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Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee
Submitted 5 July 2005; accepted in final form 15 August 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Matsunami et al. 2000). The few T2R receptors that have been characterized bind a small number of bitter ligands (Bufe et al. 2002; Chandrashekar et al. 2000) and multiple T2R receptors are expressed by the same taste bud cells (TBCs) (Adler et al. 2000). T2R-positive TBCs do not express known receptors for sweet stimuli or amino acids (Adler et al. 2000; Nelson et al. 2001). These results have been interpreted to suggest that all bitter stimuli elicit a unitary taste sensation and that the bitter taste quality is encoded by the activation of a dedicated neuronal channel (i.e., a "labeled line") (Adler et al. 2000; Mueller et al. 2005; Scott 2004; Zhang et al. 2003).
Other investigations of the gustatory processing of bitter stimuli have arrived at different conclusions than those drawn from molecular studies. Human psychophysical studies suggest that bitter substances are not a homogeneous group. Cross-adaptation experiments have shown that adaptation to quinine does not cross-generalize to other bitter stimuli such as phenylthiocarbamide or urea (McBurney et al. 1972). A similar dichotomy among bitter stimuli has been observed in bitterness rating experiments, where sensitivity to only select bitter stimuli was found to covary across individuals (Delwiche et al. 2001). In hamsters, learned aversions cross-generalize between some bitter stimuli, such as quinine and denatonium benzoate, but not others, such as quinine and caffeine, indicating that rodents may not perceive all bitter stimuli as qualitatively identical (Frank et al. 2004). Further, psychophysical studies in rodents have shown that rats are capable of discriminating among some bitter stimuli (St. John and Spector 1998) but not others (Spector and Kopka 2002). Collectively, these data suggest that some bitters may generate differential neural signals. This idea is supported by functional data from calcium-imaging studies showing selectivity of TBCs among bitter ligands (Caicedo and Roper 2001) and electrophysiological data showing that fibers of gustatory nerves differ in their sensitivities to various bitter compounds (Dahl et al. 1997). Calcium-imaging and patch-clamp electrophysiological studies have shown that a number of bitter-sensitive TBCs in mammals are responsive to stimuli of other taste qualities, such as Na+ salts and sweets (Caicedo et al. 2002; Gilbertson et al. 2001; Sato and Beidler 1997). These data challenge the specificity of bitter-sensitive TBCs proposed by molecular studies. In addition, central gustatory neurons, which ultimately give rise to perception, are typically broadly responsive across stimuli of different taste qualities; this raises questions of whether activity in purported functional groups of neurons is sufficient to unambiguously represent individual stimulus qualities (for reviews, see Scott and Giza 2000; Smith and St. John 1999). If bitter taste is indeed encoded along labeled lines, input from bitter receptors must be received by a gustatory neural type in the CNS that is selective enough to represent the qualitative features of exclusively bitter stimuli. Neurons of this type must respond differentially to bitter ligands and stimuli of other taste qualities.
Here, we recorded taste-evoked responses to a large array of intensity-equivalent concentrations of bitter and other stimuli from single gustatory neurons in the nucleus of the solitary tract (NST) to address two questions that arise from the molecular findings. We first evaluated whether physiologically defined neural types in the NST respond differentially to bitter ligands and stimuli of other taste qualities. Second, we investigated whether gustatory activity generated by different bitter stimuli in the NST is indiscriminant. These experiments attempt to relate the results of molecular studies of taste receptors to the organization of gustatory neural circuits in the brain.
These data were presented in poster form at the 27th annual meeting of the Association for Chemoreception Sciences, Sarasota, FL, April 2005.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Forty-five adult male Sprague Dawley rats, weighing 350–450 g, were used. Rats were housed in a vivarium that maintained a 12-h light/dark cycle and an ambient temperature of 
23°C. Food and water were available ad libitum. Animals 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 aspirated to expose the brain stem and allow vertical access to the NST, the first central synapse for taste information processing. Body temperature was maintained at 37°C by a heating pad.
Electrophysiological recording
The present study proceeded in two sequential phases. Phase 1 was designed to intensity-match the concentrations of stimuli that were tested. This was accomplished by identifying concentrations of stimuli that produced equipotent, integrated multi-unit taste responses in the NST. These physiologically equivalent concentrations were then used in phase 2 of this investigation, which involved assessment of single-neuron responses to taste stimuli.
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; neural activity was then verified as taste-responsive by application of various gustatory stimuli (see following text). The gustatory-responsive portion of the NST was encountered 1 mm ventral to the brain stem surface.
In phase 1, blunt tungsten microelectrodes (impedance <1 M
at 1 kHz, FHC, Bowdoinham, ME) were used to record multi-unit taste activity from the NST. An indifferent electrode was placed in nearby tissue. Differential extracellular voltages were amplified 10,000x (Grass P511), monitored using a storage oscilloscope and audio monitor, and fed into an integrator (PAVC-1, Duck Engineering Design) that rectified multi-unit activity (time constant = 500 ms). This rectified signal was then digitized (sampling rate = 25 kHz; Power 1401 RISC acquisition interface coupled with Spike 2 software, CED, Cambridge, UK) and downloaded to storage media for off-line analysis.
In phase 2, etched tungsten microelectrodes, insulated except for the tip (impedance = 1–8 M at 1 kHz, FHC), were used to record extracellular action potentials from individual NST neurons. Electrophysiological activity was band-pass filtered (bandwidth = 0.3–6 kHz), differentially amplified 10,000x (Grass P511 with high-impedance probe), and subsequently routed to a storage oscilloscope and audio monitor. Neural activity was digitized (sampling rate = 25 kHz; Power 1401/Spike 2), and action potentials generated by an individual neuron were identified based on waveform consistency, which was assessed using a spike-waveform template-matching algorithm and the analysis of spike interval histograms, where the single-neuron nature of a recording is evidenced by an absence of spike intervals shorter than the refractory period (2 ms). Digital records of spike trains recorded from each neuron were downloaded to storage media for off-line analysis.
Taste stimuli
We tested a large array of taste stimuli categorized as sweet, salty, sour, or bitter (Table 1) including the common prototypes of these categories: sucrose, NaCl, HCl, and quinine-HCl. Stimuli were made with 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 data-acquisition system, regulated solution delivery. A curved, polyethylene tube extended from the output port of this valve and was directed toward the palate of each subject. Visual inspection revealed that this configuration allowed solutions to bathe both the palate and anterior tongue, as solutions were deflected downward on encountering the palate. Moreover, independent tests using methylene blue dye verified that our delivery system effectively bathed the entire anterior tongue and palate, including the nasoincisor ducts. Studies have shown that gustatory input from the VIIth cranial nerve, which innervates the anterior tongue and palate, is critical for taste discrimination, even among bitter stimuli (Spector and Grill 1992; St. John and Spector 1998).
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50 ml of deionized water and >2 min were allowed to elapse between trials. The stimulus delivery system was thoroughly rinsed with deionized water between stimulus presentations and the tongue was kept moist with deionized water during the inter-trial interval. In phase 1, a concentration series (2 log steps in half-log-step intervals in most cases) of each stimulus was presented in ascending order with steps interleaved between presentations of a standard stimulus (0.1 M NaCl); the concentrations used were the same for each animal and are given in Fig. 1. In phase 2 presentation order was completely randomized across all stimuli for each neuron.
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Each integrated response was plotted as voltage (ordinate) against time (abscissa) and the area between the voltage signal and the y-axis zero was measured during the first 5 s of the response and during the 5-s period immediately prior to stimulus onset. Responses were quantified by subtracting the prestimulus area from the peristimulus area. The net response observed on a given stimulus trial was then standardized by dividing this value by the mean of the net responses measured on the two 0.1 M NaCl trials that bracketed this stimulus; this standardization ensures that data from different electrode penetrations and different animals contributes equally to the means.
To quantify the concentration-response relationship for each stimulus, logistic curves were fit to standardized data pooled from multiple preparations as given by
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Single-neuron responses to matched concentrations (i.e., CS) of chemical stimuli were quantified as the number of action potentials that arose during the 10-s stimulus presentation minus the number of action potentials that spontaneously occurred during the 10-s period prior to stimulus onset. A measure of response profile entropy (Smith and Travers 1979) was calculated for each neuron to quantify its breadth of responsiveness to CS concentrations of sucrose, NaCl, HCl, and quinine. Entropy is defined as
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Neurons were categorized into types using hierarchical cluster analysis (HCA). This approach has been employed in other investigations to group gustatory neurons with similar response properties into classes that presumably serve a particular function in the processing of taste information (e.g., Frank et al. 1988; Scott and Giza 1990). The outcome of HCA typically suggests neuronal groupings that are similar to those that would be derived if one used a "best stimulus" classification scheme (i.e., categorizing neurons into types based on the stimulus that evokes the highest relative rate of firing) (Chang and Scott 1984; Frank 1973; Frank et al. 1988; Nakamura and Norgren 1991; Smith et al. 1983a,b). However, HCA provides a more comprehensive approach to defining neuronal groupings as neurons can be categorized based on similarities/dissimilarities among responses to each tastant under consideration. For the present analysis, input to HCA consisted of a correlation (Pearson's r) matrix representing pairwise neuronal response profile similarity. Cluster analysis was performed using SPSS (SPSS, Chicago, IL). The "within groups linkage" amalgamation schedule was used and a scree procedure determined the appropriate number of clusters (Everitt 1980; Kim and Mueller 1978).
ANOVA was used for data analysis where applicable. 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 repeated level means were performed using a dependent-samples t-test evaluated using a critical value as given by Dunn (1961) that adequately controls 
for such comparisons. ANOVA was performed using Statistica (StatSoft, Tulsa, OK).
Relationships among across-neuron patterns of response evoked by bitter and other stimuli in the rat NST were quantified using Pearson's r. Moreover, principal components factor analysis was used to examine sources contributing to the organization of these patterns of response. Factor analysis can be used for classification purposes as variables that are correlated with one another but largely independent of other variables will combine into a factor. The correlation coefficient matrix computed among across-neuron patterns of response produced by each stimulus was used as input to this analysis. A scree test was used to determine the number of factors (Kim and Mueller 1978). The factor analytic solution was simplified using orthogonal varimax rotation, which maximizes that variance accounted for by each factor (Bieber and Smith 1986; Kim and Mueller 1978). Factor analysis was performed using Statistica.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Integrated multiunit taste responses to a concentration series of each stimulus listed in Table 1 were recorded from the NST and CS concentrations of each stimulus were computed (Fig. 1). Fourteen rats were used for this experiment. Complete data were obtained from three to five preparations for each stimulus. Some stimuli produced relatively weak responses at near-saturated concentrations and could not be intensity-matched to 0.01 M NaCl. On this basis, the following stimuli were not included in single-unit experiments in phase 2 (highest concentration used is indicated in M): L-phenylalanine, 0.18; caffeine, 0.11; sucrose octaacetate, 0.0015; phenylthiocarbamide, 0.016; theophylline, 0.032; cycloheximide, 0.004; urea, 2.0.
Phase 2: general single-unit response characteristics
Trains of action potentials were recorded from 51 NST neurons in 31 rats in response to bathing the tongue and palate with intensity-equivalent concentrations of 19 different stimuli (Table 2). Given the length of the experimental protocol, each stimulus was tested once per neuron to facilitate collecting data from multiple cells in a single preparation. Three of these stimuli were classified as sweet, 2 were Na+ salts, 2 were non-Na+ salts, 2 were acidic, and 10 were categorized as bitter. Electrophysiological records showing responses to these stimuli observed in one NST neuron are shown in Fig. 2. The responses of each neuron to the 19 stimuli are shown as across-neuron patterns in Fig. 3. The mean spontaneous discharge rate observed across all neurons was 1.5 ± 0.2 (SE) spikes/s. Considering only the prototypes of each stimulus category, neurons were generally broadly responsive across CS concentrations of sucrose, NaCl, HCl, and quinine (
= 0.74 ± 0.02 SE). Broad sensitivity to these stimuli in NST neurons has been reported by several other investigators (e.g., Cho et al. 2004; Di Lorenzo and Victor 2003; Di Lorenzo et al. 2003; Giza et al. 1991; McCaughey and Scott 2000). On the basis of their largest response to these stimuli, 11 neurons responded best to sucrose, 23 responded best to NaCl, 12 responded best to HCl, and 5 quinine-best neurons were included in our sample.
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= 0.61 ± 0.05; mean spontaneous discharge rate = 0.5 ± 0.1 spikes/s) responded optimally to sucrose and other sweets but also displayed good sensitivity to Na+ salts. Type N neurons (n = 13; = 0.75 ± 0.04; mean spontaneous discharge rate = 1.4 ± 0.4 spikes/s) responded optimally to Na+ salts but were also responsive to other stimuli. Type H/Q (n = 22; = 0.84 ± 0.02; mean spontaneous discharge rate = 2.2 ± 0.3 spikes/s) consisted of HCl- and quinine-best neurons that were broadly responsive across stimuli. These groupings are similar to those reported in other studies that have used cluster analysis to categorize NST gustatory neurons (e.g., Di Lorenzo et al. 2003; Lemon et al. 2003; McCaughey and Scott 2000; Smith et al. 1983b).
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NEURONAL TYPES. Mean responses to CS concentrations of each stimulus for each neural type are shown in Fig. 5. Analyses of variance were conducted to assess whether neural types S and N responded differentially to their optimal stimuli relative to bitter ligands. Neural type S differentially responded across stimuli [single-factor repeated-measures ANOVA, F(18,270) = 21.9, P < 0.001] and responses to sugars and ethanol were greater than those elicited by bitter stimuli (comparison of mean responses between stimulus categories, Dunn post hoc test, P < 0.05). Neural type N was differentially activated across stimuli [single-factor repeated-measures ANOVA, F(18,216) = 14.1, P < 0.001]. The mean response produced by Na+ salts in this type was greater than that evoked by bitter stimuli (comparison of mean responses between stimulus categories, Dunn post hoc test, P < 0.05).
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; Nowlis et al. 1980). Under the framework of labeled-line coding, neural type H/Q could be argued to represent a neural "channel" for a qualitative sensation common to bitter ligands and acids. Whether this is the case would depend on the sensitivities of this neural type to stimuli that rats perceive as perceptually independent of bitter and acidic stimuli, such as sweets and Na+ salts (e.g., Morrison 1967; Nowlis et al. 1980). Neural type H/Q was differentially activated by gustatory stimuli [single-factor repeated-measures ANOVA, F(18,378) = 22.4, P < 0.001]. However, although mean responses to bitter ligands were greater than the average response produced by sugars and ethanol (Dunn post hoc test, P < 0.05), this neural type did not differentially respond to NaCl and quinine or HCl (Dunn post hoc tests, = 0.05). Yet rats treat the tastes of NaCl and quinine or HCl as perceptually distinct (Morrison 1967; Nowlis et al. 1980). Moreover, this cell type was not differentially sensitive to Na+ salts and bitter or acidic stimuli in general (comparisons of mean responses between stimulus categories, Dunn post hoc tests, = 0.05). Thus rate of firing in neural type H/Q alone is likely not sufficient to exclusively represent a qualitative feature common to bitter and acidic stimuli. ACROSS-NEURON PATTERNS OF RESPONSE. Multivariate analyses were employed to examine relationships among across-neuron patterns of response generated by each stimulus to determine whether all bitter stimuli were represented similarly by neural activity. A matrix of correlation coefficients (Pearson's r) computed among patterns of response produced by each stimulus served as input to each analysis. HCA was employed to group stimuli based on similarities/dissimilarities among their evoked patterns. The result of this analysis is depicted graphically in Fig. 6, which shows that stimuli fell into one of three groups. Patterns produced by the two sugars and ethanol were similar and defined a separate group. Patterns produced by the two Na+ salts were similar yet distinct from other stimuli and were linked into a separate group. Patterns produced by all bitter ligands were similar and clustered into the final group along with patterns evoked by acidic stimuli and non-Na+ salts.
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DISCUSSION |
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; Nelson et al. 2001). However, the responsiveness of neural type H/Q to other tastants, particularly Na+ salts, raises questions of whether this neuron type could function as a dedicated coding channel for the qualitative features of bitter stimuli. That these H/Q neurons responded to bitter, acidic, and non-Na+ salts is predicted by behavioral studies indicating that rats perceive commonalities among these classes of tastants. For example, conditioned taste aversion studies have shown that learned aversions generalize to a certain extent between quinine and HCl (Nowlis et al. 1980). Further, Morrison (1967), using a conditioning procedure whereby rats were trained to press one of two levers to indicate which of two taste stimuli they sampled, found that rats confused the tastes of quinine and HCl or MgCl2 on more than half of the trials. These data suggest that particular bitters, acids, and non-Na+ salts elicit similar qualitative taste sensations to rats. Thus some overlap in the neural representation of these stimuli would be expected. If neural type H/Q functioned as a dedicated coding channel for gustatory features common to bitter, acidic, and other aversive tastants, then NaCl would also be expected to prominently elicit these features as neural type H/Q does not respond differentially to Na+ salts and bitter or acidic stimuli (Fig. 5). However, rats do not generalize between quinine or HCl and NaCl in learned generalization and discrimination paradigms (Morrison 1967; Nowlis et al. 1980). In addition, rats prefer 0.01 M NaCl, whereas they clearly avoid quinine and HCl at the concentrations used in the present study (Pfaffmann 1964). Thus it is difficult to reconcile the representation of the qualitative features of bitter and other aversive stimuli by considering the output of neural type H/Q alone.
Because neurons of type H/Q are strongly responsive to sodium salts in addition to bitter stimuli and acids, it is important to consider whether these cells play a role in the representation of sodium taste. The discrimination of sodium salts is clearly dependent on input arising from taste receptors for Na+ that are inhibited by the drug amiloride (e.g., Spector et al. 1996). Studies in the rodent NST have shown that orally applied amiloride selectively diminishes taste responses to NaCl in sucrose- and NaCl-best neurons but not HCl-best cells (Boughter and Smith 1998; Smith et al. 1996; St. John and Smith 2000), which possess response profiles similar to neurons of type H/Q in the present study. The lack of amiloride sensitivity raises the possibility that neural type H/Q could provide information about NaCl taste that is unrelated to the perception of stimulus quality, but this remains to be definitively shown. On the other hand, if neural types N and H/Q exclusively give rise to Na+ salt and aversive/bitter taste, respectively, NaCl would then elicit both a sodium taste and a distinctly bitter taste given that NaCl drives neural type H/Q just as effectively as many strongly bitter stimuli. Furthermore, under this type of coding strategy, NaCl would also be expected to possess a sweet-taste component given that sucrose-best NST neurons receive significant input from amiloride-sensitive sodium receptors (Boughter and Smith 1998; Smith et al. 1996; St. John and Smith 2000). However, rodents perceive NaCl as perceptually independent of these stimuli in both generalization and discrimination paradigms (Morrison 1967; Nowlis et al. 1980). The nervous system may compare relative levels of activation across neural types S, N, and H/Q to represent the unique taste of NaCl (see St. John and Smith 2000).
The broad sensitivity of neural type H/Q to bitters, Na+ salts and acids suggests two possibilities regarding the expression of taste receptors for these stimuli: either input from TBCs exclusively sensitive to these stimuli converges onto neurons in the NST or bitter, Na+, and H+ receptor mechanisms are expressed in common subsets of TBCs. Although some investigators have postulated that T2R-expressing TBCs respond exclusively to bitter ligands (e.g., Mueller et al. 2005; Scott 2004), the latter scenario cannot logically be ruled out as it is presently unknown how the taste receptors for bitter stimuli, Na+ salts and acids are distributed relative to one another. Moreover, calcium imaging and electrophysiological studies have shown that a proportion of mammalian TBCs responds to bitter stimuli and those of other taste qualities (Caicedo et al. 2002; Gilbertson et al. 2001; Sato and Beidler 1997).
Although it has been shown that receptors for several different bitter ligands are co-expressed by the same TBCs (Adler et al. 2000), imaging studies have shown that some TBCs are more selective to bitter stimuli than would be expected on this basis, with some cells differentially responding to bitter ligands (Caicedo and Roper 2001). Differential sensitivity to bitter tastants has also been reported in fibers of gustatory nerves (Dahl et al. 1997) and in psychophysical studies in humans and rodents (Delwiche et al. 2001; St. John and Spector 1998). Similarly, some bitter stimuli cross-adapt with one another whereas others do not (McBurney et al. 1972). In the present investigation, all bitter stimuli employed in the single-unit experiments generated highly correlated across-neuron patterns of response, indicating that these particular bitter ligands do not evoke differential neural signals in the NST. However, we excluded testing an additional set of bitter tastants (L-phenylalanine, caffeine, sucrose octaacetate, phenylthiocarbamide, theophylline, cycloheximide, and urea) because integrated multi-unit responses to very high or even near-saturated concentrations of these stimuli were of relatively low magnitude and could not be matched to our reference stimulus. This low responsivity is not attributable to insensitivity to these stimuli in rats. For example, detection thresholds range from 0.2 to 2 µM for cycloheximide and 20–600 µM for phenylthiocarbamide (Richter and Clisby 1941; Tobach et al. 1974). Yet the present investigation revealed relatively weak integrated responses in the NST to 0.004 M cycloheximide, which is more than 100-fold greater than the concentration at which rats completely avoid cycloheximide relative to water (30 µM, unpublished data), and 0.016 M phenylthiocarbamide, which is near saturation. Thus here we observed similar responses to certain bitter ligands but differential sensitivity among others. However, the present study pertains only to gustatory input mediated by the VIIth nerve and cannot address the contribution of other nerves such as the IXth, which innervates taste bud fields on the posterior tongue and is relatively more responsive to bitter ligands (Frank 1991). On the other hand, the VIIth nerve input, but not the IXth, is necessary for gustatory discriminations in rats, even among bitter stimuli (St. John and Spector 1998).
This differential neural sensitivity to bitter tastants must be interpreted in the context of the concentrations of the stimuli that were used and behavioral sensitivity toward these concentrations. For example, although rats would find the taste of 0.007 M quinine aversive, rats readily avoid quinine at lower concentrations (e.g., 0.0001 M) (Pfaffmann 1964). Neural responses to lower yet behaviorally averse concentrations of quinine would, accordingly, be of lesser magnitude and could more closely approximate the response produced by 0.004 M cycloheximide. Nonetheless, it is possible that the differential responding to these stimuli that was observed in the present study reflects asymmetry in the distribution of receptor mechanisms for quinine and cycloheximide across gustatory epithelia. This idea is also supported by data from mice showing good sensitivity to quinine in both the chorda tympani (CT, a branch of cranial nerve VII) and IXth nerves yet strong responding to cycloheximide in the IXth and almost a complete lack of sensitivity to this stimulus in the CT nerve across a range of concentrations (Danilova and Hellekant 2003). The differential sensitivity of nerve VII to these and other bitter stimuli could present a basis for further investigation of the question of whether all bitters are perceived alike given the importance of the VIIth nerve for taste discriminations (Spector and Grill 1992; St. John and Spector 1998). The bitter ligands quinine and denatonium benzoate are similarly effective for this nerve as indexed by high correlation among evoked across-neuron patterns of response (Fig. 3) and, accordingly, cannot be discriminated by rats (Spector and Kopka 2002).
Multivariate analyses of the present data indicated that similarities among across-neuron patterns of response categorized stimuli into groups that would be generally predicted based on prior work. First, sugars and ethanol evoked correlated activity patterns that are distinct from those evoked by salts, acids and bitter ligands (Figs. 6 and 7). This correspondence between responses to ethanol and sugars replicates previous work showing ethanol taste selectively stimulates neural substrates that underlie the processing of sweet taste (e.g., Lemon et al. 2004) and that ethanol elicits a sweet taste sensation in rats (e.g., Di Lorenzo et al. 1986). Sodium salts evoked patterns of response that are unique relative to non-Na+ salts, acids, bitter, and sweet stimuli (Figs. 6 and 7), which corresponds with data showing that rats perceive the tastes of Na+ salts as independent of these other stimulus categories (Morrison 1967; Nowlis et al. 1980). Across-neurons patterns of response produced by all the bitter ligands tested in the present study were similar and were also similar to patterns produced by acidic stimuli and non-Na+ salts (Figs. 6 and 7), which would be expected to a certain extent as indicated previously.
Although across-neuron patterns of activity appear to categorize stimuli generally according to predictions arising from behavioral studies, some complications arise in the interpretation of patterns evoked by stimuli that elicit common but not identical qualitative features, such as bitters and acids. For example, although behavioral studies predict some degree of correlation between neural responses to quinine and HCl, patterns evoked by these stimuli were found to be highly correlated in multivariate analyses (Figs. 6 and 7); this has also been reported in other investigations (e.g., Giza et al. 1996; Scott and Giza 1990; St. John and Smith 2000). It is possible that such high correlations among patterns generated by bitter and acidic tastants reflect hedonic in addition to qualitative attributes of these stimuli as both categories of tastants are readily avoided by rats (Pfaffmann 1964). Future investigations aimed at understanding how qualitative and hedonic information is carried by neural activity represent a logical next step toward unraveling the neural code for taste (see Katz et al. 2001; Nishijo et al. 1998; Sewards 2004; Yamamoto et al. 1994).
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FOOTNOTES |
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1 We use the terms sweet, salty, sour, and bitter in this manuscript to refer to different categories of taste stimuli as a convenience. These categorizations relate only to human perceptual experience, and we do not mean to imply that rodents, for example, perceive quinine as "bitter". 
Address for reprint requests and other correspondence: D. V. Smith, Dept. of Anatomy and Neurobiology, University of Tennessee Health Science Center, 855 Monroe Ave., Suite 515, Memphis, TN 38163 (E-mail: dvsmith{at}utmem.edu)
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, stimulus onset. Spikes that arose from individual neurons were identified using a waveform template-matching algorithm. Inset: autocorrelogram depicting the cumulative distribution of interspike intervals between template-matched spikes across the 19 responses. Each time bin along the abscissa is 100 µs wide with the number of occurrences represented along the ordinate. Autocorrelation analysis was used to verify the single-neuron nature of recordings, evidenced by an absence of spike intervals shorter than the refractory period (
