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1 Department of Anatomy and Neurobiology, University of Tennessee College of Medicine, Memphis, Tennessee 38163, Japan 2 Division of Integrative Physiology, Department of Functional, Morphological, and Regulation Science, Faculty of Medicine, Tottori University, Yonago 683-0826, Japan
Submitted 6 March 2003; accepted in final form 9 April 2003
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ABSTRACT |
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50%
relative to control. On average, attenuation was statistically significant
only in class S and N neurons. Although the magnitude of gurmarin-induced
response suppression did not differ across sucrose concentration, responses to
different sweet-tasting compounds were differentially affected. Responses to
NaCl, HCl, or quinine were not suppressed by gurmarin. Results suggest that
information from gurmarin-sensitive and -insensitive receptor processes
converges onto single NST neurons. |
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INTRODUCTION |
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), significantly attenuates integrated chorda tympani (CT)
nerve responses to sweet-tasting substances in rats
(Imoto et al. 1991;
Miyasaka and Imoto 1995) and
C57BL mice (Ninomiya and Imoto
1995; Ninomiya et al.
1997,
1998). Additionally, palatal
gurmarin treatment suppresses integrated responses to sugars, sodium
saccharin, and sweet-tasting amino acids recorded from the greater superficial
petrosal (GSP) nerve in rats (Harada and
Kasahara 2000). Gurmarin does not affect responses to nonsweet
stimuli representative of other basic taste quality classes (e.g., NaCl, HCl
and quinine) in the CT (Imoto et al.
1991; Miyasaka and Imoto
1995) and GSP (Harada and
Kasahara 2000) nerves in rats and the CT nerve in mice
(Ninomiya and Imoto 1995;
Ninomiya et al. 1997,
1998).
Lingual gurmarin treatment does not affect integrated responses to
sweeteners recorded from the glossopharyngeal nerve of C57BL mice
(Ninomiya et al. 1997). Data
obtained from this species suggest the existence of two different types of
receptors for sweets, gurmarin-sensitive and -insensitive, that are
differentially distributed across the tongue. Moreover, a recent extension of
these findings shows that gurmarin application inhibits responses to sucrose
in only a subset of sugar-best CT fibers in this strain of mouse
(Ninomiya et al. 1999). Those
fibers that are affected exhibit absolute or near absolute suppression, with
responses to 0.5 M sucrose suppressed to 
10% of control on average. This
differential effect is somewhat analogous to the influence of amiloride on the
neural processing of salt information as lingual application of amiloride
suppresses responses to salts only in NaCl-best CT fibers
(Hettinger and Frank 1990;
Ninomiya and Funakoshi 1988)
and NaCl-best (Boughter and Smith
1998; Boughter et al.
1999; Giza and Scott
1991; Scott and Giza
1990; Smith et al.
1996; St. John and Smith
2000) and sucrose-best (Smith
et al. 1996; St. John and
Smith 2000) neurons in the nucleus of the solitary tract (NST).
Therefore it is possible that input arising from gurmarin-sensitive and
-insensitive sweet transduction mechanisms is segregated to particular classes
of taste-responsive neurons in the brain stem.
To test the hypothesis that gurmarin sensitivity is differentially distributed across gustatory neuron types in the rat NST, we recorded trains of actions potentials evoked by various taste stimuli, including sucrose and other sweeteners, from single NST neurons prior to and after lingual/palatal application of gurmarin. These experiments attempt to relate specific transduction mechanisms to the organization of gustatory neural circuitry within the CNS.
A portion of these results was presented at the 2002 meeting of the Association for Chemoreception Sciences, Sarasota, FL.
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METHODS |
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Thirty-four adult male Sprague Dawley rats, weighing 210–550 g, were
used as subjects. Rats were housed individually in a vivarium, which
maintained a 12-h light/dark schedule and ambient temperature of
23°C. Food and water were available ad libitum. Subjects were deeply
anesthetized with urethan (1.5 g/kg ip) and prepared for electrophysiological
recording. Each rat was tracheotomized and secured in a nontraumatic head
holder that deflected its snout 27° downward: this configuration
served to minimize brain stem movements associated with breathing. The
occipital bone was removed and parts of the cerebellum were gently aspirated
to expose the brain stem and allow access to the NST. Body temperature was
maintained at 37°C by a heating pad.
Single-unit electrophysiology
Etched tungsten microelectrodes, insulated except for the tip (impedance =
0.5–8 M
at 1 kHz, FHC, Bowdoinham, ME), were used to record
extracellular action potentials from single NST neurons. For each preparation,
the area of the brain stem where the rostral pole of the solitary tract
resided was visually located using vascular landmarks present on the dorsal
surface of the exposed tissue. A hydraulic micromanipulator was then used to
slowly advance the microelectrode through the brain stem. The portion of the
NST that contained neurons responsive to lingual stimulation was initially
identified by a change in neural activity associated with the passage of
anodal current (10 µA/500 ms) across the anterior tongue; cells were then
verified as taste-driven by application of various gustatory stimuli (see
following text). The gustatory-responsive portion of the NST was located
1 mm ventral to the brain stem surface.
Electrophysiological activity was band-pass filtered (bandwidth = 0.3–10 kHz), differentially amplified (Grass P511 with high-impedance probe) and subsequently routed to various monitors and analytic devices. Spikes that arose from single neurons were identified based on waveform consistency, which was continuously observed throughout each recording session using a storage oscilloscope and, following analog to digital conversion (sampling rate = 25 kHz), a template-matching algorithm (Power 1401 RISC acquisition interface coupled with Spike 2 software, CED, Cambridge, UK). Well-isolated neurons with robust responses to 0.5 M sucrose were used for experimentation. Trains of action potentials that arose during recording sessions were pulse-code modulated and stored, along with voice and trial marker cues, on VHS tape. Digital records of aggregate electrophysiological activity, including template-matched spikes, were downloaded to storage media for off-line quantitative analysis.
Taste stimuli
Generally, neurons were tested with two of three groups of taste stimuli. Tastants within each group were presented individually to, and subsequently rinsed from, the oral cavity of each preparation under normal (i.e., control) conditions and after oral application of gurmarin. Stimulus trials of interest were replicated as many times as possible. Every neuron was tested with a set of stimuli that consisted of representatives of the four basic taste qualities (herein referred to as the basic stimulus set), which were presented in random order. Some cells were then subjected to a half-logarithmic step ascending concentration series of sucrose, whereas others were tested using a randomized array of various other sweet-tasting compounds (see Table 1).
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Tastants were made from reagent grade stock dissolved in deionized water.
Solutions were delivered at room temperature to the anterior tongue and palate
via a gravity flow system at a rate of 2.5 ml/s. A three-way solenoid
fluid valve, which was controlled by the acquisition system, regulated
solution delivery. A curved, polyethylene tube that extended from the output
port of this valve was directed toward the palate of each subject. Visual
inspection revealed that this configuration allowed solutions to effectively
bathe both the palate and anterior tongue as solutions were deflected downward
on encountering the palate. Moreover, a test using methylene blue dye was
performed on one preparation to verify that this method of taste stimulus
delivery adequately bathed both the anterior tongue and palate. Using the
described flow system, dye was delivered to the oral cavity for 10 s. The
distribution of stain was then assessed with a dissecting microscope, which
revealed that the entire anterior tongue and soft palate were dyed. A
subsequent test verified that the nasoincisor ducts (NIDs) were also
stained.
During data acquisition, taste stimuli were presented to each subject using
a specific protocol. The tongue and palate were first rinsed with deionized
water for 10 s followed immediately by a taste stimulus for 10 s. The tongue
and palate were then rinsed with 50 ml of deionized water and >2 min
was allowed to elapse between trials. The stimulus delivery system was
thoroughly rinsed with deionized water between presentation trials.
Experimental protocol and gurmarin application
Once applied, gurmarin is a difficult substance to remove from gustatory
epithelia. Integrated CT responses to sucrose required >4 h for complete
recovery after gurmarin treatment despite repetitive distilled water rinses of
the tongue (Miyasaka and Imoto
1995). Although methods do exist by which gurmarin removal may be
chemically facilitated (Ninomiya et al.
1998), only slight recovery of mouse CT fiber responses to sucrose
was observed at 10 min post anti-gurmarin treatment
(Ninomiya et al. 1999).
Moreover, repeated anti-gurmarin reactions, which required up to 100 min, were
necessary for complete recovery of CT sucrose responses to baseline levels
(Miyasaka and Imoto 1995).
Therefore we chose a simple pre-/postgurmarin design, assuming that responses
to nonsweet stimuli would serve as controls for the viability of the neurons
from which we recorded, as responses to NaCl, HCl, and quinine in the CT
(Imoto et al. 1991;
Miyasaka and Imoto 1995) and
GSP (Harada and Kasahara 2000)
nerves in rats and the CT nerve in mice
(Ninomiya and Imoto 1995;
Ninomiya et al. 1997,
1998) are not altered by
gurmarin treatment.
Once single-unit responses evoked during control stimulus presentations
were recorded, 10 µg/ml gurmarin (dissolved in deionized water; 2.4
µM) was applied to both the tongue and palate of each subject using a
blunt-tipped syringe (2–4 ml total volume). For each preparation, the
application of gurmarin to the tongue and palate was visually verified.
Moreover, a test using methylene blue dye was performed on one preparation to
verify that our gurmarin application procedure did indeed adequately bathe
both the anterior tongue and palate, including the NIDs.
Treatment of the tongue with 10 µg/ml gurmarin has been shown to produce
significant and near maximal attenuation of integrated CT nerve responses to
0.5 M sucrose in the rat (Miyasaka and
Imoto 1995). When applied to the rat palate, 10 µg/ml gurmarin
yielded significant and substantial suppression of integrated greater
superficial petrosal nerve responses to sucrose and other compounds described
as sweet tasting by humans (Harada and
Kasahara 2000). However, the effect of gurmarin is not
instantaneous after application, with 4.8 µM gurmarin requiring 5 min
to produce maximal suppression of sucrose responses recorded from single mouse
CT fibers (Ninomiya et al.
1999). Therefore we allowed 10–15 min to elapse after
gurmarin treatment before proceeding with experimentation. After this time,
each component of the stimulus set used under control conditions was presented
as previously described and evoked responses recorded. After completion of
this phase, an additional experiment was conducted on some neurons using a
stronger concentration of gurmarin to determine if effects, or lack thereof,
observed at the standard concentration were indeed limits. For these cells, we
doubled the concentration of gurmarin (20 µg/ml; 4.8 µM), applied
it as previously described and retested the stimulus set 10–15 min
posttreatment.
Data analysis
The basic metric used to quantify gustatory responses in NST neurons was net response magnitude, expressed in spikes/s. This measure was calculated as the average number of spikes that occurred each s during the first 5 s of taste stimulus presentation minus the average firing rate per s during the 5-s water rinse period that immediately preceded this epoch (i.e., spontaneous discharge). These data served as input to all subsequent statistical analyses. For each neuron, responses evoked by chemical stimuli were considered significant if the net response exceeded the mean ± 2.54 SDs of the spontaneous discharge rate.
To describe the breadth of tuning of each neuron, a measure of response
profile entropy (Shannon and Weaver
1949; Smith and Travers
1979) was calculated using net responses to each component of the
basic stimulus set. Entropy is defined as
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0. Cells were categorized into types based on the stimulus within the basic set that evoked the maximal net response (i.e., their best stimulus) prior to gurmarin treatment. Additionally, hierarchical cluster analysis (HCA; conducted using Statistica, StatSoft, Tulsa, OK) was performed to quantitatively and objectively identify sets of neurons with pregurmarin tuning profiles (i.e., net responses to only basic set stimuli) that were most similar. Input to HCA consisted of a distance matrix representing pairwise neuronal tuning profile similarity/dissimilarity, where 1– Pearson's product-moment correlation (r) served as the distance metric. The unweighted pair-group average amalgamation schedule was used.
The effect of gurmarin on taste responses was statistically evaluated by
using appropriate ANOVAs. Significant interactions were sometimes explored
using planned interaction comparisons as our general hypothesis dictated a
priori predictions with regard to the outcome of specific experiments (i.e.,
postgurmarin stimulus responses were compared with only their respective
controls). For each ANOVA, degrees of freedom and P values for
within-subject tests were corrected using the Greenhouse-Geisser adjustment to
protect against violations of sphericity. Although these corrections were made
prior to establishing P levels, only the uncorrected degrees of
freedom and P values are reported. Post hoc comparisons among
within-subject level means were accomplished through the use of paired samples
t-test in which each observed score was evaluated using a Dunn
critical value. This sort of multiple comparison procedure (MCP) is the only
sort of post hoc test for repeated level means that has adequate control of
for all pairwise comparisons
(Toothaker 1991).
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RESULTS |
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Trains of action potentials were recorded from 35 NST neurons with robust
responses to 0.5 M sucrose [mean response to 0.5 M sucrose across all neurons
= 10.5 ± 1.0 (SE) net spikes/s]. Our conservative statistical criterion
indicated that 34 of these cells significantly responded to 0.5 M sucrose. Two
neurons in our sample were recorded simultaneously from one preparation.
Eleven neurons were found to be sucrose-best, 18 were classified as NaCl-best,
and 6 were typed as HCl-best. No quinine-best units were observed in our
sample. Overall, these neurons were broadly responsive to the basic stimuli
(
= 0.79 ± 0.02 SE). Nineteen
neurons significantly responded to all components of the basic stimulus set,
13 significantly responded to only three of these stimuli, 2 significantly
responded to only two tastants, and 1 cell significantly responded to only one
stimulus. Figure 1 displays the
across neuron pattern of response evoked by each basic stimulus and the
average spontaneous discharge observed for each neuron prior to gurmarin
treatment.
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HCA was used to objectively classify neurons into heterogeneous types based
on similarities/dissimilarities among their tuning profiles measured in
response to the control presentation of each component of the basic stimulus
array. The final HCA solution is represented graphically by the dendrogram in
Fig. 2. HCA suggested three
classes of neurons. All sucrose-best neurons were linked into class S
(n = 14; = 0.77 ±
0.03 SE); three NaCl-best units with robust responses to sucrose were also
bound to this cluster. Class N (n = 15;
= 0.78 ± 0.04 SE) was
composed almost entirely of NaCl-best neurons, the exceptions being a pair of
HCl-best cells with strong NaCl responses. Class H (n = 6;
= 0.89 ± 0.02 SE) neurons
responded strongly to HCl, NaCl, and quinine.
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Effects of gurmarin on neuronal responses to sucrose
Trains of action potentials that were evoked by presentation of each component of the basic stimulus set were recorded before and after gurmarin (10 µg/ml) application from all 35 neurons in our sample. Gurmarin treatment was found to attenuate sucrose-evoked spike discharge for the majority of these cells although the degree of suppression varied across affected neurons. Many cells displayed residual responses to sucrose after gurmarin treatment; postgurmarin responses to sucrose remained significantly greater than average spontaneous discharge for 22 neurons. Complete gurmarin-induced inhibition (i.e., 0 net spikes/s) of a response to 0.5 M sucrose was observed during one trial for one neuron within our sample. Gurmarin treatment did not activate NST neurons, implying that 10 µg/ml gurmarin does not produce a gustatory quality sensation in the rat. Figure 3 shows digital oscilloscope records for two NST neurons in which the magnitude of the postgurmarin response to 0.5 M sucrose was either almost completely (Fig. 3A) or partly (Fig. 3B) suppressed relative to within-neuron control (i.e., unadulterated sucrose response). For the neuron in Fig. 3A, the response to sucrose was almost fully inhibited by gurmarin (96% suppression). For the unit in Fig. 3B, the magnitude of the postgurmarin response to sucrose was attenuated by 68%. For both neurons, the magnitude of the response evoked by 0.1 M NaCl was not altered by gurmarin treatment.
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The orotopic receptive field of the neuron depicted in
Fig. 3B was at least
partially composed of the fungiform papillae as this cell was driven by anodal
stimulation of the anterior tongue. As methylene blue dye tests indicated that
our gurmarin application procedure effectively bathed both the anterior tongue
and palate, and taste stimulus delivery was limited to these same regions, the
partial effect observed for this neuron could not be attributed to inadequate
gurmarin application. Moreover, we acquired data from five neurons that
clearly received input from the fungiform papillae but for which gurmarin
treatment failed to attenuate responses to 0.5 M sucrose (criterion = 35%
suppression relative to within-neuron control; mean suppression = 0 ±
3.42%; maximum suppression = 11%).
Assuming a criterion of 35% attenuation relative to within-neuron
control, gurmarin application suppressed responses to 0.5 M sucrose for 22
(63%) neurons in our sample. If the criterion was raised to 50% attenuation,
16 (45%) cells were affected. For three (9%) neurons, postgurmarin sucrose
responses were attenuated by 90% relative to control. These descriptors are
summarized in Fig. 4, which
displays across-neuron patterns of response to 0.5 M sucrose measured under
control conditions (Fig.
4A) and after oral application of 10 µg/ml gurmarin
(Fig. 4B). The
difference between these patterns is shown in
Fig. 4C. Moreover,
these data indicate that sucrose responses recorded from neurons
representative of each HCA-determined neuronal class were affected by
gurmarin. However, a disproportionate percentage of cells in each class
displayed sucrose responses that were substantially attenuated after gurmarin
treatment. Eight (57%) class S, six (40%) class N, and two (33%) class H
neurons exhibited postgurmarin responses to sucrose that were suppressed by
50% relative to control. Figure
4C further describes the magnitude of the effect as
observed across neuronal type.
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The null hypothesis that gurmarin application did not differentially affect
basic taste response magnitudes across neuronal class was statistically
evaluated using a neuron (35 cases) by neuronal group (3 levels; each
HCA-defined neuronal class served as a level) by gurmarin treatment (2 levels)
by stimulus (4 levels) mixed ANOVA. This hypothesis was not accepted as a
significant neuronal group by gurmarin treatment by stimulus interaction was
found [F(6,96) = 3.34, P = 0.005]. Planned interaction
comparisons revealed that responses to sucrose were significantly attenuated
after oral application of gurmarin in class S [F(1,32) = 18.12,
P = 0.0002] and N [F(1,32) = 12.14, P = 0.001]
neurons whereas sucrose responses in class H cells were unaffected ( =
0.01). For each neuronal class, gurmarin treatment did not affect responses to
NaCl, HCl, or quinine relative to control ( =0.01).
Figure 5 graphically summarizes
these data.
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To determine if the observed effects, or lack thereof, of 10 µg/ml gurmarin approximated limits, taste responses were recorded from three neurons following oral application of 20 µg/ml gurmarin. Data regarding responses to sucrose that were recorded from these cells are graphed in Fig. 6. For the neuron depicted in Fig. 6A, gurmarin suppressed responses to 0.1, 0.32, 0.5, and 1.0 M sucrose, although concentration-response functions measured following oral application of 10 and 20 µg/ml gurmarin were not different. Moreover, the response evoked by 0.5 M sucrose after 10 µg/ml gurmarin treatment was nearly identical to that measured after application of 20 µg/ml gurmarin (difference = 1 net spike / 5 s), which indicated that 10 µg/ml gurmarin produced a maximal effect. As seen in Fig. 6B, 10 or 20 µg/ml gurmarin did not affect responses to 0.5 M sucrose measured from two other neurons. Both of these cells received verified input from the fungiform papillae but were unaffected by gurmarin treatment. As observed with the standard concentration, oral application of 20 µg/ml gurmarin did not activate NST neurons.
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Sucrose concentration-response functions
To determine if the effect of gurmarin was differential across sucrose
concentrations, a half-logarithmic step ascending concentration series of
sucrose, which ranged from 0.01 to 1.0 M, was presented to 19 neurons (class
S: n = 7; class N: n = 10; class H: n = 2) both
before and after gurmarin application. A neuron by gurmarin treatment (2
levels) by sucrose concentration (5 levels) mixed ANOVA revealed that sucrose
responses recorded from these cells were significantly influenced by gurmarin
application and stimulus concentration [gurmarin by sucrose concentration
interaction: F(4,60) = 7.74, P = 0.00004]. Planned
interaction comparisons indicated that postgurmarin responses to 0.1 M
[F(1,15) = 9.58, P = 0.007], 0.32 M [F(1,15) =
8.64, P = 0.01] and 1.0 M [F(1,15) = 10.35, P =
0.006] sucrose were significantly attenuated relative to control. Although
measured responses were negligible, responses to 0.01 and 0.032 M sucrose were
not significantly suppressed after gurmarin application ( = 0.05).
Considering only those concentrations where significant suppression was noted,
the amount by which postgurmarin sucrose responses were attenuated did not
significantly differ across sucrose concentration (pairwise comparisons of
0.1, 0.32, and 1.0 M sucrose pre-/postgurmarin response magnitude differences,
Dunn MCP, = 0.05; see Fig.
7).
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Effects of gurmarin on neuronal responses to various sweet-tasting compounds
The effect of oral application of 10 µg/ml gurmarin on responses to
various sweet-tasting compounds was explored in seven neurons. Given the small
n and the sometimes disparate effects observed across the cells, we
present data from experiments conducted on individual neurons in
Fig. 8. For neuron
N14, gurmarin treatment attenuated responses to sucrose, fructose,
glycine, D-asparagine, and D-histidine by 50%
relative to control. Given the presence of residual postgurmarin sweet
responses, these data suggest that this neuron received input from
gurmarin-sensitive and -insensitive receptor mechanisms that were rather
broadly tuned. In contrast, the overall lack of a gurmarin treatment effect
noted for neuron N7 implied that sweet responses in this cell were
mediated by input derived entirely from gurmarin-insensitive sweet
transduction processes. However, data obtained from neurons N15, S5, and H5
were most interesting. For neuron N15, gurmarin treatment did not
affect the response to sucrose or fructose. However, postgurmarin responses to
glucose and maltose were suppressed by 50%. Moreover, the postgurmarin
response to galactose was fully inhibited. Sweet responses were also
differentially affected by gurmarin in neuron H5. For this cell, responses to
sucrose and fructose were resistant to gurmarin treatment. However, responses
to Na-saccharin, D-asparagine, and D-histidine were
suppressed by 50% relative to control, whereas the postgurmarin glucose
response was fully inhibited. These data suggest that some NST neurons may
receive convergent input from gurmarin-sensitive and -insensitive receptor
mechanisms that are differentially tuned. Data obtained from neuron
S5 further exemplify this point. This cell received information regarding
the presence of maltose on gustatory epithelia from exclusively
gurmarin-sensitive sweet transduction processes as the response to maltose was
completely inhibited after gurmarin treatment. Although the postgurmarin
response to sucrose was attenuated by 50% relative to control, the
residual indicates that both gurmarin-sensitive and -insensitive receptors
contributed to this response. Therefore the tuning characteristics of the
gurmarin-sensitive and -insensitive receptor processes that initiated the
transmission of sucrose and maltose information to this neuron differed, as
the gurmarin-insensitive component was not responsive to 0.5 M maltose.
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DISCUSSION |
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50% relative to control. Based on this
criterion, the largest number of affected neurons was found in class S,
followed by classes N and H, respectively. On average, postgurmarin responses
to 0.5 M sucrose were found to be significantly suppressed only in class S and
N neurons. Additionally, the effect of gurmarin was sometimes differential
across different sweet-tasting compounds within individual neurons, which
implied that some NST cells may receive convergent input from
gurmarin-sensitive and -insensitive receptor mechanisms that are
differentially tuned to various sweet-tasting ligands. Responses to NaCl, HCl
and quinine were not affected by gurmarin treatment. Information from gurmarin-sensitive receptors is not restricted to a single NST neuronal type
The existence of gurmarin-sensitive and -insensitive receptor mechanisms
was first suggested by data concerning the effects of gurmarin on whole-nerve
responses to sweeteners recorded from rodents. Although integrated CT
(Imoto et al. 1991;
Miyasaka and Imoto 1995;
Ninomiya and Imoto 1995;
Ninomiya et al. 1998) and GSP
(Harada and Kasahara 2000)
responses to sucrose were found to be significantly suppressed after gurmarin
treatment, a residual postgurmarin response to this stimulus was evident in
these recordings. Moreover, the inhibitory effect of gurmarin on CT responses
to 0.5 M sucrose became asymptotic, though not absolute, at 5 µM,
which produced 80% suppression relative to control; no further
suppression was observed even if the tongue was treated with 240 µM
gurmarin (Miyasaka and Imoto
1995). For C57BL mice, the effects of gurmarin have been shown to
be nerve specific. Whereas integrated CT responses to various sweeteners were
suppressed by gurmarin to 50% of control in this species
(Ninomiya and Imoto 1995;
Ninomiya et al. 1997), those
recorded from the glossopharyngeal nerve were recalcitrant to gurmarin
treatment (Ninomiya et al.
1997). As gurmarin is believed to act on an apical receptor
binding site (Miyasaka and Imoto
1995; Yoshie et al.
1994), these data suggest that at least two receptor processes for
sweeteners exist in rodents, classified on the basis of their sensitivity to
gurmarin (Ninomiya et al.
1999). Moreover, single sucrose-best CT neurons can be segregated
into types on the basis of their susceptibility to lingual gurmarin treatment
(Ninomiya et al. 1999),
suggesting selective synaptic coupling between taste receptor cells (TRCs)
that express gurmarin-sensitive or -insensitive receptors and particular
subsets of sucrose-best CT fibers.
Selective coupling between specific types of TRCs and peripheral gustatory
neurons has been shown by the effects of lingual amiloride treatment on
responses to NaCl in single CT fibers. Salt responses recorded only from those
fibers responding best to NaCl were susceptible to amiloride treatment
(Hettinger and Frank 1990;
Ninomiya and Funakoshi 1988),
suggesting that this fiber type exclusively innervates TRCs that express
amiloride-sensitive Na+ transduction processes. This segregation of
amiloride-sensitive salt information to a particular type of CT neuron is
similar to the observation that gurmarin-sensitive sweet information is
selectively distributed to a particular type of sucrose-best CT fiber
(Ninomiya et al. 1999).
Because amiloride-sensitive salt information is predominantly relayed to
NaCl-best (Boughter and Smith
1998; Boughter et al.
1999; Giza and Scott
1991; Scott and Giza
1990; Smith et al.
1996; St. John and Smith
2000) and sucrose-best (Smith
et al. 1996; St. John and
Smith 2000) NST neurons, it could be hypothesized that
gurmarin-sensitive sweet information is also differentially distributed across
physiologically defined NST neuronal types. Such organization may have
implications for how sweet information is encoded by neural activity in the
CNS, as the arrangement of amiloride-sensitive input contributes to the neural
representation of salt information in the NST
(Boughter et al. 1999;
Giza and Scott 1991;
Scott and Giza 1990;
St. John and Smith 2000) and
gustatory behavioral discrimination between salts
(Spector et al. 1996).
To our knowledge, the present study is the first to explore the
distribution of gurmarin sensitivity across taste-driven neurons in the CNS.
Although gurmarin-sensitive information was not restricted to a particular
class of neuron, a disproportionate percentage of cells from each neuronal
class exhibited sucrose responses that were substantially attenuated following
gurmarin treatment. Over half of the class S neurons exhibited postgurmarin
sucrose responses that were attenuated by 50% relative to control; the
same could be said for less than half of the N or H class cells. Overall,
postgurmarin responses to sucrose were significantly attenuated only in class
S and N neurons. In some respects, the distribution of gurmarin sensitivity
across NST neuronal types relative to those in the periphery
(Ninomiya et al. 1999) is
similar to that observed for amiloride-sensitive salt input as the apparent
restriction of information derived from a particular receptor process to a
specific peripheral neuron class is not absolute in the CNS. However,
amiloride is more effective at reducing responses to Na+ salts in
those NST cells that respond maximally to these stimuli relative to the
average gurmarin-induced attenuation of responding to sucrose observed in
class S cells in the present study (see
Boughter and Smith 1998;
Boughter et al. 1999;
Giza and Scott 1991;
Scott and Giza 1990;
Smith et al. 1996;
St. John and Smith 2000).
Although an amiloride-insensitive component is apparent, salt responses in
NaCl-best NST neurons are predominantly derived from amiloride-sensitive salt
input (Boughter and Smith 1998;
Boughter et al. 1999;
Giza and Scott 1991;
Scott and Giza 1990;
Smith et al. 1996;
St. John and Smith 2000),
whereas gurmarin-sensitive and -insensitive receptor mechanisms contributed
almost equally, on average, to sucrose responses in class S cells (see
Fig. 5). Although they are
somewhat differently organized, the neuronal circuits that underlie
amiloride-sensitive salt and gurmarin-sensitive sweet input to the brain
distribute information to more than one category of NST neuron.
Convergence of neural information in the gustatory NST has been directly
demonstrated (Ogawa et al.
1984; Sweazey and Smith
1987; Travers et al.
1986; Vogt and Mistretta
1990) and implied (Boughter and
Smith 1998; Doetsch and
Erickson 1970; Hill et al.
1983; Smith et al.
1996; St. John and Smith
2000; Travers and Smith
1979) by a number of studies. Moreover, many NST neurons in the
present study appeared to receive convergent input from gurmarin-sensitive and
-insensitive receptor mechanisms. This was partially suggested by the
observation that, for the majority of these cells, residual postgurmarin
responses to 0.5 M sucrose were found that exceeded statistical threshold,
whereas the effect of gurmarin on sucrose responses recorded from single mouse
CT fibers is purportedly more absolute
(Ninomiya et al. 1999);
companion rat single-fiber data do not presently exist. However, the presence
of these residual responses by themselves may not necessarily reflect input
derived from gurmarin-insensitive receptor components
(Ninomiya et al. 1999) as
factors such as inadequate gurmarin concentration and/or treatment could also
result in such responses. However, our experimental measures taken to address
these possibilities suggest otherwise.
We encountered taste-driven NST neurons during experimentation that did not
appreciably respond to sucrose and thus were not included in our study. The
composition of our neuronal sample reflects this: we recorded from many
neurons that were subsequently typed as class S (40% of sample) and N (43% of
sample) that responded well to sucrose. However, only 6 (17% of sample)
sucrose-responding class H cells were found. Many cells with strong responses
to HCl, NaCl, and quinine were encountered that did not respond to sucrose,
suggesting that some cells may not receive sucrose-mediated input from the
anterior tongue and palate, areas known to be populated with TRCs that express
sweet receptor mechanisms (Gilbertson et
al. 2001). Moreover, this low n may have influenced our
findings regarding the effect of gurmarin on responses to sucrose across
neuronal class as failure to observe a significant effect of gurmarin in class
H neurons may be attributable to low statistical power. Assuming this caveat
to be true and that further investigation would yield a sizable number of
sucrose-responsive and gurmarin-sensitive class H NST neurons, our analogy
between the differential distribution of gurmarin-sensitive sweet and
amiloride-sensitive salt information across NST neuronal types could be
rendered less appropriate, although a recent report showed that amiloride
significantly attenuated responses to NaCl in some HCl-best NST neurons
(St. John and Smith 2000).
Effects of gurmarin varied across sweeteners within individual neurons
Although neurophysiological data are limited, various psychophysical
studies have suggested multiple receptor mechanisms for sweet compounds.
Intensity matching experiments in humans indicated that concentrations of
fructose, glucose, and sucrose could be found that rendered these stimuli
indiscriminable (Breslin et al.
1996). However, higher concentrations of maltose could not be
matched using this procedure, suggesting that maltose activates a separate
receptor process. It was recently demonstrated that whole-mouth adaptation to
fructose increased discriminability between fructose and glucose in humans,
indicating that these two sugars possibly stimulate separate receptor sites
(Tharp and Breslin 2002).
Other human cross-adaptation experiments have reported similar findings of
incomplete cross-adaptation among various sweet stimuli
(Faurion et al. 1980;
Froloff et al. 1998;
Schiffman et al. 1981).
Moreover, discrimination experiments, in which intensity was rendered an
irrelevant cue (Spector et al.
1997), have shown that rats can discern sucrose from maltose,
suggesting independent receptor mechanisms for these stimuli in the rodent.
Our data complement these findings by showing that some neurons received input
from gurmarin-sensitive and -insensitive receptor processes that responded
differentially to sweet compounds (see Fig.
8), suggesting that some receptor mechanisms are sensitive to only
a subset of, and not all, sweet-tasting ligands. Moreover, our data indicate
that some neurons may receive input from TRCs expressing gurmarin-sensitive
receptor processes that are unresponsive to sucrose (see
Fig. 8). Although our low
n with regard to this type of data warrants further investigation, a
complementary differential effect of gurmarin on responses to a sweetener
array has been observed in integrated GSP nerve recordings in rats. Phasic
responses to sucrose, fructose, lactose, and maltose were significantly
inhibited, whereas responses to galactose and glucose were unaffected
following palatal gurmarin treatment
(Harada and Kasahara 2000).
The convergence of peripheral fibers that are driven by differentially tuned
gurmarin-sensitive and -insensitive receptor processes onto NST neurons could
account for the observed idiosyncratic effects of gurmarin on neuronal
responses to various sweeteners.
Recent advances in molecular biology suggest the existence of a single
mammalian receptor for sweets, T1R2/T1R3, as this candidate responded to all
sweet taste stimuli tested (Li et al.
2002). However, other reports suggest that T1R2/T1R3 is extremely
selective, recognizing only a limited range of sweet compounds
(Nelson et al. 2001).
Moreover, T1R2 is purportedly undetectable in most fungiform taste papillae
(Hoon et al. 1999), yet some
fungiform TRCs clearly respond to sucrose
(Gilbertson et al. 2001).
Although our understanding of sweet taste reception is far from complete, it
is generally agreed that sweet stimuli activate TRCs through at least two
transduction pathways: one involves the generation of cyclic nucleotides, the
other modulates levels of inositol triphosphate
(Herness and Gilbertson 1999;
Lindemann 1996,
2001). Receptor and
transduction processes are the initial steps toward gustatory perception,
which is ultimately a product of neural information processing in the brain.
Further understanding of the neural mechanisms underlying sweet perception
will necessitate the derivation of the relationships between receptor
mechanisms and the physiology of neurons in the CNS.
Implications for gustatory neural information processing
Although scant, data regarding the effects of gurmarin on gustatory
behavioral tasks do exist. Gurmarin was found to suppress the avoidance of
sucrose for C57BL mice trained in a conditioned taste aversion paradigm
(Nakashima et al. 2001).
Additionally, rats fed a diet containing G. sylvestre exhibited a
transient reduction in preference for sucrose: intake decreased and
subsequently recovered several days later at a time when gurmarin binding
proteins, which suppress the activity of gurmarin, appeared in the saliva of
these subjects (Katsukawa et al.
1999). The present study demonstrated that sucrose responses
recorded from class S and N neurons were significantly attenuated after
gurmarin treatment, indicating that these cells processed gurmarin-sensitive
sweet input. Therefore activity generated by class S and N neurons
possibly contributes to such sucrose-mediated behavioral tasks. This correlate
reinforces the notion of a distributed neural code for taste in the CNS, where
activity generated by a network of individual cells of different
physiologically defined types underlies the neuronal representation of
multiple stimulus qualities and parameters
(Erickson 1968;
Pfaffmann 1959;
Scott and Giza 2000;
Smith and St. John 1999).
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests: C. H. Lemon, Dept. of Anatomy and Neurobiology, University of Tennessee College of Medicine, 855 Monroe Ave., Suite 515, Memphis, TN 38163. (E-mail: chris{at}utmem.edu).
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, stimulus onset.
