1Department of Oral Physiology,
Ninomiya, Yuzo,
Toshiaki Imoto, and
Tadataka Sugimura.
Sweet taste responses of mouse chorda tympani neurons: existence of
gurmarin-sensitive and -insensitive receptor components. Inhibitory effects of gurmarin (gur) on responses to sucrose and other
sweeteners of single fibers of the chorda tympani nerve in C57BL mice
were examined. Of 30 single fibers that strongly responded to 0.5 M
sucrose but were not or to lesser extent responsive to 0.1 M NaCl, 0.01 M HCl, and 0.02 M quinine HCl (sucrose-best fibers), 16 fibers showed
large suppression of responses to sucrose and other sweeteners by
lingual treatment with 4.8 µM (~20 µg/ml) gur (suppressed to
4-52% of control: gur-sensitive fibers), whereas the remaining 14 fibers showed no such gur inhibition (77-106% of control:
gur-insensitive fibers). In gur-sensitive fibers, responses to sucrose
inhibited by gur recovered to ~70% of control responses after
rinsing the tongue with 15 mM Gurmarin (gur) is a peptide (mol wt, 4,209, isolated from the plant Gymnemma sylvestre) that is known to
specifically inhibit responses to sweeteners (substances taste sweet to
humans) but not to other basic taste stimuli, such as NaCl, HCl, and
quinine, in the rat (Imoto et al. 1991 Similar mouse strain and nerve specificities have been reported in
inhibition of salt responses by amiloride, a sodium channel blocker,
which suppressed NaCl responses of the chorda tymapni in C57BL but not
BALB mice (Ninomiya et al. 1989 To investigate this possibility, we examined single-fiber responses of
the chorda tympani nerve to various taste stimuli including sweeteners
before and after lingual treatment with gur in gur-sensitive C57BL mice
(Ninomiya and Imoto 1995 Subjects were adult male and female mice of C57BL/6CrSlc strain
(age 8-25 wk, n = 24 males and 12 females), ranging in
weight from 20 to 35 g. Each mouse was anesthetized with an
injection of sodium pentobarbital (40-50 mg/kg ip) and maintained at a
surgical level of anesthesia with supplemental injections of sodium
pentobarbital. The trachea was cannulated, and the mouse was then fixed
in the supine position with a head holder to allow dissection of the chorda tympani nerve. The chorda tympani nerve was exposed at its exit
from the lingual nerve and cut near its entrance to the bulla. Single
or a few fibers of the nerve were dissected apart with a pair of
needles and lifted on a silver wire electrode. An indifferent electrode
was placed in nearby tissue. Impulse discharges resulting from chemical
stimulations of the tongue were fed into an amplifier (Iyodenshikogaku
K-1), monitored on an oscilloscope and audiomonitor, recorded on a
recorder (Nihon-kohden, WS-641G), and stored on magnetic tape for
future analysis.
The anterior one-half of the mouse's tongue was enclosed in a flow
chamber. Solutions were delivered into the flow chamber by gravity flow
and flowed over the tongue for a controlled period. Solutions used were
(in M) 0.1 NaCl, 0.01 HCl, 0.02 quinine HCl, 0.03-1.0 sucrose, 0.5 fructose, 0.5 glucose, 0.02 saccharin Na, 0.03 D-tryptophan, and 0.1 D-phenylalanine. These
chemicals were dissolved in distilled water at ~23°C. Methods for
chemical stimulation were the same as those described in our previous
reports (Ninomiya and Imoto 1995 Single fibers were identified by uniform spike height, singular wave
form, and examination of latencies between contiguous spikes. In total,
30 single fibers responding to sucrose were obtained from 26 mice (1 each from 22 mice and 2 each from 4 mice). Frequency-time histograms of
impulse discharges before, during, and after chemical stimulation of
the tongue were made by means of a spike-analysis-system
(Iyodenshikogaku, SAS-1). For data analysis, we used the net average
frequency for the first 5 s after the stimulus onset obtained by
subtracting the spontaneous frequency for a 5-s period before stimulation.
Thirty chorda tympani fibers responding to sucrose were classified
into two groups according to gur sensitivity of sucrose responses. In
16 of 30 fibers, responses to 0.5 M sucrose were suppressed by gur to
<50% of control (Figs. 1, A
and B, and 2, Gur-sensitive).
Percent control responses of this group to 0.5 M sucrose after gur
ranged from 4.3 to 48.3% with a mean of 20.3 ± 13.1% (SD; Fig.
1A, Gur-sensitive). Mean number of impulses/5 s of 16 fibers
to 0.5 M sucrose after gur (12.5 ± 8.2) was significantly smaller
than that before gurmrin (66.8 ± 30.3, t-test,
P < 0.001, Fig. 1B, Gur-sensitive, A-P).
In the remaining 14 fibers, sucrose responses were only sightly if at
all inhibited by gur (Figs. 1, A and B, and 2,
Gur-insensitive). Their percent control responses ranged from 76.9 to
105.6% with a mean of 91.2 ± 8.1% (SD; Fig. 1B,
Gur-insensitive). The mean number of impulses/5 s of the 14 fibers
before gur (62.6 ± 28.6) was not significantly different from
that after gur (57.2 ± 26.7, t-test, P > 0.05, Fig. 1B, Gur-insensitive, a-n). Figure 2 shows
sample records of responses of gur-sensitive and -insensitive fibers to
four taste stimuli (0.1 M NaCl, 0.01 M HCl, 0.02 M quinine HCl, and 0.5 M sucrose) before and after gur. Both fibers strongly responded to 0.5 M sucrose but only slightly if at all to other stimuli, indicating
typical sucrose-best fibers (Frank 1973
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin and were almost
abolished by further treatment with 2% pronase. In gur-insensitive
fibers, sucrose responses were not inhibited by gur, but were largely
suppressed by pronase. These results suggest existence of two different
receptor components for sweeteners with different susceptibilities to
gur in mouse taste cells, one gur sensitive and the other gur
insensitive. Taste cells possessing each component may be specifically
innervated by a particular type of chorda tympani neurons.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
) and mouse chorda
tympani nerve (Ninomiya and Imoto 1995
; Ninomiya
et al. 1997
, 1998
) and gerbil taste cells
(Uchida and Sato 1997
). In mice, it has been shown that
the gur inhibition of sweetener responses is strain and nerve specific
(Ninomiya and Imoto 1995
; Ninomiya et al.
1997
), that is, responses of the chorda tympani nerve
innervating taste buds in the fungiform papillae to various sweeteners
were suppressed by gur to ~50% of control in C57BL mice, whereas no
such suppression was observed in BALB mice. Even in C57BL mice,
responses of the glossopharyngeal nerve innervating taste buds in the
circumvallate and foliate papillae were not suppressed by gur. These
results suggests that there may exist two different receptor types for sweeteners in mice, one gur sensitive and the other gur insensitive. The C57BL strain possesses both types in the taste buds innervated by
the chorda tympani nerve, whereas BALB mice almost exclusively have the
gur-insensitive type. However, because this evidence is based on the
analysis of relative magnitudes of whole nerve responses of peripheral
taste nerves, more convicing evidence differentiating the gur-sensitive
and -insensitive response components is required.
), and no such suppression was observed in the glossopharyngeal nerve even in C57BL
mice (Ninomiya et al. 1991
). Furthermore, in
single-fiber response analysis of the chorda tympani in C57BL mice
(Ninomiya 1996
, 1998
), rats (Ninomiya and
Funakoshi 1988
), and hamsters (Hettinger and Frank
1990
), it has also been shown that amiloride primarily inhibits
NaCl responses of fibers that selectively respond to sodium salts
(labeled N type), whereas it hardly affects NaCl responses of fibers
that are broadly sensitive to electrolytes (labeled H or E type). This
suggests the existence of selective synapse formation between
particular classes of taste fibers and amiloride-sensitive and
-insensitive taste cells in those animals. Therefore by analogy, it is
possible that, if gur-sensitive and -insensitive receptor components
for sweeteners in mice actually exist, they would differentially
localize in taste cells and might be innervated by different groups of
taste fibers. If this were the case, chorda tympani fibers innervating
sweetener-responsive taste cells might be devided into two groups,
according to their susceptibilities to gur.
). The results suggested the existence of two groups of sucrose-responsive chorda tympani fibers that would selectively innervate taste cells possessing gur-sensitive and -insensitive receptor components for sweeteners.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
; Ninomiya et al.
1997
). The order of chemical stimulation during the first
survey to find fibers responding to sweeteners was used as (in M) 0.5 sucrose, 0.1 M NaCl, 0.01 HCl, and 0.02 quinine HCl. If the fiber
clearly responded to sucrose, we continued and further applied other
sweet substances, such as (in M) 0.02 saccharin Na; 0.03, 0.1, 0.3, and
1.0 M sucrose; 0.03 D-tryptophan, and 0.1 D-phenylalanine. Then 0.5 M sucrose was applied once more to check the reproducibility of the response before the lingual treatment with gur. In some fibers, we did not try 0.03, 0.1, 0.3, and
1.0 M sucrose. During chemical stimulation of the tongue, the test
solution flowed at the same flow rate as the distilled water used for
rinsing the tongue (~0.5 ml/s). The tongue was rinsed for >1 min
during the intervals between successive stimulations. To examine gur
inhibition of chorda tympani responses, the tongue was treated with 4.8 µM (~20 µg/ml) gur dissolved in 5 mM phosphate buffer (pH 6.8)
for 5 min in the similar manner as that described by our previous study
(Ninomiya and Imoto 1995
). Gur is reported to be very
stable against selective cleavage by proteases, even under conditions
such as high temperature, low pH, and the presence of urea (Arai
et al. 1995
). Inhibitory effects of gur on rat chorda tympani
responses to sucrose were hardly changed when gur solution was heated
to 90°C, and stored for
2 mo (Imoto et al. 1991
). To
obtain similar potencies of gur throughout the experiments, we used
aliquots of the same stock solution of gur that were stored at
20
°C and warmed to the room temperature immediately before application
to the tongue. We chose a concentration of gur (4.8 µM) that
exhibited the maxmum suppressive effect on chorda tympani responses in
C57BL strain (Ninomiya and Imoto 1995
). The time for the
gur treatment (5 min) was shorter than that (10 min) used in our
previous studies. However, our pilot experiments showed that the
lingual treatment with 4.8 µM gur for 5 min produced the maximum
suppressive effect of gur on sucrose responses (~50% of control). In
the fibers whose sucrose responses were suppressed by gur, to
facilitate the recovery of the suppressed sweetener responses,
animal's tongue was rinsed for 10 min with 15 mM
-cyclodextrin (
-CD), which could remove the effect of gur (Ninomiya et al. 1998
). In most animals, gur was applied to the tongue only
once, and thereby data from only one preparation were obtained from each animal. In some animals, we tested the second gur treatment for
the second preparation. However, we did not use the data from the
second trial for the current analysis except in those cases when the
first preparation of the animal was sensitive to gur and its suppressed
response frequencies to sucrose by gur recovered to >85% of control
(monitored by a spike counter) after rinsing the tongue with
-CD. In
some fibers, 2% pronase E (dissolved in 50 mM phosphate buffer at pH
6.8), a specific inhibitor for sweetener responses in rats (Hiji
1975
) and mice (Ninomiya et al. 1997
), was
further applied for 10 min to the tongue after gur and
-CD
experiments to check its inhibitory effects on sweetener responses.
RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
). Responses to
sucrose in the gur-sensitive fiber was almost abolished by gur, whereas
no such suppression was observed in the gur-insensitive fiber. The
suppressed sucrose response after gur in the gur-sensitive fiber
recovered to some extent after rinsing the tongue with 15 mM
-CD for
10 min, which by itself has no effect on sucrose response of the
gur-insensitive fiber. The more or less complete recovery of sucrose
responses after rinsing the tongue with 15 mM
-CD was observed in
all 11 gur-sensitive fibers tested. Percent sucrose responses after
-CD among the 11 fibers were ranging from 40.0 to 88.1% of control (mean = 66.3 ± 16.0%). This mean percent sucrose response
(~66%) of the gur-sensitive fibers after
-CD was comparable with
that presumed on a basis of possible interaction between 4.8 µM gur and 15 mM
-CD [~20% of free gur (~0.96 µM) remained]
(Ninomiya et al. 1998
). The mean response (impulses/5 s)
of the 11 gur-sensitive fibers tested with
-CD, which were 69.9 ± 23.9 before gur and 12.1 ± 9.0 after gur, were recovered to
45.9 ± 17.2 after
-CD. As shown in Fig. 2, further treatment
with 2% pronase for 10 min almost abolished sucrose responses in both
types of fibers. The mean response (impulses/5 s) of six
gur-sensitive and six gur-insensitive fibers tested, which were
69.0 ± 18.3 and 60.7 ± 16.5 before gur, reduced to 3.2 ± 1.7 and 3.3 ± 4.1 after pronase, respectively. Concentration-response relationships for sucrose before and after gur
for two groups are shown in Fig.
3A. Responses of gur-sensitive fibers to 0.03-1.0 M sucrose decreased to ~15% of control
(10.4-22.2%) after gur [ANOVA, F(1,8) = 23.0, P < 0.01), whereas those of gur-insensitive fibers
were not significantly changed after gur [ANOVA, F(1,8) = 0.1, P > 0.05]. Response profiles of the two groups
of sucrose-best fibers to eight stimuli are shown in Fig.
3B. In gur-sensitive fibers, responses to not only sucrose
but other sweeteners were significantly suppressed by gur
(t-test, P < 0.05-0.001), whereas no such
inhibition was obsered in responses to any stimulus in gur-insensitive
fibers (t-test, P > 0.05). Comparing
responses to six sweeteners before gur between two groups, a
significant difference was observed in response to 0.1 M
D-phenylalanine, which was larger in gur-sensitive than
-insensitive fibers (t-test, P < 0.05).
View larger version (55K):
[in a new window]
Fig. 1.
A: distribution of mouse chorda tympani fibers according
to their percent control responses to 0.5 M sucrose after 4.8 µM
gurmarin (gur) (control, before gur = 100%). Fibers were
classified into gur-sensitive and -insensitive groups.
B: responses of gur-sensitive (n = 16, A-P) and gur-insensitive types (n = 14, a-o)
of mouse chorda tympani fibers to 0.5 M sucrose before
( ) and after gur (
).
View larger version (36K):
[in a new window]
Fig. 2.
Sample recordings of 2 types (Gur-sensitive and Gur-insensitive) of
single chorda tympani fibers to 0.1 M NaCl, 0.01 M HCl, 0.02 M quinine
HCl, and 0.5 M sucrose before (top 4 traces) and after
4.8 µM gur for 5 min (middle 4 traces), to 0.5 M
sucrose after rinsing the tongue with 15 mM -cyclodextrin for 10 min
(second trace from the bottom), and after further
treatment with 2% pronase for 10 min (bottom trace).
Stimulation started at the point indicated by the arrow.
View larger version (28K):
[in a new window]
Fig. 3.
A: concentration-response relationships for sucrose
before ( ) and after 4.8 µM gur (
) in
2 types of mouse chorda tympani fibers [Gur-sensitive
(n = 6) and Gur-insensitive (n = 5)]. Vertical bars indicate SDs. B: responses
profiles to 0.1 M NaCl (Na), 0.01 M HCl (H), 0.02 M quinine HCl (Q),
0.5 M sucrose (Suc), 0.5 M fructose (Fru), 0.5 M glucose (Glc), 0.02 M
saccharin Na (Sac), 0.03 M D-tryptophan
(D-Try), and 0.1 M D-phenylalanine
(D-Phe) before (
) and after 4.8 µM gur
(
) in 2 types of mouse chorda tympani fibers
[Gur-sensitive (n = 9-16) and Gur-insensitive
type (n = 9-14)]. Vertical bars indicate SDs.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In our previous study measuring whole-nerve responses of the mouse
chorda tympani (Ninomiya and Imoto 1995), we found that in C57BL mice gur at the most effective range of concentration suppressed chorda tympani responses to various sweeteners to ~50% of
control. Further, we hypothesized that this strain possesses two
different response components for sweeteners, one gur sensitive and the
other gur insensitive. The current single fiber study revealed the
existence of both gur-sensitive and -insensitive types of
sucrose-responsive and pronase-sensitive chorda tympani fibers in this
strain and thereby clearly supported the previously mentioned
hypothesis. Responses to 0.03-1.0 M sucrose in gur-sensitive fibers
were suppressed to ~20% of control by gur (Figs. 1-3), whereas no
such suppression was evident in gur-insensitive fibers (~90% of
control after gur) (Figs. 1-3). In total of both gur-sensitive and
-insensitive fibers, the mean response (impulses/5 s) to 0.5 M sucrose
(64.9 ± 29.6, n = 30) was reduced to 51.5%
(impulses/5 s: 33.4 ± 29.4, n = 30) by gur, which
was comparable with that found in whole-nerve experiments. In
single-fiber studies on selective inhibitors, especially whose effects
are long lasting such as gur, 2-3 h in rats and mice (Imoto et
al. 1991
; Ninomiya and Imoto 1995
), and gymnemic
acid, 1 h in humans (Kurihara 1969
), there might
generally be difficulty in determining if the reduction of response
after the inhibitor was caused by the inhibitor or by innevitable
gradual decrease in basic activity of fibers during a long-duration
recording. To overcome this difficulty, we used
-CD, which reduced
the effect of gur (Ninomiya et al. 1998
), and found that
in all fibers tested the suppression of sucrose responses by gur was to
some extent (mean: ~70% of control) reversed by rinsing the tongue
with
-CD. This suggests that reduction of impulses of the fibers
after gur observed in this study mostly was due to the effect of gur.
-CD is known to contain seven
-1,4-linked D-glucopyranose units and have a hydrophobic cavity leading
to specific binding of guest molecules, such as amino acids with phenyl
rings (e.g., tyrosine and phenylalanine) (Li and Purdy 1992
). Our recent studies (Ninomiya et al. 1998
)
have demonstrated that the inhibitory effect of
-CD on gur
suppression of sweetener responses is due to formation of inclusion
complexes between
-CD and tyrosine residues of gur. Therefore the
observed effect of gur may be due to its action on extracellular
domains of sweetener receptors of taste cells, and
-CD may prevent
gur from binding with receptor sites by forming the inclusion complexes
with gur on the taste cell membrane.
Existence of more than one type of the sweet taste receptor in mammals
has been suggested by many studies, including human psychophysical
(Faurion et al. 1985; Schiffman et al.
1981
) and neurophysiological studies recording whole chorda
tympani responses in chimpanzees (Hellekant et al.
1985
), dogs (Anderson et al. 1962
), gerbils
(Vlahopoulous and Jakinovich 1986
), rats (Yamada and Imoto 1987
; Yamamoto and Kawamura
1971
), hamsters (Yamada and Imoto 1987
),
and mice (Iwasaki and Sato 1986
). In gerbils, previous
studies with sweetener inhibitors (Vlahopoulous and Jakinovich 1986
) suggested the existence of at least two different
receptor sites for sucrose because the gerbil chorda tympani responses to sucrose were inhibited by three different competitive inhibitors with different structural characteristics (methyl
4,6-dichloro-4,6-dideoxy-
-D-galacto-pyranoside,
-nitrophenyl
-D-glucopyranoside, and
chloramphenicol). In chimpanzees, gymnemic acid did not completely
abolish the chorda tympani responses to sucrose, and 20-30% of the
sucrose response remained after gymnemic acid, suggesting the existence
of two different sucrose sites, one gymnemic acid sensitive and the
other gymnemic acid insensitive (Hellekant et al. 1985
).
Incomplete suppression of whole chorda tympani responses to sucrose by
inhibitors has also been reported in cases of zizyphin in rats and
hamsters (Yamada and Imoto 1987
), Zn and/or Cu ions in
rats (Yamamoto and Kawamura 1971
), hamsters
(Myers et al. 1993
), and mice (Iwasaki and Sato 1986
), and gur in rats (Imoto et al.
1991
) and mice (Ninomiya and Imoto 1995
).
However, because the magnitude of suppression of responses might be
depend on the potency of inhibitors or many other possible factors
(i.e., concentration dependencies, competitive or noncompetitive), the
existence of the residual responses to sucrose after treatment with
such inhibitors may not always indicate the existence of two different
sucrose receptor sites. For example, recent studies analyzing
single-fiber responses in chimpanzee chorda tympani (Hellekant
et al. 1998
) demonstrated that gymnemic acid exclusively
inhibited responses to sweeteners in S-type (sucrose-best) fibers that
were classified by a hierarhical cluster analysis but not those in
other types of fibers. Therefore the residual responses after gymnemic
acid might be derived from those of fibers other than S-type cluster,
which may mainly relate to recognition for taste substances other than
sweeteners. The current results showing that two groups of sucrose-best
chorda tympani fibers are classified by their gur sensitivities may
thus be the first clear-cut evidence suggesting the existence of two
different sucrose and/or sweetener sites in mice.
This study revealed that two groups of sucrose-responsive chorda
tympani fibers selectively innervate taste cells whose sweetener responses were inhibited or unaffected by gur. This possible matching between taste cells possessing particular receptor mechanisms and their
corresponding types of fibers has previously been suggested in the
cases between amiloride-sensitive and -insensitive taste cells and
axons in rats (Ninomiya and Funakoshi 1988), hamsters (Hettinger and Frank 1990
), and mice (Ninomiya
1998
) and two different types of amiloride-sensitive fibers
with different temperature dependencies in mice (Ninomiya
1996
). A recent study demonstrated that such selective synaptic
formation between taste cells and axons with different amiloride
sensitivities was not altered even after cross-regeneration between the
chorda tympani and glossopharyngeal nerves in mice (Ninomiya
1998
). This finding suggests that regenerated taste axons
selectively recouple with the appropriate type of receptor cell whether
they innervate the front or the back of the tongue. Existence of
selective synapse formation between the amiloride-sensitive cell types
was also suggested in the relay from the peripheral taste axons to the
second-order neurons in the nucleus tractus solitarius (Scott
and Giza 1990
). Olfactory receptor neurons are known to express
a particular type of receptor protein (Chess et al.
1994
) and send their axons to any one or a few of some 2,000 modules, called glomeruli, in the olfactory bulb (Ressler et al.
1994
; Vassar et al. 1994
). For this
highly specific mechanism of axon guidance, existence of guidance
molecules (i.e., receptor proteins) (Singer et al. 1995
)
has been postulated. It is therefore possible that guidance molecules
may exist also in taste cells and/or axons to effect the matching.
Future extensive studies including molecular genetic analysis may lead
to answer to this question on taste axon-cell matching specificity.
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank Dr. Bruce P. Bryant of the Monell Chemical Senses Center for helpful discussions.
This work was supported in part by Grants-in-Aid 9470407 and 9557147 for Scientific Research from the Ministry of Education, Science, Sport, and Culture of Japan.
![]() |
FOOTNOTES |
---|
Address for reprint requests: Y. Ninomiya, Dept. of Oral Physiology, Asahi University School of Dentistry, Hozumi, Motosu, Gifu 501-0296, Japan.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 January 1999; accepted in final form 25 February 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|