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Continuous trophic influence of chromatolysed gustatory neurons on taste buds.

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Continuous Trophic Influence of Chromatolysed
Gustatory Neurons on Taste Buds
ANDREW A. ZALEWSKI
Laboratory of Neuropathology and Neuroanatomical Sciences, National
Institute of Neurological Diseases and Stroke, National Institutes of
Health, Public Health Service, U. S . Department of Health,
Education and Welfare, Bethesda, Margland 20014
ABSTRACT
Taste buds are believed to be morphologically dependent on a neurohumor that is supplied by the intact gustatory nerve because after nerve transection
the buds disappear. Since taste buds regenerate after reinnervation by peripheral or
central fibers of the nodose ganglion, the present experiment was performed to determine whether chromatolysed gustatory neurons could support taste buds. The
denervated vallate papillae o f adult rats was reinnervated by the central fibers of
the nodose ganglion and, after a time sufficient to allow for nerve and bud regeneration, the peripheral fibers of the ganglion were cut. Despite undergoing the typical
chromatolytic reaction, the gustatory neurons still maintained the taste buds. The
number and appearance of the buds found was similar to that seen i n control centrally reinnervated papillae. Transection o f the central fibers did, however, cause
the disappearance of the buds. The results demonstrate that chromatolysed gustatory
neurons can support taste buds, and it is concluded that the metabolic changes which
occur i n chromatolysed gustatory neurons do not interfere with their trophic function.
Taste buds disappear from the gustatory
papillae after transection of their nerve
supply. It has been suggested that the buds
disappear because denervation eliminates
a neurohumor that is needed to support
the buds (Olmsted, '20; Torrey, '34). This
neurohumor was suggested to originate
in the nerve cell body and pass via axoplasmic flow to the periphery where it
causes taste bud development, maintenance, or regeneration. Furthermore, the
neurohumor is transmitted by peripheral
and central fibers of the gustatory neurons
because reinnervation of a denervated
papilla by either of these fibers causes
bud regeneration (Zalewski, '69b). The
present experiment was performed to determine whether chromatolysed gustatory
neurons could continue to provide the
trophic neurohumor needed to maintain
the taste buds. In order to test the trophic
function of chromatolysed neurons, the
denervated vallate papilla was first reinnervated by the central fibers of the
nodose ganglion; the peripheral fibers
were left intact. After a time designed to
allow for nerve and bud regeneration
(Zalewski, '69b), the peripheral fibers
were cut and the papilla examined for
taste buds. Since a number of metabolic
changes occur in neurons after transecANAT. REC., 167: 165-174.
tion of their peripheral fibers (Bodian and
Mellors, '45; Nandy, '68), the chromatolytic reaction could affect the taste buds
in various ways depending on what happened to the hypothetical neurohumor.
First, the neurons could stop producing
it so that all the buds would degenerate
and disappear, or secondly, increased
amounts of the neurohumor could be produced so that more buds would appear,
and finally no change could occur so that
the number and appearance of the buds
would be similar to that of a control centrally reinnervated papilla.
The present experiment was designed
therefore to test the trophic function of
chromatolysed gustatory neurons on taste
buds.
MATERIALS AND METHODS
Taste buds in the rat's vallate papilla
are innervated by the right and left glossopharyngeal nerves and in order to cause
the disappearance of all taste buds both
nerves have to be transected. Since reininervation by one gustatory nerve can
cause the appearance of many buds
(Zalewski, '69a), the following unilateral
reinnervation was performed. While under
chloral hydrate anesthesia (40 mg/100
Received Dec. 15, '69. Accepted Jan. 28, '70.
165
166
ANDREW A. ZALEWSKI
gm ip), the vallate papilla of OsborneMendel male rats (225-250 gm) was
denervated by bilaterally transecting the
glossopharyngeal nerve beneath the stylohyoid muscle. The left nodose ganglion
was exposed and the nerve fibers cut as
far as possible central to the ganglion. An
arterial graft technique (Zalewski, '69a)
was then employed to join the central
fibers that were attached to the nodose
ganglion to the distal glossopharyngeal
nerve stump and thereby effect a unilateral
reinnervation of the papilla. The central
ends of the glossopharyngeal nerves were
implanted into adjacent neck muscle in
order to prevent their regeneration back
to the tongue. Six months later some animals again were anesthetized and either
the peripheral ( 9 animals) or central ( 3
animals) fibers of the ganglion cut. Either
peripheral or central nerve transection was
performed 5-7 mm from the ganglion, and
the nerve end attached to the ganglion
implanted into adjacent muscle. A schematic drawing of the operative procedures
is shown in figure 1. The papillae of three
animals with peripherally and one animal
with centrally transected nerve fibers
were studied after 5, 10, and 30 days.
Two normal, two chronically denervated,
and two control centrally reinnervated
papillae were also studied.
At the appropriate time, the animals
were anesthetized and exsanguinated. The
tongues were removed, and the region
containing the vallate papilla quenched
in isopentane that was cooled to - 70°C
by dry ice. Six-micron thick frozen crosssections were cut and incubated to detect
adenosine triphosphatase or cholinesterase
activity. In addition, the left nodose
ganglion of each animal was removed,
and 8 p thick frozen sections incubated
to detect acid phosphatase activity. Details
of all the histochemical procedures have
been reported (Zalewski, '68). Control
enzyme sections were incubated without
added substrate.
OBSERVATIONS
Taste buds
Taste buds in the normal, chronically
denervated, and centrally reinnervated
vallate papilla. Taste buds were identified by their adenosine triphosphatase ac-
POST-OPERATIVE
PRE-OPERATIVE
Tongue
1 ongue
Chest,Abdomen
Chest,Abdomen
Fig. 1 Schematic drawing of the operative procedures. The pre-operative figure shows the normal neuroanatomical relationship of the glossopharyngeal and vagus nerves. The glossopharyngeal
nerve (left and right) was transected at A, while the vagus nerve (left only) was cut at B. The
post-operative figure demonstrates the arterial sleeve and the method used to join the central fibers that are attached to the vagal nodose ganglion to the distal peripheral fibers of the glossopharyngeal nerve. After six months, the peripheral vagal fibers were cut at C to induce chromatolysis
in the neurons of the vagal nodose ganglion.
CHROMATOLYSED GUSTATORY NEURONS
tivity. In the normal papilla, the buds
were found only in the epithelium of the
lower portions of the trench walls (fig. 2).
Each bud extended through the entire
width of the epithelium, and all the taste
bud cells exhibited adenosine triphosphatase activity (fig. 6). Since dermal nerve
fibers and blood vessels also had adenosine triphosphatase activity, nerve fibers
were examined by a cholinesterase reaction. Cholinesterase staining revealed a
plexus of nerve fibers immediately beneath the epithelium where the taste buds
were found. Control sections of all papillae
that were incubated without added substrate did not reveal any enzyme staining.
No taste buds were found in the chronically denervated papillae (fig. 4 ) . The epithelium that previously contained the buds
was atrophied, and none of the epithelial
cells present exhibited the intense adenosine triphosphatase activity characteristic
of taste bud cells. Cholinesterase staining
was absent beneath the epithelium.
Taste buds were found randomly distributed throughout all papillae after reinnervation by the central fibers of the
nodose ganglion (fig. 5). Although smaller
numbers of buds were present, those found
clearly resembled the buds of a normal
papilla. The regenerated buds extended
through the entire width of the epithelium
and seemed to contain a normal number
of taste bud cells. All the bud cells had
intense adenosine triphosphatase activity
(fig. 6 ) . Intense cholinesterase activity
was demonstrated beneath the trench epithelium supporting the buds, whereas
faint activity was present beneath regions
1acking them.
E f f e c t of central nerve fiber transection
on the regenerated taste buds. The taste
buds that were found five days after cutting the central reinnervating fibers of
the nodose ganglion were in various stages
of degeneration (figs 3 , 7 ) . The buds
varied in size and shape, and they appeared to be moving away from the basement membrane toward the epithelial
surface. The degenerating bud was not
lost as a unit; individual taste cells seemed
to be lost so that the bud became progressively smaller as it moved toward the
epithelial surf ace. The adenosine triphosphatase activity of the degenerating taste
167
cells was still intense and only slightly
reduced from that of normal taste cells.
Cholinesterase activity was virtually absent beneath the regions of degenerating
taste buds. By ten days, all traces of taste
buds (fig. 4 ) and cholinesterase activity
had disappeared. No cells with intense
adenosine triphosphatase activity remained
anywhere in the epithelium (fig. 8). The
only added change found 30 days after
cutting the central nerve fibers was a
slight atrophy of the epithelium.
E f f e c t of peripheral nerve fiber transection o n the regenerated taste buds. Normal appearing taste buds were found at
all time intervals after cutting the peripheral nodose nerve fibers (figs. 5,9).
There was no obvious decrease or increase
in the number of buds when compared to
control centrally reinnervated papillae. All
the buds were comparable in size, shape,
number of taste cells, and enzyme activity
to normal or regenerated buds, Cholinesterase activity was intense at all times
beneath the epithelium containing the
buds.
Nodose ganglion
Nerve cells in the normal and centrally
reinnervating nodose ganglia. The neurons in the nodose ganglion of normal as
well as control centrally reinnervated animals had a similar appearance (fig. 10).
Neurons of various sizes were present,
and almost all had a centrally located
nucleus. Acid phosphatase activity was intense in some neurons, but in most it was
moderate to faint. The enzyme seemed
to have predominantly perinuclear distribution.
E f f e c t of peripheral nerve fiber transection o n t h e nerve cells of the nodose
ganglion. Transection of the peripheral
fibers caused an increase in acid phosphatase activity of all the neurons in the
ganglion. Enzyme activity was only slightly
increased after five days, but after ten
days, it was markedly increased (fig. 11).
In addition, the neurons by ten days had
a peripherally located nucleus. Acid phosphatase activity was still elevated after
30 days, but considerably less so than
that found at ten days.
E f f e c t of central nerve fiber transection
on the nerve cells of the nodose ganglion.
Transection of the central fibers did not
168
ANDREW A. ZALEWSKI
cause any displacement of the nerve cell's
nucleus, but it did cause a moderate increase in acid phosphatase activity. The
increase in the enzyme was first noted
after ten days, but it was not as marked
as the increase found after peripheral
nerve transection. Enzyme activity was
normal or perhaps slightly elevated after
30 days.
Although quantitative counts were not
made, it appeared that all ganglia of centrally reinnervated animals (control and
experimental) had a smaller number of
neurons.
occur in the nerve cell body could be
unrelated to peripheral metabolic events.
It should not be inferred, however, that
chromatolysis did not cause any changes
in the taste bud cells. Biochemical and
physiological changes could well have occurred but been insufficient to cause bud
degeneration.
The observations on the degenerating
buds seen after cutting the central fibers
of the reinnervated papillae confirm the
previous suggestion that degenerating
taste buds are lost by desquamation from
the epithelial surface (Guth, '57). It
DISCUSSION
should be stressed that the degenerating
An increase in acid phosphatase activity bud is not lost as a unit, but rather indiand a peripheral displacement of the nerve vidual cells are lost until the bud struccells nucleus are two of the many bio- ture completely disappears. Furthermore,
chemical and morphological changes since no cells with intense adenosine triwhich occur in chromatolysed neurons phosphatase activity remain after dener(Bodian and Mellors, '45; Nandy, '68). vation, it is probable that residual generalYet, despite undergoing chromatolytic lingual epithelial cells of the papilla rather
changes, the gustatory neurons still main- than "taste cell rests" are transformed
tained the taste buds. If chromatolysis did into taste buds after gustatory nerve reaffect the neuron's trophic function, the generation. The demonstration that taste
most likely result would have been a buds regenerate after excision of the encessation or decrease in the production tire gustatory papilla indicates that cells
of the neurohumor and a disappearance other than those covering the papilla can
of the taste buds. However, no evidence give rise to taste buds (Zalewski, '69c).
of bud degeneration was found. It is unAlthough chromatolytic changes (morlikely that affected buds would have com- phological and biochemical) regularly
pletely disappeared before five days since occur in sensory neurons after transecvarious remnants of degenerating buds tion of their peripheral fibers, no such
were readily found five days after cutting changes have yet to be demonstrated after
the central fibers of the control reinner- transection of their central fibers (Carmel
vated papillae. Furthermore, it would be and Bennett, '69; Lieberman, '69). Howdifficult to imagine why inadequate ever, the finding in the present study that
amounts of the neurohumor should exist acid phosphatase activity increased in the
prior to five days when the chromatolytic nodose ganglion neurons ten days after
reaction did not reach a peak until ten transection of their central fibers suggests
days after peripheral nerve transection. that biochemical changes do occur. A
If buds or bud cells were lost, some evi- more comprehensive biochemical study of
dence of their degeneration should have neurons should therefore be performed
been found at the five or ten days inter- before the chromatolytic reaction can be
vals. Since the number of buds and cells considered a reaction specific to periphfound after inducing chromatolysis was eral and not central nerve fiber transection.
similar to that of control centrally reinLITERATURE CITED
nervated papillae, it can be concluded that
the metabolic changes which occurred in Bodian, D., and R. C. Mellors 1945 The rethe cell body of the gustatory neurons did
generative cycle of motoneurons, with special
reference t o phosphatase activity. J. Comp.
not interfere with their trophic function,
Med., 81: 4 6 9 4 8 8 .
This result may indicate that the trophic
P. W., and B. M. Stein 1969 Cell
neurohumor is produced in the nerve fibers Carmel,
changes in sensory ganglia following proximal
rather than in the cell body. If this were
and distal nerve section in the monkey. J.
the case, the chromatolytic changes which
Comp. Neur., 135: 145-166.
CHROMATOLYSED GUSTATORY NEURONS
Guth, L. 1957 The effects of glossopharyngeal
nerve transection on the circumvallate papilla
of the rat. Anat. Rec., 128: 715-732.
Lieberman, A. R. 1969 Absence of ultrastructural changes i n ganglionic neurons after
supranodose vagatomy. J. Anat., 104: 49-54.
Nandy, K. 1968 Histochemical study on chromatolytic neurons. Arch. Neurol., 18: 425-434.
Olmsted, J. M. D. 1920 The results of cutting
the seventh cranial nerve in Amiurus nebulosus (Lesueur). J. Exp. Zool., 31: 369-401.
Torrey, T. W. 1934 The relation of taste buds
to their nerve fibers. J. Comp. Neur., 59:
203-220.
169
Zalewski, A. A. 1968 Changes i n phosphatase
enzymes following denervation of the vallate
of the rat. Exa. Neurol.. 22: 40-51.
.aaailla
*
1969a Combined effects of testosterone
and motor, sensory, or gustatory nerve reinnervation on the regeneration of taste buds i n
the rat. Exp. Neurol., 24: 285-297.
196913 Regeneration of taste buds after
reinnervation by peripheral or central fibers of
vagal ganglia. Exp. Neurol., 25: 429-437.
-1969c Regeneration of taste buds i n
the lingual epithelium after excision of the
vallate papilla. Exp. Neurol., i n press.
PLATE 1
E X P L A N A T I O N OF FIGURES
All figures are cross-sections of the vallate papilla. Adenosine triphosphatase stain. x 70.
170
2
Normal vallate papilla. Taste buds are present only in the epithelium
of the lower portions of the trench walls.
3
Reinnervated vallate papilla five days after cutting the central fibers
of the nodose ganglion. Degenerating tatse buds are seen i n the
trench walls.
4
Reinnervated vallate papilla ten days after cutting the central fibers
of the nodose ganglion. No taste buds are present. The chronically
denervated papillae similarity lacked taste buds.
5
Reinnervated vallate papilla ten days after cutting the peripheral fibers of the nodose ganglion. Normal appearing taste buds are present.
A similar number and appearance of buds were found in control
centrally reinnervated papilla.
CHROMATOLYSED GUSTATORY NEURONS
Andrew A. Zalewski
PLATE 1
171
PLATE 2
EXPLANATION OF FIGURES
6
Trench wall of control centrally reinnervated papilla. The regenerated buds extend through the entire width of the epithelium, and
all the taste cells exhibit adenosine triphosphatase activity. Buds of
a normal papilla have a similar appearance. Adenosine triphosphatase stain. x 300.
7
Trench wall five days after cutting the central reinnervating fibers.
The degenerating buds have moved away from the basement membrane and contain very few taste cells. Adenosine triphosphatase
stain. x 300.
8
Trench wall ten days after cutting the central reinnervating fibers.
No taste bud cells are present. Adenosine triphosphatase stain. x 300.
9 Trench wall ten days after cutting the peripheral reinnervating fibers. The buds have a normal appearance. Adenosine triphosphatase
stain. x 300.
10 Nerve cells of control central reinnervating nodose ganglion. Most
neurons have moderate to faint acid phosphatase activity similar to
that of a normal nodose ganglion. Acid phosphatase stain. x 300.
11
172
Nerve cells ten days after cutting the peripheral fibers of the central
reinnervating nodose ganglion. All the neurons exhibit a marked
increase i n acid phosphatase activity. Acid phosphatase stain. x 300.
CHROMATOLYSED GUSTATORY NEUROiYS
PLATE 2
Andrew A. Zalewski
173
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