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The distribution of alkaline phosphatase activity in normal and cross-species regenerated rat and mouse taste buds.

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The Distribution of Alkaline Phosphatase Activity in
Normal and Cross-species Regenerated
Rat and Mouse Taste Buds
ANDREW A. ZALEWSKI
Laboratory of Neurochemistry, National Znstitute of Neurological and Communicative
Disorders and Stroke, National Institutes of Health, US.Department of Health,
Education, and Welfare, Bethesda, Maryland 20014
ABSTRACT
Alkaline phosphatase (ALK Pase) activity can be detected histochemically in the taste buds of rats but not mice. Since taste buds develop, regenerate and are maintained under the influence(s1 of the sensory nerve it was
decided to study cross-species regenerated buds of these two animals to determine whether the nerve also regulated ALK Pase development in taste cells.
Grafts of rat sensory ganglion and mouse tongue or mouse ganglion and rat
tongue were combined in the anterior chamber of the eyes of immunologicallydeficient nude mice and the cross-species buds that developed a t 35 days were
examined histochemically for ALK Pase. The results revealed that the rat
nerve did not cause ALK Pase to appear in any buds found in mouse tongue
grafts and t h a t mouse nerve could support buds containing ALK Pase in r a t
tongue tissue. Because the cross-species regenerated buds were histochemically
characteristic of those normally found in r a t or mouse tongue, there is no evidence that the foreign nerve altered gene expression for ALK Pase in the target
organ, and the action of the nerve on gustatory epithelium appears to be that of
activation and maintenance.
The development, maintenance and regeneration of taste buds requires the presence of
sensory nerve fibers (Oakley and Benjamin,
'66; Guth, '71; Zalewski, '74). It is believed but
not proven that the taste buds develop because the nerve produces a substance which
interacts with and transforms epithelial cells
into taste bud cells (Torrey, '34; Guth, '71;
Gutmann, '76). The action of sensory nerves
appears to be non-specific since different taste
nerves can cause taste bud regeneration in the
same tongue region (Guth, '58; Zalewski, '69;
Oakley, '74). Moreover, the influence of the
nerve seems limited because Oakley ('67)
showed t h a t the taste responses of a tongue
area remained the same even though the buds
were reinnervated by a different taste nerve.
The failure of a foreign sensory nerve to convert taste responses contrasts with foreign
motor nerves which can alter both the speed of
contraction (see review by Close, '72) and the
histochemical appearance of skeletal muscle
fibers (Romanul and van der Meullen, '66;
Yellin, '67; Karpati and Engel, '67; Brooke et
ANAT. REC. (1979) 194: 283-292.
al., '71; Gutmann, '76). Since it is possible that
different neural influences may regulate the
speed of contraction and the enzyme content
of muscle, it is pertinent to determine whether
enzymes change in taste buds which are innervated by a foreign taste nerve. Unfortunately,
the histochemical presence and distribution of
enzymes in taste buds in the front and back of
the tongue of the same animal are similar
(Iwayama, '71; Zalewski, unpublished data on
rat, mouse and monkey). On the other hand,
the taste buds of different animals differ with
respect to the presence or distribution of the
enzyme ALK Pase (Bourne, '48; Iwayama and
Nada, '67; Zalewski, '68; Vij et al., '72). It
would be possible to determine whether a
foreign taste nerve might alter enzyme activity in taste buds if one could use the taste
nerve of one animal species to cause the development of taste buds in the tongue tissue of
another animal species. Such a cross-species
development of taste buds has been reported
Received Nov. 2, '77. Accepted Dec. 11, '78.
283
284
ANDREW A. ZALEWSKI
and the tongue graft placed over the peripheral end of the ganglion.
Recipient nude mice were anesthetized
after 35 days and the eye containing the
graft(s) enucleated. The grafts were removed
from the eyes and because they were small
they were placed between slabs of skeletal
muscle to facilitate sectioning them. The muscle containing the graft(s) was frozen in liquid
nitrogen and 8-pm-thickserial frozen sections
of the grafts cut at - 20°C in a cryostat. When
tongue and ganglion grafts were combined
MATERIALS AND METHODS
they were sectioned together rather than
All animals were obtained from the Small being separated and cut individually. AdjaAnimal Section, National Institutes of cent sections of the grafts were mounted on
Health, Bethesda, Maryland. The recipient different glass slides so that different histoanimals were 7-week-old male mice which chemical reactions could be performed on the
were homozygous for the mutation nude (nu1 same taste buds. Sections of normal (i.e., nonnu). The nude mice were anesthetized with transplanted) rat and mouse vallate papillae
chloral hydrate (400 mg/kg/ip) and groups of were cut after being placed alone or together
them received into the anterior chamber of between muscle (8 each way) or after being
one eye either: (1)a rat tongue graft alone (4 cut directly from the tongue (4 each). The tisanimals), (2) a mouse tongue graft alone (4 sue sections were stained to detect adenosine
animals), (3) a rat ganglion combined with a triphosphatase (ATPase) (Wachstein and
mouse tongue graft (8 animals) or (4) a mouse Meisel,' '571, acetylcholinesterase (AChE)
ganglion combined with a rat tongue graft (8 (Hanker et al., '73) or ALK Pase (Burstone,
animals). The ganglion used was the sensory '61) enzyme activities. The recommended invagal nodose ganglion while the tongue graft cubation protocols for ATPase and AChE deconsisted mainly of the vallate papilla and tection were followed but since several variasome underlying connective tissue. All grafts tions of the naphthol AS method of Burstone
were obtained from adult male C57 BLl6 mice for ALK Pase are possible it is important to
and 21-day-old male Sprague-Dawley rats. mention that AS-MX phosphate was used as
The tongue grafts were obtained from animals substrate, Fast Blue RR was used as dithat were first anesthetized and then killed by azonium salt and 0.2 M Tris (pH 8.3) was used
opening up the chest and removing the heart. as a buffer. It is noteworthy that ALK Pase
After exsanguination, the tongue was re- localization was the same in normal taste
moved and that portion which contained the buds regardless of whether sections were unvallate papilla was fashioned into a 2-mm- fixed, fixed in ice cold acetone or fixed in 10%
square graft. When a ganglion was to be com- (ice cold or room temperature) neutral forbined with a tongue graft in the eye, the malin. Consequently ALK Pase was detertongue graft was placed between slabs of mus- mined in grafts of unfixed sections that were
cle (obtained from the donor animal) to keep it incubated for 20 minutes a t room temperature
clean and moist. Ganglia were obtained from (i.e., 22°C). All ALK Pase sections were counanesthetized donors. Surgical instruments terstained with 1%
methyl green after enzyme
were sterilized before use whereas the grafts incubation.
were not since no eye infections due to conRESULTS
taminated grafts were encountered in our preNormal taste buds
vious study (Zalewski et al., '77). The technique of introducing the grafts into the eye
Taste buds were identified by their ATPase
consisted of making a stab-wound with a No. activity (Iwayama and Nada, '67; Zalewski,
11 scalpel blade in the center of the cornea, '68; Akisaka and Oda, '77). In the rat (fig. 1)
sliding the grafts through this opening, and and mouse (fig. 6), the buds were present in
pushing them laterally until they rested on the epithelium of the inner and outer trench
top of the iris a t the iris-corneal junction. walls of the papilla. Each taste bud extended
When tongue and ganglion grafts were com- through the entire thickness of the epithelium
bined in the eye the ganglion was put in first and all taste cells exhibited ATPase activity.
by Zalewski et al. ('77) who showed that buds
appeared when grafts of rat nodose ganglion
and mouse tongue tissue were combined in the
anterior chamber of the eyes of immunologically-deficient nude mice. Since ALK Pase
is present in rat but not mouse taste buds,
cross-species combinations of ganglia and
tongue grafts (i.e., rat ganglion and mouse
tongue graft) were made in order to determine
whether the nerve regulates ALK Pase in
taste buds {Iwayama and Nada, '69).
285
TASTE BUD HISTOCHEMISTRY
The apical ends of the taste bud cells which
are composed of microvilli (see review by Murray, ’73, for electron microscopical details of
taste buds) also stained for ATPase (fig. 2).
The innervation of the taste buds was detected by AChE staining which revealed the
presence of nerve fibers immediately beneath
the epithelium which contained the buds. The
presence or distribution of ALK Pase activity
was different in the buds of the rat and the
mouse. In the rat, ALK Pase activity was present in the superficial epithelium of the trench
walls (fig. 3). This localization of ALK Pase,
however, does not indicate whether the enzyme is present in the taste cells, in the superficial cells of the surrounding epithelium, or in
both types of cells; the use of shorter incubation times seemed to resolve this question.
When five minute incubation times were used
it was observed that ALK Pase reaction product appeared in regions corresponding to the
microvilli of the taste buds (fig. 4). After ten
minutes, reaction product began to spread out
between microvilli of adjacent buds (fig. 5) so
that after the standard time of 20 minutes incubation ALK Pase activity appeared to be
present along the superficial epithelium of the
trench walls where the buds were located (fig.
3). These results demonstrate that the ALK
Pase activity seen in the trench walls of the
rat vallate papilla is associated with taste bud
cells. In addition, a few taste cells stained intensely for ALK Pase activity throughout (fig.
5). In contrast to the rat, no ALK Pase activity was seen in mouse taste buds (fig. 7) even
when thicker sections, longer incubation
times, or papillae from different mouse strains
(i.e., DBA or BALB/c) were examined. The
above results regarding ALK Pase pertained
to sections of rat and mouse buds which were
cut from papillae that were “sandwiched” between muscle separately or cut directly from
the tongue. Similarly no ALK Pase appeared
in mouse taste buds when mouse and rat
papilla were blocked together in the same
muscle and cut and stained.
of the present study was therefore to determine whether rat nerve caused ALK Pase t o
appear in mouse taste buds in which no enzyme is normally present or whether mouse
nerve could support taste buds with ALK Pase
when normally mouse nerve does not. No taste
buds were found in rat or mouse tongue grafts
transplanted alone ke., without ganglion) t o
the eyes of nude mice. Taste buds were found,
however, in five of the eight mouse tongue
grafts that were combined with rat ganglia
and in four of the eight rat tongue grafts that
were combined with mouse ganglia. A total of
60 and 46 regenerated buds were present respectively in the two types of cross-species
graft combinations. In the mouse tongue
grafts, the cross-species regenerated taste
buds were found in the trench walls and in two
cases some additional buds were present in the
epithelium covering the top of the papilla (fig.
8).All buds extended through the thickness of
the epithelium and all cells in them had
ATPase activity (fig. 9). None of the buds
found in the trench wall (fig. 12) or in the epithelium on the top of the papilla (figs. 10, 11)
exhibited ALK Pase. On the other hand, the
cross-species buds that developed after combining mouse ganglion and rat tongue tissue
(fig. 14) did contain ALK Pase (fig. 15). The
regenerated buds in the rat tongue were found
only in the trench walls of the papilla (fig. 14).
AChE staining showed that neurons survived
in all ganglia (see example in fig. 13) in the
eyes of nude mice and that nerve fibers were
present beneath the epithelium which contained the regenerated taste buds (fig. 13). No
AChE was seen beneath the epithelium of the
trench walls of the papillae which lacked
buds.
DISCUSSION
The present results demonstrate that rat
nerve can cause the development of taste buds
in mouse tongue tissue and that mouse nerve
can do likewise in rat tongue tissue. These
findings indicate that a similar molecular
mechanism can act through the nerve and epiCross-species regenerated taste buds
thelium of tissues from different animal spePrevious studies in the rat have demon- cies to initiate the development and maintestrated that nerve fibers of the sensory vagal nance of taste buds. It is interesting though
nodose ganglion can substitute for the normal that the neural influence on taste buds did not
glossopharyngeal nerve innervation of the extend to regulating their histochemical charvallate papilla and cause taste buds with ALK acter. Thus ALK Pase activity appeared in
Pase activity to develop both in situ (Za- cross-species regenerated rat taste buds even
lewski, ’69) and in the anterior chamber of the though these buds were innervated by mouse
eye (Zalewski, unpublished data). The essence nerve fibers which normally support buds
286
ANDREW A. ZALE WSKI
showing no ALK Pase. Similarly, cross-species
regenerated buds in mouse tongue tissue did
not develop ALK Pase activity despite their
being innervated by rat nerve fibers which
normally subserve taste buds with ALK Pase.
The present data together with the results of
Oakley (‘67), which show respectively that
neither enzyme nor taste responses change in
taste buds after reinnervation by a foreign
taste nerve, support the notion that the action
of the sensory nerve on gustatory epithelium
is solely that of activation and maintenance.
ACKNOWLEDGMENTS
The author wishes to thank Mr. George F.
Creswell and Mrs. Janina D. Ziemnowicz for
excellent histological assistance and Mrs. Sylvia Z. Levinson for manuscript preparation.
LITERATURE CITED
Akisaka, T., and M. Oda 1977 The fine structural localization of adenosine triphosphatase activity on the taste bud
in the fungiform papillae of t h e rat. Arch. Histol. Jap., 40:
63-72.
Bourne, G. H. 1948 Alkaline phosphatase in taste buds
and nasal mucosa. Nature, 161: 445-446.
Brooke, M. H., E. Williamson and K. K. Kaiser 1971 The behavior of four fiber types in developing and reinnervated
muscle. Arch. Neurol., 25: 360-366.
Burstone, M. S. 1961 Histochemical demonstration of
phosphatases in frozen sections with Naphthol AS-phosphates. J. Histochem. Cytochem., 9: 146-153.
Close, R. I. 1972 Dynamic properties of mammalian skeletal muscles. Physiol. Rev., 52: 129-197.
Guth, L. 1958 Taste buds on t h e cat’s circumvallate
papilla after reinnervation by glossopharyngeal, vagus,
and hypoglossal nerves. Anat. Rec., 130: 25-37.
1971 Degeneration and regeneration of taste
buds. In: Handbook of Sensory Physiology. Vol. 4, Chemical Senses. Part 2, Taste. L. M. Beidler, ed. Springer-Verlag, Berlin, pp. 63-74.
Gutmann, E. 1976 Neurotrophic relations. Ann. Rev.
Physiol., 38: 177-216.
Hanker, J. S., L. P. Thornburg, P. E. Yates and H. G. Moore,
111 1973 The demonstration of cholinesterases by the for-
mation of osmium blacks a t the sites of Hatchett’s brown.
Histochemie, 37: 223-242.
Iwayama, T. 1971 Histochemical observations of alkaline phosphatase activity of t h e lingual epithelium after
t h e suppression of salivation. Histochemie, 28: 351-354.
Iwayama, T., and 0. Nada 1967 Histochemical observations on the phosphatases of the tongue, with special reference to taste buds. Arch. Histol. Jap., 28: 151-163.
1969 Histochemical observation on phosphatase
activities of degenerating and regenerating taste buds.
Anat. Rec., 163: 31-38.
Karpati, G., and W. K. Engel 1967 Transformation of the
histochemical profile of skeletal muscle by “foreign” innervation. Nature, 215: 1509-1510.
Murray, R. G. 1973 The ultrastructure of taste buds. In:
The Ultrastructure of Sensory Organs. I. Friedman, ed.
Elsevier Publishing Company, Inc., New York, pp. 1-81.
Oakley, B. 1967 Altered temperature and t a s t e responses from cross-regenerated sensory nerves in the
rat’s tongue. J. Physiol., 188: 353-371.
1974 On the specification of tasteneurons in the
r a t tongue. Brain Res., 75: 85-96.
Oakley, B., and R. M. Benjamin 1966 Neural mechanisms
of taste. Physiol. Rev., 46: 173-211.
Romanul, F. C. A,, and J. P. van der Meullen 1966 Reversal
of enzyme profiles of muscle fibers in fast and slow muscles by cross-innervation. Nature, 212: 1369-1370.
Torrey, T. W. 1934 The relation of taste buds to their
nerve fibers. J. Comp. Neur., 59: 203-220.
Vij, S., R. Kanagasuntheram and A. Krishnamurti 1972
Enzymic changes in taste buds of monkey following transection of glossopharyngeal nerve. J. Anat.,113: 425-432.
Wachstein, M., and E. Meisel 1957 Histochemistry of hepatic phosphatases a t a physiologic pH. Am. J. Clin.
Pathol., 27: 13-23.
Yellin, H. 1967 Neural regulation of enzymes in muscle
fibers of red and white muscle. Exp. Neurol., 19: 92-103.
Zalewski, A. A. 1968 Changes in phosphatase enzymes
following denervation of t h e vallate papilla of t h e rat.
Exp. Neurol., 22: 40-51.
1969 Combined effects of testosterone and
motor, sensory or gustatory nerve reinnervation on t h e
regeneration of taste buds. Exp. Neurol., 24: 285-297.
1974 Neuronal and tissue specifications involved in taste bud formation. Ann. N. Y. Acad. Sci., 228:
344-349.
Zalewski, A. A., G. F. Creswell, H. G. Goshgarian and T. H.
Oh 1977 The nude mouse: An in uiuo model for demonstrating cross-species trophic nerve function. Exp. Neurol., 54: 397-402.
PLATES
PLATE 1
EXPLANATION OF FIGURES
All figures are 8 - r m frozen cross-sections of normal or transplanted tissues which were “sandwiched” between muscle to facilitate sectioning. Figures 1, 2, 6, 8, 9 and 14 ATPase stain; figures 3-5, 7, 10-12 and 15
ALK Pase stain; figure 13 AChE stain.
1 Normal rat vallate papilla. Taste buds are present only in the epithelium of the outer (arrow OT) and inner (arrow IT) trench walls. x 70.
2 Normal rat taste buds. All cells in the buds stain for ATPase. Note that the taste cells end in their apical
region as microvilli (arrows). x 300.
3 ALK Pase activity in a normal rat vallate papilla. The reaction product (arrows, the dark homogeneous
precipitate) of the enzyme is present in the superficial regions of the trench walls which support the taste
buds. This 20-minute incubation preparation does not indicate whether the enzyme is present in taste or
epithelial cells. X 70.
4 ALK Pase activity in normal rat taste buds. After five minutes of incubation reaction product is seen
around the microvilli (arrows). x 300.
5 ALK Pase activity in normal rat taste buds. After ten minutes of incubation reaction product coalesces
between microvilli of adjacent taste buds to form a linear distribution in the superficial region of the
trench wall. Note the intense ALK Pase throughout most of the extent of a taste cell (arrow). x 300.
6 Normal mouse taste buds. Buds are present in both trench walls as in the rat (fig. 1). x 60.
7 ALK Pase activity of a normal mouse vallate papilla. No enzyme activity is present in the trench walls.
x 70.
288
TASTE BUD HISTOCHEMISTRY
Andrew A. Zalewski
PLATE 1
289
PLATE 2
EXPLANATION OF FIGURES
8 Taste buds t h a t developed after combining a r a t ganglion with a mouse tongue graft. Regenerated taste
buds (region bounded by arrows) are present in t h e epithelium covering the top of the papilla. X 70.
9 A taste bud t h a t developed after combining a rat ganglion with a mouse tongue graft. All taste cells in
t h e regenerated bud exhibit ATPase staining. X 300.
10 ALK Pase activity in taste buds t h a t developed after combining a rat ganglion with a mouse tongue
graft. No enzyme is present in t h e region (bounded by arrows) which corresponds to t h e location of the
regenerated taste buds t h a t were identified by ATPase staining in figure 8. X 70.
11 ALK Pase activity in a taste bud t h a t developed after combining a rat ganglion with a mouse tongue
graft. The three arrows outline t h e taste bud which is located under t h e short-arrow in figure 10. Note
that no enzyme is present in the taste cells. X 300.
12 ALK Pase activity in taste buds t h a t developed after combining a rat ganglion with a mouse tongue
graft. No ALK Pase was seen in buds found in t h e trench wall. The arrows point to the apical ends of
taste buds which were distinguished by ATPase staining in a n adjacent section.
13 AChE staining of t h e rat ganglion which caused t h e development of the taste buds in the mouse tongue
graft illustrated in figures 8 and 10. AChE staining is present in the cytoplasm of the neurons which
survived in t h e transplanted ganglion (arrow GA). AChE staining can also be seen in the nerve trunk
(arrow NT) coming from the ganglion and in nerve fibers in t h e subepithelial region (arrow SE) where
t h e regenerated taste buds (arrow TB) are located. X 70.
14 Taste buds t h a t developed after combining a mouse ganglion with a r a t tongue graft. Several regenerated buds are present in one outer trench wall of t h e papilla (long arrow) while only one bud is present in
each inner trench wall (short arrows). X 70.
15 ALK Pase activity in taste buds t h a t developed after combining a mouse ganglion with a r a t tongue
graft. ALK Pase is localized to the superficial regions of the outer (long arrow) and inner (short arrows)
trench wall where regenerated buds appeared.
290
TASTE BUD HISTOCHEMISTRY
Andrew A. Zalewski
PLATE 2
291
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