Department of Anatomy and Neurobiology and Program in Neuroscience, University of Maryland School of Medicine, Baltimore, Maryland 21201-1509
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ABSTRACT |
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John, Steven J. St. and David V. Smith. Neural Representation of Salts in the Rat Solitary Nucleus: Brain Stem Correlates of Taste Discrimination. J. Neurophysiol. 84: 628-638, 2000. One mechanism of salt taste transduction by gustatory receptor cells involves the influx of cations through epithelial sodium channels that can be blocked by oral application of amiloride. A second mechanism is less clearly defined but seems to depend on electroneutral diffusion of the salt through the tight junctions between receptor cells; this paracellular pathway is insensitive to amiloride. Because the first mechanism is more sensitive to sodium salts and the second to nonsodium salts, these peripheral events could underlie the ability of rats to discriminate sodium from nonsodium salts on the basis of taste. Behavioral experiments indicate that amiloride, at concentrations that are tasteless to rats, impairs a rat's ability to discriminate NaCl from KCl and may do so by making both salts taste like KCl. In the present study, we examined the neural representation of NaCl and KCl (0.05-0.2 M), and mixtures of these salts with amiloride (0, 3, and 30 µM), to explore the neural correlates of this behavioral result. NaCl and KCl were represented by distinct patterns of activity in the nucleus of the solitary tract. Amiloride, in a concentration-dependent manner, changed the pattern for NaCl to one more characteristic of KCl, primarily by reducing activity in neurons responding best to NaCl and sucrose. The effect of amiloride concentration on the response to 0.1 M NaCl in NaCl-best neurons was virtually identical to its effect on behavioral discrimination performance. Modeling the effects of blocking the amiloride-insensitive pathway also resulted in highly similar patterns of activity for NaCl and KCl. These results suggest that activity in both the amiloride-sensitive and -insensitive pathways is required for the behavioral discrimination between NaCl and KCl. In the context of published behavioral data, the present results suggest that amiloride-sensitive activity alone is not sufficient to impart a unique signal for the taste of sodium salts.
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INTRODUCTION |
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Two transduction
mechanisms have been described for sodium salts: an amiloride-sensitive
and -insensitive pathway (see Herness and Gilbertson
1999; Lindemann 1996
; Stewart et al.
1997
). Amiloride-sensitive transduction occurs when sodium
enters taste receptor cells directly via apical epithelial sodium
channels similar to those found in kidney and colon (Benos et
al. 1996
). The influx of sodium can be competitively inhibited
by the lingual application of the diuretic drug amiloride
(DeSimone and Ferrell 1985
). Amiloride-insensitive transduction, in contrast, is believed to begin with electroneutral diffusion of the salt across the tight junctions between taste receptor
cells and sodium entry into the cells via unspecified basolateral ion
channels (DeSimone and Ferrell 1985
; Elliott and Simon 1990
; Ye et al. 1993
). This paracellular
pathway is not blocked by mucosal application of amiloride. Sodium
salts with large anions, such as sodium gluconate, have less access to
the paracellular pathway, making their evoked responses smaller and more amiloride sensitive than the response to NaCl (Elliott and Simon 1990
; Formaker and Hill 1988
; Ye et
al. 1991
).
Taste responses evoked by nonsodium salts, such as KCl or
NH4Cl, are predominantly insensitive to amiloride
treatment, although there is evidence in both rats (Minear et
al. 1996) and hamsters (Boughter et al. 1999
)
that amiloride reduces the response to KCl. In rodent taste tissue,
however, the amiloride-sensitive channel is far more permeable to
Na+ than K+ (Brand
et al. 1985
; Heck et al. 1984
; Herness
1987
; Ninomiya and Funakoshi 1988
). Nonsodium
salts are thought to stimulate taste receptors predominantly through
the paracellular pathway (Kloub et al. 1997
;
Stewart et al. 1997
), although the cellular mechanisms
of this transduction pathway are not completely known. In behavioral
generalization studies, nonsodium salts and acids are categorized
similarly by both rats and hamsters (Nowlis et al. 1980
;
Smith et al. 1979
) and tend to generate similar patterns of activity across gustatory afferent neurons (Perrotto and
Scott 1976
; Smith et al. 1983
); both are
perceptually and neurally distinct from sodium salts (Erickson
1963
; Krieckhaus and Wolf 1968
; Morrison 1967
; Nowlis et al. 1980
).
Input from the amiloride-sensitive and -insensitive transduction
mechanisms remains largely segregated in fibers of the chorda tympani
(CT) nerve (Hettinger and Frank 1990; Ninomiya
and Funakoshi 1988
) and in cells of the CNS of rodents
(Boughter and Smith 1998
; Boughter et al.
1999
; Giza and Scott 1991
; Scott and Giza
1990
; Smith et al. 1996
). If amiloride is added
to an ongoing response to NaCl (N), the response of N-best neurons in
the hamster CT nerve (Hettinger and Frank 1990
) and in
the nucleus of the solitary tract (NST) (Boughter and Smith
1998
; Boughter et al. 1999
) is suppressed to
prestimulus levels. These data suggest that the sodium responses of
N-best cells are completely amiloride sensitive. Conversely, there is
absolutely no effect of amiloride on the responses of HCl (H)-best CT
fibers or NST neurons (Boughter and Smith 1998
;
Boughter et al. 1999
; Hettinger and Frank
1990
). The response to NaCl is partially reduced in sucrose
(S)-best NST neurons of the hamster (Boughter and Smith
1998
; Boughter et al. 1999
). Because these two
receptor mechanisms are differentially sensitive to sodium and
nonsodium salts and because their inputs remain segregated in the CNS,
the relative activation of these two pathways could underlie the
ability of rodents to discriminate among salts.
Direct behavioral support for the involvement of the
amiloride-sensitive pathway in salt discrimination was recently
presented (Spector et al. 1996; see also Hill et
al. 1990
). Water-restricted rats were trained to lick a
drinking spout to obtain NaCl or KCl (0.05, 0.1, or 0.2 M) and to press
one of two levers associated with each stimulus (Spector et al.
1996
). Discrimination performance (the percentage of correct
lever responses) was then measured over sessions when amiloride (1, 3, 10, 30, or 100 µM) was mixed with the salt solutions. Amiloride at
the two highest doses completely prevented rats from discriminating
NaCl from KCl. Because amiloride is tasteless to rats at 100 µM
(Markison and Spector 1995
), Spector et al.
(1996)
concluded that amiloride reduced discrimination performance by virtue of its selective effect on the
amiloride-sensitive transduction pathway.
The present study was conducted to examine the relative contribution of
these two transduction pathways to salt discrimination. We recorded
responses to NaCl and KCl from cells in the NST, the first central
synapse for gustatory information, to determine the central neural
representation of the taste of these salts after blocking the
amiloride-sensitive or -insensitive pathway. We used the same
concentrations of salts and amiloride as in the aforementioned
behavioral work (Spector et al. 1996). In addition, given our knowledge of the relative distribution of these two transduction inputs to different cell types, we modeled the neural response patterns that would occur if we had the appropriate agent to
block the amiloride-insensitive transduction pathway. The present experiment suggests that activity in both the amiloride-sensitive and
-insensitive pathways subserves the behavioral discrimination between
NaCl and KCl.
A portion of these results was presented at the 1999 meeting of the Association for Chemoreception Sciences, Sarasota, FL.
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METHODS |
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Subjects and recording
Twenty-nine male Sprague Dawley rats, weighing 210-574 g, were deeply anesthetized with urethan (1.7 g/kg ip) and prepared for electrophysiological recording. Supplemental injections of anesthesia were occasionally given as necessary. Following bilateral hypoglossal neurectomy, the rat was tracheotomized and secured in a nontraumatic headholder that deflected the head downward at a 27° angle. These preparations served to minimize brain stem movements associated with breathing. The brain stem was exposed for recording by removal of a portion of the occipital bone and aspiration of the posterior part of the cerebellum. Throughout the procedure, body temperature was monitored and maintained at 37 ± 1°C with a heating pad.
Extracellular recordings from single neurons in the gustatory zone of
the NST were made using tungsten microelectrodes (0.4-0.8 M). The
search for taste-responsive neurons typically began 2.8 mm anterior to
obex, 1.8 mm lateral to the midline, and approximately 1 mm ventral to
the surface of the brain stem. Initially taste-responsive neurons were
identified by a change in neural activity evoked by anodal current
pulses (40 µA, 0.5 s) applied to the anterior tongue and then
confirmed with chemical stimulation of the tongue. Action potentials
were amplified (Grass P511), discriminated with a dual time-amplitude
window discriminator (Bak DDIS-1), displayed on a storage oscilloscope,
and monitored with an audio monitor. The amplified action potentials
were recorded along with voice cues on digital PCM-VCR tape. An
IBM-compatible computer configured with a Lab Master DMA board
(Scientific Solutions; Solon OH) and custom software controlled
chemical stimulus delivery and on-line data acquisition and analysis.
Stimulus protocol
Once a single neuron was isolated, it was tested with three groups of stimuli: group A, basic stimuli; group B, a standard array of 18 salt-amiloride mixtures, and group C, an array of six 1.0 M salt-amiloride mixtures (Table 1). The three groups of stimuli were always tested sequentially, but presentation within these groups was random for any given neuron. If a neuron did not remain viable throughout the testing of group A and B stimuli, it was excluded from analysis. Thus every cell was tested with group A and B stimuli, whereas a subset was tested also with group C.
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Tastants were delivered at room temperature to the anterior tongue via
gravity flow at a rate of 2.6 ml/s. During a trial, the tongue was
first rinsed with distilled water for at least 15 s (the last
5 s of which were used to determine the cell's spontaneous rate),
followed immediately by the taste stimulus for 10 s. The tongue
was then rinsed with at least 50 ml of distilled water and greater than
2 min were allowed to elapse between trials to preclude adaptation
effects from confounding interpretation of neuronal responses
(Smith et al. 1975, 1978
). To more fully characterize
the concentration-dependent effects of amiloride on single NST cells,
five N-best neurons were tested for their responses to 0.1 M NaCl mixed
with all five amiloride concentrations used in the Spector et
al. (1996)
experiment: 1, 3, 10, 30, and 100 µM.
Whereas amiloride added to the stimuli would be effective in blocking
the amiloride-sensitive pathway, there is currently no comparable drug
known that will effect a reversible block of the paracellular pathway.
Although lanthanum chloride will presumably block the tight junctions
(Holland et al. 1989), it has nonspecific effects on
both amiloride-sensitive channels and calcium channels, and its effects
on taste responses are varied and not readily reversible (unpublished
observations). However, since we know the relative contribution of the
amiloride-sensitive and -insensitive pathways to the various neuron
types from our previous work on the hamster NST (Boughter and
Smith 1998
; Boughter et al. 1999
), which are
paralleled in the effects of amiloride on rat NST cells in the current
data, we are able to model the effects of such a blocker. For this
modeling, we assume that the hypothetical blocker of the
amiloride-insensitive pathway produces the same effect on H-best cells
that amiloride does on N-best cells (i.e., a 75.5% reduction of the
response to NaCl with the mixture protocol used in the current study),
whereas it has half that effect (37.75%) on S-best cells. This
assumption is based on data showing that responses to NaCl and KCl are
completely unaffected by amiloride in H-best NST cells and that about
half or less of the response to these stimuli is blocked by amiloride
in S-best neurons. Although the effect of amiloride is less when it is
applied before or mixed with NaCl (Scott and Giza 1990
;
Smith et al. 1996
), as in the present data, the complete
inhibition that occurs when amiloride is added to an ongoing NaCl
response (Boughter and Smith 1998
; Boughter et
al. 1999
; Hettinger and Frank 1990
) suggests
that all of the input to N-best cells arises from the
amiloride-sensitive transduction pathway. In addition, NaCl and KCl
produce comparable responses in the non-N-best neurons (Boughter
et al. 1999
) (see also RESULTS), suggesting a
common mechanism. We assume that the amiloride-insensitive mechanism is
the paracellular pathway described by Ye et al. (1993)
,
although there could be other, yet unknown, transduction mechanisms for
these salts. In summary, for this hypothetical treatment we reduced the
responses to both NaCl and KCl in H-best neurons by 75.5% (as in the
mixture protocol with amiloride, see RESULTS) and in S-best
neurons by 37.75%, mimicking the effect of a specific blocker of the
amiloride-insensitive pathway.
Data analysis
The window-discriminated action potentials were converted into frequency counts. Net responses were derived by subtracting the number of impulses to water alone during the last 5 s prior to stimulus onset (multiplied by 2) from the frequency count during 10 s of taste stimulation.
For some analyses, neurons were classified into best-stimulus
categories based on which of the group A (basic) stimuli
evoked the greatest net response (Frank 1973). A two-way
ANOVA (salt concentration × amiloride concentration) was
conducted separately for NaCl and KCl to determine whether the
responses of neurons responding best to NaCl (N-best), sucrose
(S-best), or HCl (H-best) were significantly modified by salt or
amiloride concentration. The breadth of tuning of cells in each
best-stimulus category was measured using the formula for entropy
introduced by Smith and Travers (1979)
. Entropy, which
ranges from 0 (i.e., a neuron responding exclusively to 1 of the 4 stimuli) to 1 (a neuron responding equally to all 4) was determined
using the four group A stimuli.
To examine the neural representation of the group A and
B stimuli, across-neuron patterns were derived. Two measures
were used to quantify the similarity of across-neuron patterns for pairs of stimuli: across-neuron correlations (Erickson
1963) and neural mass differences (NMDs) (Erickson
1986
; Gill and Erickson 1985
). Across-neuron
correlations were Pearson product-moment correlations between the 10-s
net firing rates to stimulus pairs across all neurons. Across-neuron
correlations have been shown to be high between stimuli that animals
treat as qualitatively similar and low between stimuli that can be
behaviorally discriminated on the basis of taste quality
(Erickson 1963
; Smith et al. 1979
).
The NMD is the sum of the differences in firing rates (over 10 s)
to two stimuli across all neurons. NMDs are high between easily
discriminated stimuli and low between stimuli that are behaviorally
similar to one another (Dahl et al. 1997; Gill
and Erickson 1985
). The NMD may be a more reliable metric than
across-neuron correlations when low levels of activity are generated
(e.g., as with 30 µM amiloride), because low correlations resulting
from low response levels can sometimes give a misleading implication of
high discriminability (Erickson 1986
; Gill and
Erickson 1985
). However, the NMD is not merely a reflection of
qualitative differences between stimuli. Large NMDs can be generated
between stimuli of different qualities or between stimuli of the same
quality at different intensities.
Each measure has its strengths and weaknesses. The across-neuron correlation is thought to be an index of qualitative differences between taste stimuli, whereas the NMD reflects differences in either quality or intensity. Because both quality and intensity may be relevant in the behavioral discrimination task being modeled, both measures are presented. Both measures were also used in multidimensional scaling routines (SPSS for Windows, v. 9.0; SPSS, Chicago IL), which produced stimulus spaces representing the relative differences in the across-neuron patterns among the group A and B stimuli.
For five cells tested with the wider array of amiloride concentrations,
the concentration-response data for amiloride's effects on 0.1 M NaCl
were compared with the corresponding behavioral data from
Spector et al. (1996). For the electrophysiological measure, a sigmoidal curve was fit to the average percent response of
the cells (net spikes in 10 s) when NaCl was mixed with amiloride relative to the no amiloride condition. The same function was applied
to the behavioral data, where the "response" was taken as overall
performance (percent of correct responses) during behavioral sessions
with amiloride relative to the no amiloride condition. The sigmoidal
function was
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RESULTS |
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Group A stimuli
Action potentials were recorded from 37 NST neurons in response to
both group A and B stimuli (see Table 1). Based
on the response to group A stimuli, the neurons were
classified as 21 N-best, 7 S-best, and 9 H-best; none of the cells
responded best to 0.02 M QHCl. Figure 1
shows representative responses from an H-best neuron (Fig.
1A) and an N-best neuron (Fig. 1B) to the group A stimuli and two group B stimuli (0.1 M
NaCl +30 µM amiloride and 0.1 M KCl). As previously shown
(Boughter and Smith 1998; Boughter et al.
1999
; Giza and Scott 1991
; Scott and Giza
1990
; Smith et al. 1996
), N-best NST neurons are
characterized by amiloride sensitivity, whereas H-best neurons are
predominantly amiloride insensitive. That is, adding 30 µM amiloride
reduced the response to NaCl in the N-best neuron (B), but
not in the H-best cell (A). In general, all neurons were
quite broadly tuned to the group A stimuli (Fig.
1C), with mean entropy of 0.80, 0.83, and 0.86 for S-, N-,
and H-best neurons, respectively.
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Group B stimuli
Concentration-response functions for NaCl and KCl demonstrate that N-best neurons are far more responsive to NaCl (Fig. 2A) than KCl (Fig. 2B). Two separate ANOVAs (salt concentration × amiloride concentration) confirmed that, in N-best neurons, NaCl responses were significantly modified by both NaCl concentration (F[2,40] = 57.2, P < 0.001) and amiloride concentration (F[2,40] = 68.9, P < 0.001) and that KCl responses were affected by KCl concentration (F[2,40] = 43.8, P < 0.001) and amiloride concentration (F[2,40] = 48.8, P < 0.001). Preplanned contrasts indicated that all levels of amiloride and salt concentration differed significantly from one another, except that 3 and 30 µM amiloride did not differentially suppress responses to KCl.
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In contrast, H-best neurons were approximately equally responsive to isomolar concentrations of NaCl (Fig. 2C) and KCl (Fig. 2D). The responses of H-best neurons were affected by NaCl concentration (F[2,16] = 25.8, P < 0.001) and slightly by amiloride concentration (F[2,16] = 5.0, P = 0.021). Preplanned contrasts revealed that 3 µM amiloride differed significantly from 0 µM (F[1,8] = 5.5, P = 0.042), but there was no difference in the effects of 3 and 30 µM amiloride. It is clear, however, that the effects of amiloride were far less dramatic on NaCl responses in H-best cells than in N-best cells (Fig. 2, A and C; Table 2). H-best neurons were also modified by KCl concentration (F[2,16] = 16.8, P < 0.001), but KCl responses were unaffected by amiloride (F[2,16] = 0.4, P = 0.69) in these cells.
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Salts were not strong stimuli for S-best neurons (Fig. 2, E and F); nevertheless, this neuron type responded to both NaCl (F[2,12] = 21.0, P < 0.001) and KCl (F[2,12] = 14.6, P = 0.001) in a concentration-dependent manner. Amiloride significantly reduced both NaCl responses (F[2,12] = 12.6, P = 0.001) and KCl responses (F[2,12] = 7.8, P = 0.007) in S-best cells. All levels of salt concentration and amiloride concentration differed significantly from one another.
In summary, N-best neurons, unlike H- and S-best neurons, are driven by NaCl to a much greater degree than by KCl. Amiloride reduced NaCl responses in all three neuron types, but the effect of amiloride was far more pronounced in N-best cells than in S-best cells and in S-best cells than in H-best cells (Fig. 2, A, C, and E; Table 2). Likewise, amiloride inhibited KCl-evoked responses in N-best neurons to a greater degree than in S-best neurons, whereas the KCl response of H-best neurons was unaffected by amiloride (Fig. 2, B, D, and F; Table 2).
Neural representation of salt stimuli
Across-neuron patterns for two pairs of stimuli are presented in
Fig. 3, which depicts () the
differences between the patterns for NaCl and KCl. Neurons are arranged
along the abscissa by best-stimulus classification; the S-best cells on
the left, the N-best cells in the middle, and the H-best cells on the
right. Within these groups, the neurons are arranged in order of their
response to 0.1 M NaCl (Fig. 3A); the arrangement is the
same in each graph. In Fig. 3A, isomolar NaCl and KCl
responses are shown, and Fig. 3B shows responses to 0.2 M
NaCl and 0.05 M KCl, concentrations which evoke the greatest difference
in across-neuron patterns among the group B stimuli. NaCl
and KCl evoke very different patterns of activity across NST neurons
(Fig. 3, A and B, top). However, amiloride
produces a pronounced, concentration-dependent reduction in the
distinction between these neural patterns. At the highest dose of
amiloride (30 µM; bottom), the across-neuron patterns are
virtually indistinguishable.
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The NMDs (Fig. 3, ) for some selected stimulus pairs are shown in
Fig. 4. The absolute value of the NMD is
not meaningful, but as a relative measure, the higher the NMD the more
easily discriminated are two stimuli. Thus the NMDs between 0.1 M NaCl and sucrose, HCl, and QHCl (Fig. 4A; open circles) indicate
the level of NMD characteristic of stimuli that rats can easily
discriminate on the basis of taste. This range of NMDs is highlighted
in the figure by the upper shaded zone. The NMD for a replication of NaCl (Fig. 4A; solid circle) gives an indication of the
biological and experimental variability in the experiment and reflects
the value of NMD characteristic of stimuli that cannot be behaviorally discriminated. The lower shaded zone highlights this indiscriminable range in the NMD. As seen in Fig. 4B, the NMD between
isomolar pairs of NaCl and KCl are all within the discriminable range
(open circles). However, amiloride has a concentration-dependent effect on the NMD, so that at 30 µM (triangles), it is within the
indiscriminable range. This level of NMD suggests that NaCl and KCl
would not be discriminable on the basis of either qualitative or
intensive differences (compare 0.1 M NaCl with itself, filled circle,
Fig. 4A). At the intermediate concentration of amiloride
(squares), there was also an effect of salt concentration, such that
the NMD for 0.05 M salts was closer to the indiscriminable zone and for
0.1 and 0.2 M salts closer to the discriminable zone.
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Although it is clear from the NMD measure that amiloride reduces the
neural discrimination between NaCl and KCl, an analysis of the
across-neuron correlations between stimuli provides additional information (Fig. 5). Across-neuron
correlations are relatively low between stimuli that taste different
and high between stimuli with similar taste. Figure 5 shows the effect
of amiloride on the correlation of 0.1 M NaCl with itself () and
with 0.1 M KCl (
). With no amiloride (0 µM), NaCl correlates only
moderately with KCl (r = +0.54) but highly with an
independent presentation of NaCl (r = +0.85). At the
highest concentration of amiloride (30 µM), the situation is
reversed: NaCl in amiloride correlates poorly with an unadulterated
presentation of NaCl (r = +0.30) and actually
correlates better with unadulterated KCl (r = +0.76). In other words, when mixed with amiloride, NaCl is
represented by an across-neuron pattern progressively more similar to
that evoked by KCl and less like the pattern for NaCl (cf. Fig. 3).
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The NMDs were used to create a multidimensional space for all 22 group A and B stimuli (Fig.
6). Although presented here in three
separate panels, these relationships were derived from a single
multidimensional scaling analysis of all 22 stimuli. Such spaces
represent the similarities and differences in the across-neuron patterns evoked by these stimuli (Bieber and Smith
1986). KCl and NaCl were initially separated from one another
(Fig. 6A), but amiloride moved the NaCl stimuli (filled
triangles) toward the KCl stimuli (open triangles) in a
concentration-dependent manner (Fig. 6, B and C).
At 30 µM, the KCl and NaCl stimuli were grouped together in this
two-dimensional space, reflecting the similarities in the patterns of
neural activity depicted in Fig. 3. A taste space based on the
across-neuron correlations showed a similar dramatic effect of
amiloride on the neural representation of NaCl and KCl (see Fig. 7).
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Whereas treatment with amiloride produced across-neuron patterns for NaCl and KCl that were different from those produced by unadulterated NaCl stimuli (Figs. 3 and 6), modeling the effects of a paracellular pathway blocker resulted in across-neuron patterns for both NaCl and KCl that were highly similar to those produced by unadulterated NaCl. A multidimensional space representing both the effects of amiloride and the results of modeling the effects of blocking the amiloride-insensitive pathway is shown in Fig. 7. This space was based on the across-neuron correlations among the stimuli. As in the NMD space, treatment of the stimuli with 30 µM amiloride produced similar across neuron correlations among NaCl and KCl stimuli (Fig. 7, triangles), which were grouped away from unadulterated NaCl. Mimicking the effects of a paracellular blocker, however, resulted in across-neuron patterns for both NaCl and KCl stimuli (Fig. 7, squares) that were virtually indistinguishable from unadulterated NaCl. Thus whether the amiloride-sensitive or -insensitive pathway is blocked, cells in the rat NST cannot distinguish between NaCl and KCl.
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One subtle difference in the taste spaces based on NMD and correlation
is worth noting. As shown in Fig. 6A, KCl grouped near QHCl
(Q) in the NMD-generated space. In the taste space based on
correlations, KCl (open circles) did not appear as near to QHCl,
whereas QHCl was located closer to HCl. This difference reflects the
way NMDs and correlations quantify differences in the across-neuron
patterns: NMDs reflect both differences in intensity and in the
patterns of responses across neurons, whereas correlations are
insensitive to intensity differences. Indeed, across this sample of NST
neurons, HCl and QHCl correlated +0.83 but maintained a relatively
large NMD because, in general, the neurons were much more responsive
to HCl than QHCl. However, the NMDs between QHCl and KCl were
quite low (Fig. 6A), reflecting the lower response rates to
these two stimuli relative to the others. In that regard, it is
noteworthy that rats can discriminate between KCl and concentrations of
QHCl somewhat lower than those used in the current study (0.01-0.1 mM)
(St. John and Spector 1998).
Amiloride: extended concentration range
Amiloride inhibited the responses of N-best cells to 0.1 M NaCl
across the extended amiloride concentration range. Figure 8A shows the mean response of
five N-best neurons to 0.1 M NaCl mixed with different concentrations
of amiloride relative to the response to 0.1 M NaCl alone. There was a
striking similarity between this effect and the efficacy of amiloride
in disrupting behavioral responses in the Spector et al.
(1996) study (Fig. 8B). Where comparable, the values
of the parameters defining the curves fit to the concentration-response
data are remarkably similar. That is, both have virtually the same
slope (b) and half-maximum (c). The only
difference was in the minimum response (d), but this
difference is due to the scale of the ordinate. For the
neurophysiological measure, the minimum that could be expected is zero
spikes in 10 s (without considering the possibility of inhibitory
responses). On the other hand, the minimal behavioral performance that
could be expected is 50%, indicating rats performing at chance levels in the discrimination task (see Spector et al. 1996
for
further details).
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Group C stimuli
Although N-best neurons respond differentially to NaCl and KCl at midrange concentrations (Fig. 2, A and B), higher concentrations of KCl drive N-best neurons quite well. For the subset of neurons tested with group C stimuli, 1.0 M KCl correlated strongly with 0.1 M NaCl in N-best neurons (Fig. 9; r = +0.85), showing that these neurons alone cannot provide a unique signal for sodium taste. Moreover, the overall across-neuron correlation between NaCl and KCl remains moderate (r = +0.60) at these concentrations due to the differential input provided by the other neuron types (H- and S-best neurons). Whether rats can behaviorally discriminate these particular concentrations of NaCl and KCl has not been tested, but if they can, it seems unlikely that the activity of N-best neurons alone could provide the discriminative signal.
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Salt responses across neuron types
The contribution of each neuron type to the overall patterns of
response to each stimulus under both the amiloride and the amiloride-insensitive treatment is depicted in Fig.
10. Here, the mean response of each
neuron type is shown for 0.1 M NaCl and 0.1 M KCl in the untreated
condition (), when mixed with amiloride (
), and after
the hypothetical block of the amiloride-insensitive pathway (
). As
reflected in the MDS analyses (Figs. 6 and 7), amiloride makes the
across-neuron type patterns for both NaCl and KCl more similar to
untreated KCl than to NaCl. On the other hand, the
amiloride-insensitive treatment makes these patterns more similar to
untreated NaCl than to KCl. These shifts in pattern are due to the
reduction primarily of the responses of N- and S-best cells after
amiloride and of H- and S-best cells after the model treatment.
Nevertheless, the responses to both stimuli are characterized by
substantial activity in all three neuron types. The similarity between
NaCl and KCl, for example, after the model treatment (as reflected in
the MDS space of Fig. 7) is not due to the large response in N-best
cells but in the relative response among the three cell types. The
response of the N-best cells to KCl after this model treatment is
actually no greater than the response of these cells to untreated KCl,
yet KCl after this treatment produces an across-neuron pattern highly
similar to untreated NaCl (Fig. 7). Similarly the response of H-best
neurons to NaCl after amiloride is no different from the response
of these neurons to untreated NaCl, yet the across-neuron pattern for
NaCl after amiloride is like that evoked by KCl either untreated or after amiloride (Figs. 3 and 7).
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DISCUSSION |
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Segregation of amiloride-sensitive and -insensitive responses in the rat NST
Consistent with studies of peripheral taste fibers (Brand
et al. 1985; Formaker and Hill 1988
; Heck
et al. 1984
; Herness 1987
; Hettinger and
Frank 1990
; Ninomiya and Funakoshi 1988
), NaCl
responses in the NST were partially inhibited by amiloride. Amiloride
predominantly affected responses in N-best neurons (Fig. 2 and Table
2). These results are consistent with other work demonstrating the
segregation of amiloride-sensitive and -insensitive activity in the NST
(Boughter et al. 1999
; Giza and Scott
1991
; Scott and Giza 1990
; Smith et al.
1996
) and extend earlier findings in the rat by demonstrating amiloride effects across a dosage range (3-30 µM) substantially lower than used previously (500 µM) (Giza and Scott
1991
; Scott and Giza 1990
).
In the present study, the segregation of amiloride-sensitive and
-insensitive input was not absolute. Average responses to NaCl were
significantly reduced by amiloride in all neuron types, suggesting some
convergence of amiloride-sensitive and -insensitive peripheral fibers
onto H- and S-best NST neurons (Fig. 2). Nonetheless it is also
apparent that the bulk of amiloride-sensitive input is to N- and S-best
neurons (Fig. 2 and Table 2); the input to H-best cells is minimal at
best. Using a different cell classification scheme, Giza and
Scott (1991) found that amiloride inhibited NaCl responses in
two groups of neurons that responded best to NaCl but not in two other
groups that were relatively more responsive to acids. Thus there is
general agreement that amiloride predominantly affects N-best neurons.
A striking difference to the results of Giza and Scott
(1991)
was that amiloride significantly reduced responses to
KCl in the present experiment (Fig. 2). An effect of amiloride on KCl
responses has been reported in studies on the rat chorda tympani nerve
(Lundy and Contreras 1997
; Minear et al.
1996
; Ninomiya and Funakoshi 1988
) and the
hamster NST (Boughter et al. 1999
).
Relationship to discrimination behavior
A primary goal of the present study was to relate the neural
representation of NaCl and KCl to the behavioral results of
Spector et al. (1996). In that experiment, rats were
trained to press one lever after tasting NaCl and another one after
tasting KCl. There were several significant results in the behavioral
study: discrimination performance deteriorated when increasing
concentrations of amiloride were mixed with NaCl and KCl; rats
performed better on trials with higher salt concentrations; rats
responded to NaCl mixed with high concentrations of amiloride as if it
was KCl (i.e., they pressed the KCl lever); and the concentration of
amiloride with a half-maximal effect on discrimination performance was
in the range of the inhibition constant for amiloride's effects on NaCl-evoked activity in the CT.
The current neurophysiological results relate directly to the
behavioral findings. First, amiloride had a concentration-dependent effect on the neural representation of NaCl and KCl (Figs. 3-6). Across-neuron patterns for NaCl and KCl are very different in the NST,
which presumably allows the qualitative discrimination of these salts
behaviorally (Erickson 1963). The difference in these
across-neuron patterns declines when the salts are mixed with 3 µM
amiloride and virtually disappears at 30 µM (Figs. 3-6).
Second, the neurophysiological data suggest why behavioral performance improved at higher salt concentrations. The NMD measure, which takes into account both quality and intensity information to provide an overall index of discriminability, decreases as a function of amiloride concentration but increases slightly with salt concentration (Fig. 4B). More dramatically, the taste spaces based on the NMD (Fig. 6) clearly separate NaCl (filled triangles) from KCl (open triangles) in the no amiloride condition (A) but not at 30 µM amiloride (C). However, at 3 µM amiloride (B), 0.2 M NaCl is located closer to unadulterated NaCl, whereas 0.05 M NaCl is located nearer to KCl. This suggests that higher concentrations of NaCl, even mixed with amiloride, should be easier for rats to recognize than lower concentrations.
The third behavioral finding was that rats tended to press the KCl-appropriate lever when the stimulus was NaCl mixed with high concentrations of amiloride. In the derived taste spaces (Figs. 6 and 7), amiloride shifted NaCl stimuli closer to unadulterated KCl rather than vice versa. This shift reflects the fact that amiloride had a relatively larger effect on responses to NaCl than to KCl (Figs. 2 and 3), which changed the across-neuron pattern evoked by NaCl to one more characteristic of KCl (Figs. 3 and 5). This result is consistent with the interpretation that NaCl, when mixed with amiloride, tastes like KCl.
Finally, Spector et al. (1996) found that the effect of
amiloride on discrimination could be described by a sigmoidal function (Fig. 8B), with amiloride producing a half-maximal effect on
behavior at 3.93 µM. This value was in the range of the inhibition
constant for amiloride's effect on NaCl responses in the rat chorda
tympani nerve (Brand et al. 1985
; DeSimone and
Ferrell 1985
) and in rat and hamster taste receptor cells
(Avenet and Lindemann 1991
; Gilbertson et al.
1992
) when stimulated with midrange concentrations of NaCl. When a subset of N-best neurons was tested with the full range of
amiloride concentrations, we found the concentration-response function
for amiloride's effect on 0.1 M NaCl responses to be well described by
a similar function (Fig. 8A), with an inhibition constant of
3.49 µM. Thus there is a striking correspondence in amiloride's
action on taste receptor cells, peripheral nerve fibers, N-best cells
in the NST and the rat's ability to make a behavioral discrimination
between sodium and nonsodium salts.
Other investigators have shown that amiloride reduces the neural
differentiation of sodium and nonsodium salts (Boughter et al.
1999; Giza and Scott 1991
; Scott and Giza
1990
). However, comparison of earlier rat electrophysiological
data to the Spector et al. (1996)
experiment is limited
by the salt concentrations used and by the very high amiloride
concentration employed (500 µM). Amiloride has a half-maximal effect
on taste receptor cells near 1 µM (Avenet and Lindemann
1991
; Gilbertson et al. 1992
); such strong
concentrations could produce nonspecific effects (Lindemann 1996
; Lundy and Contreras 1997
; Lundy et
al. 1997
; Smith and Benos 1991
).
Implications for taste quality coding
Our results are consistent with the interpretation that amiloride makes NaCl taste like KCl to rats. In addition, when we modeled the effect of blocking the amiloride-insensitive transduction pathway (see METHODS and Fig. 7), the neural distinction between NaCl and KCl was reduced. Given that this manipulation caused KCl and NaCl to cluster near unmodified NaCl in the multidimensional space (Fig. 7), it is indeed possible that the perceptual effect of such a blocker would be to make KCl taste like NaCl.
Does this mean that N-best cells, which are substantially inhibited by
amiloride, are responsible for coding the "sodium taste" (cf.
McCaughey and Scott 1998), whereas H-best neurons are
responsible for coding the "nonsodium taste" of salts? In other
words, are N- and H-best cells labeled lines for these two qualities of
salt taste?
We believe such an interpretation ignores several salient features of
the rodent taste system, as well as available information on behavioral
responses to NaCl and KCl. For one, a labeled-line interpretation
ignores the fact that both salts stimulate all neuron types. As shown
in Fig. 10, information about NaCl is indeed carried predominately
through N-best cells, but it is difficult to support the opposite claim
for KCl. That is, KCl evokes roughly equivalent levels of activity in
N- and H-best neurons and certainly more activity in N- and S-best
cells combined than in H-best cells. Taken to its logical conclusion, a
labeled-line interpretation (that N-best neuron activity signals a
sodium taste and H-best neuron activity a "potassium taste") would
imply that both NaCl and KCl are complex tastes, perceptually
representing mixtures of sodium and potassium taste to rats. Since it
is known that rats will avoid a conditioned stimulus in proportion to
its concentration in a mixture (Smith and Theodore
1984), the labeled-line notion would predict (by virtue of
the broad tuning displayed in Fig. 10) that taste aversions conditioned
to NaCl should generalize to KCl and vice versa. However, there is
strong behavioral evidence that NaCl and KCl do not cross-generalize
(Hill et al. 1990
; St. John et al.
1997
).
Second, interpreting the effects of amiloride as due to the specific
inhibition of N-best neuronal activity is an oversimplification at
best. As summarized in Fig. 10, amiloride () does not simply reduce
the N-best neural activity to zero while having no effect on H- or
S-best units. Instead, N-best and S-best responses are considerably reduced, but activity in neither neuron type is completely eliminated. It is important to note that when an identical stimulus protocol is used in behavioral experiments, rats cannot distinguish NaCl from KCl (Spector et al. 1996
).
Why then does amiloride cause NaCl to taste like KCl? We believe the
most straightforward explanation, and one that accounts for the
greatest amount of neural and behavioral data, is that the relative
activity across all neurons is responsible for encoding the taste of
these salts. Note the similarity in the across neuron patterns after
amiloride in Fig. 3. In Fig. 10, where across-neuron patterns have been
simplified as "across neuron type patterns," the same case can be
made. It is not the amount of activity in any one neuron type that
conforms to the predicted results, otherwise unadulterated NaCl (),
which has the greatest activity in H-best units of the six stimuli
presented, should taste the most like KCl. Instead it is the relative
activity that appears critical: amiloride treatment renders the
patterns of both NaCl and KCl (
) similar to that of unadulterated
KCl and dissimilar to that of NaCl. Behaviorally (Spector et al.
1996
) and neurally, the results of amiloride treatment are
consistent with the perceptual effect of making both salts taste like
KCl. In addition, after the paracellular treatment (
) NaCl and KCl
are similar in their patterns across cell types, but the amount of
activity in the N-best cells is dramatically different. These data,
along with the lack of behavioral generalization between NaCl and KCl,
suggest strongly that it is not the absolute activity in any one neuron type but the relative activity across neurons that represents the taste
of these salts.
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ACKNOWLEDGMENTS |
---|
We thank Dr. John D. Boughter, Jr., for valuable comments on the manuscript.
This research was supported in part by National Institute on Deafness and Other Communication Disorders Grant DC-00353 to D. V. Smith.
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FOOTNOTES |
---|
Address for reprint requests: S. J. St. John, Dept. of Anatomy and Neurobiology, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore, MD 21201-1509 (E-mail: sstjo001{at}umaryland.edu).
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 4 November 1999; accepted in final form 19 April 2000.
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REFERENCES |
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