saliva. In the taste bud of the fungiform
papilla of the rat, however, Farbman ('65)
noticed the absence of intracellular secretory droplets which are usually present in
taste buds of the circumvallate and foliate
Fig. 5 ATPase activity in control. The activity is specifically found on the membrane of taste
bud cells. In the lamina propria, a strong activity
is present along the nerve fibers running in parallel with the gutter, as well as on the blood vessels.
Fig. 6 ATPase seven days after the operation.
Taste buds, which are shown with ATPase stain,
exist in the lower portions of the gutters only.
The reaction is still present on the membrane of
all the survived taste bud cells.
Fig. 7 ATPase 20 days after the operation. All
the taste bud cells have already disappeared and
therefore no reaction products are observed in the
epithelial layer. But in the lamina propria, the
intense activity is still present not only in blood
vessels but also in subgemmal nerve plexus.
Fig. 8 ATPase 37 days after the operation.
Taste buds reappear in the lower parts of the
gutters and indicate strong activity of ATPase in
the epithelial layer. The poor regeneration of
taste bud cells are probably related to poor regeneration of the severed nerve.
HISTOCHEMISTRY OF DE- AND REGENERATING TASTE BUD
Figures 5-8
35
36
TAKASHI IWAYAMA AND OSAMI NADA
Figures 9-12
HISTOCHEMISTRY OF DE- AND REGENERATING TASTE BUD
papillae. This difference might coincide
with the absence of alk. Pase on the fungiform papilla. A small amount of alk. Pase
activity might also be derived from lingual
glands, distributed on the trench wall and
sometimes condensed around the openings
of their excretory ducts (fig. 3 ) .
ATPase of taste buds was so specific and
intense that this reaction could clearly
stain taste bud cells in non-stained squamow epithelia as far as they exist. The
activity persisted on the membrane of taste
bud cells until they disappeared in degeneration, and reappeared at the beginning
of their differentiation in regeneration.
During the degeneration and regeneration,
the existence of taste bud cells, which
was apt to be overlooked in the preparation of hematoxylin-eosin stain, could be
easily recognized by this staining. In fact,
ATPase-reactive cells can be observed even
in the epithelium of new-born rat circumvallate papillae, although Torrey ('40)
could not recognize taste buds until the
ninth postnatal day. The results will be
published elsewhere in detail. In lamina
propria, as shown in figure 7, the reaction
of ATPase activity was found along the
subgemmal nerve plexus of the animal of
the twentieth postoperative day and no discernible changes were found on the plexus
even 60 days after the operation. This finding presumably demonstrates that most of
the activity associated with the nerve fibers
Fig. 9 Acid Pase i n control. The activity is
more intense i n the taste bud cells than in the
other epithelial cells, which also show diffuse reaction, but some of taste bud cells are devoid of
the activity. The reaction is localized to the supranuclear cytoplasm of the taste bud cells. Ganglion
cells with the reaction are present at the base of
the papilla.
Fig. 10 Acid Pase seven days after the operation. Taste bud cells apparently diminish i n nurnber, but acid Pase is strongly reactive i n all the
surviving taste bud cells, mostly i n their supranuclear regions.
Fig. 11 Acid Pase 20 days after the operation.
The activity of the epithelial layers shows a weak
and diffuse staining. Some reaction products are
observed in the epithelium covering the bottom of
the gutters and might indicate an infiltration of
leukocytes. A n intense staining is present at the
lower left, indicating a lingual gland.
Fig. 12 Acid Pase 37 days after the operation.
Some of the taste bud cells indicate the intense
activity of acid Pase in the diffusely reacted epithelium, although the activity is not distinct i n
earlier stage.
37
is derived from their Schwann cells, which
survive i n their original sites for a long
time.
Acid Pase was found as one of the lysosoma1 enzymes by de Duve ('55, '63). This
enzyme is also reported to be associated
with the Golgi apparatus and the secretory
granules of the pancreas (Sobel and Avrin,
'65; Lazarus et al., '66). As already discussed, taste bud cells seem to have some
secretory functions and therefore might
have the activity related to secretory function. But taste bud cells, with a life span
of 250 & 50 hours (Beidler and Smallman,
'65), are usually undergoing degeneration
and lysosomes would increase, as a matter
of course, in old degenerating cells. In the
present experiments, the activity of this
enzyme was intense in all the surviving
cells after the operation but weak in newly
induced ?ells. These facts may indicate
that mosi: of the activity is of lysosomal
origin. Goldfisher et al. ('64) reported the
existence of acid Pase in endoplasmic reticulum and Golgi saccules of some neurones as well as in lysosome. The accurate
localization remains to be determined with
submicror;copichistochemistry.
LITERATURE CITED
1951 Theory
Baradi, A. F., and G. H. Bourne
of tastes and odors. Science, 113: 660-661.
1953 Gustatory and olfactory epithelia.
Int. Rev. Cytol., 2: 289-330.
1959 New observations on the alkaline
glycerophosphatase reaction in the papilla foliata. J . biophys. biochem. Cytol., 5: 173-174.
Baradi, A. F., and D. Brandes 1963 Electron
microsco2ic localization of alkaline phosphatase in pi;pilla foliata. J. Histachem. Citochem.,
1 1 : 815-:317.
Beidler, L. M., and R. L. Smallman 1965 Renewal of cells within taste buds. J. Cell Biol.,
27: 263-272.
Bourne, G. H. 1948 Alkaline phosphatase in
taste buds and nasal mucosa. Nature, 161:
445-446.
Burstone, N [ . S. 1961 Histochemical demonstration of phosphatases in frozen sections with
naphthol AS-phosphates. J. Histochem. Cytochem., 9: 146-156.
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deDuve, C. 1963 The lysosome concept. In:
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38
TAKASHI IWAYAMA AND OSAMI NADA
de Lorenzo, A. J. 1958 Electron microscopic
observations on the taste buds of the rabbit.
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1963 Studies on the ultrastructure and
histophysiology of cell membranes. In: Olfaction and Taste, ed. by Y. Zotterman. Pergamon
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1921 Effects of cutting the lingual
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Rakhawy, M. T. E. 1962 The histochemistry of
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its probable function i n taste. Acta anat., 51:
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Sobel, H. J., and E. Avrin 1965 Localization of
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