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Taste Responsiveness of Fungiform Taste Cells With Action Potentials

¹Section of Oral Neuroscience, Graduate School of Dental Sciences, Kyushu University, Fukuoka; and ²Department of Brain Science and Engineering, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu, Japan

Submitted 18 April 2006; accepted in final form 8 September 2006

	ABSTRACT

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It is known that a subset of taste cells generate action potentials in response to taste stimuli. However, responsiveness of these cells to particular tastants remains unknown. In the present study, by using a newly developed extracellular recording technique, we recorded action potentials from the basolateral membrane of single receptor cells in response to taste stimuli applied apically to taste buds isolated from mouse fungiform papillae. By this method, we examined taste-cell responses to stimuli representing the four basic taste qualities (NaCl, Na saccharin, HCl, and quinine-HCl). Of 72 cells responding to taste stimuli, 48 (67%) responded to one, 22 (30%) to two, and 2 (3%) to three of four taste stimuli. The entropy value presenting the breadth of responsiveness was 0.158 ± 0.234 (mean ± SD), which was close to that for the nerve fibers (0.183 ± 0.262). In addition, the proportion of taste cells predominantly sensitive to each of the four taste stimuli, and the grouping of taste cells based on hierarchical cluster analysis, were comparable with those of chorda tympani (CT) fibers. The occurrence of each class of taste cells with different taste responsiveness to the four taste stimuli was not significantly different from that of CT fibers except for classes with broad taste responsiveness. These results suggest that information derived from taste cells generating action potentials may provide the major component of taste information that is transmitted to gustatory nerve fibers.

	INTRODUCTION

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The gustatory pathways begin with taste receptor cells that detect chemical signals within the oral cavity. Their messages are transmitted to the gustatory afferent fibers and sent on to higher-order neurons. Responses of individual taste receptor cells to stimuli representing different taste qualities were first examined by recording receptor potentials of the rat and hamster taste cells in the fungiform papilla on the anterior tongue innervated by the chorda tympani (CT) nerve (Kimura and Beidler 1961; Ozeki 1971; Ozeki and Sato 1972; Sato and Beidler 1982, 1997). These results suggested that individual taste receptor cells respond to more than one stimulus representing the four basic taste qualities (sweet, sour, salty, and bitter to human). Recent experiments using a patch-clamp technique measured changes in current and voltage in response to taste stimuli from single receptor cells in an in situ preparation (Gilbertson et al. 2001). The results also demonstrated that the majority (70%) of receptor cells of rat fungiform and palate papillae responded to multiple taste stimuli.

In mice, however, the taste selectivity of individual receptor cells examined varies according to the experimental method employed and region of the tongue examined. For example, an earlier study using intracellular recordings of receptor potentials reported that most (90%) taste receptor cells of the fungiform papillae on the anterior tongue showed multiple sensitivity to four basic taste stimuli (Tonosaki and Funakoshi 1984). In contrast, molecular studies using in situ hybridization analysis demonstrated that most taste cells in taste buds on all tongue regions and palate expressing bitter receptors T2Rs do not coexpress a component of sweet and umami receptors, T1R3 (Adler et al. 2000; Nelson et al. 2001), suggesting separate cellular systems for bitter versus sweet and umami taste signaling. A subsequent Ca²⁺ imaging study using a tissue slice preparation containing circumvallate taste papilla, however, revealed that although the population of receptor cells responding to only one of the four basic stimuli in the circumvallate taste buds on the posterior tongue was much larger (60%) than that reported in intracellular recording experiment on the fungiform taste bud cells (10%), still many (40%) individual taste cells responded to multiple taste qualities with some taste cells responding to both sweet and bitter stimuli (Caicedo et al. 2002). At the nerve fiber level, previous studies demonstrated that single CT fibers innervating fungiform and foliate taste bud cells in mice showed narrower breadth of responsiveness to the four basic taste stimuli (higher response selectivity which was expressed by lower entropy value) (entropy value = 0.22) (Ninomiya et al. 1982, 1984) than that in rats (0.53–0.59) (Ogawa et al. 1968; Yamamoto et al. 1984). This breadth of responsiveness of the CT fibers in mice may fit well with that of taste cells of circumvallate taste buds from the Ca²⁺ imaging study but differ from that of the same fungiform taste bud cells obtained from the intracellular recording experiment.

It is well known that taste cells generate action potentials in response to various taste stimuli (Avenet and Lindemann 1991; Béhé et al. 1990; Cummings et al. 1993; Furue and Yoshii 1997; Gilbertson et al. 1992; Kashiwayanagi et al. 1983; Roper 1983). Recent studies suggested that action potentials, or their underlying voltage-gated Ca²⁺ and Na⁺ currents, of a subset of taste cells may be important for transmitter release at the synapse with taste nerve fibers (Béhé et al. 1990; Medler et al. 2003).

In the present study, therefore, by using a newly developed extracellular recording technique, we recorded action potentials from single taste receptor cells in response to taste stimuli applied to the apical side of the taste bud isolated from the mouse fungiform papillae and examined their response profiles to various stimuli including: NaCl, Na saccharin, HCl, and quinine-HCl as the prototypical stimuli representing four basic tastes to human. A special attempt was made to compare taste responsiveness of single fungiform taste cells with that of single CT fibers. The results demonstrated that 60% of cells responded to one of four taste stimuli. The breadth of responsiveness of mouse fungiform taste bud cells generating action potentials obtained was very similar not only to that of mouse circumvallate taste bud cells previously reported in a Ca²⁺ imaging study (Caicedo et al. 2002) but also to that of single CT fibers. The observed consistency in response characteristics of taste cells and fibers suggests that information derived from receptor cells generating action potentials may form a major component of taste information that is transmitted to gustatory nerve fibers.

	METHODS

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Isolation of single taste bud

All experimental procedures were approved by the committee for Laboratory Animal Care and Use at Kyushu University, Japan. Subjects were adult male and female C57BL/6N mice at >8 wk of age. Animals (n = 65) were anesthetized with ether and killed by cervical dislocation. The anterior part of tongues was removed and 100 µl of Tyrode solution containing 1 mg/ml elastase (Roche, Mannheim, Germany) were injected between epithelium and underlying tissue. After injection, tongues were incubated for 20 min at 26°C in Tyrode solution and bubbled with 95% O₂-5% CO₂ gas. The lingual epithelium was peeled from underlying tissue after incubation, pinned out in a silicone elastomer (Sylgard)-coated culture dish with mucosal side down and washed by Tyrode solution. Individual taste buds of fungiform papilla with a piece of epithelium were excised from the epithelial sheet and transferred to a recording chamber. The residual sheet was stored in the refrigerator for another series of experiments.

Experimental setup for recording

The schematic drawing of our experimental setup is shown in Fig. 1A. The recording chamber (bath volume: 0.7 ml) containing excised taste buds was mounted on the stage of an inverted microscope (Eclipse TE 300; Nikon, Tokyo). Two manipulators were installed on the stage of the inverted microscope, one for recording and another for the manipulation of the stimulating pipette. The stimulating pipette was pulled with a vertical puller (PE-3; Narishige, Tokyo) from a borosilicate glass capillary of 1.5 mm OD with filament (GD-1.5; Narishige). The orifice of a stimulating pipette was 4050 µm. The stimulating pipette was fitted into the microelectrode holder and a thin polyethylene tube (100150 µm ID) was introduced into the stimulating pipette for taste stimulation. A single taste bud with a piece of epithelium was drawn into the orifice of the stimulating pipette in the orientation shown (Fig. 1B). Gentle suction on the pipette was maintained by a peristaltic pump (MP-3N; Tokyo Rikakikai, Tokyo) to perfuse taste solutions and to hold the taste bud in place. Tyrode solution was always perfused inside the stimulating pipette except during the period of recording. We confirmed by visual observation of dye solution that stimulating solutions did not leak into the bath. Diffusion through the tight junction might occur, but we could not confirm this by the visual observation. The bath solution (Tyrode solution) was continuously perfused with a gravity-fed perfusion system at 2 ml/min.

View larger version (53K): [in this window] [in a new window]   FIG. 1. The experimental setup for patch recording. A: schematic drawing of our experimental setup. The mucosal side of an isolated taste bud was attached to the stimulating pipette with gentle suction. The receptor membranes of taste cells were adapted to deionized water 30 s before stimulation. Taste stimuli were applied by exchanging the solution in the stimulating pipette. The bath solution (on the basolateral side) was continuously irrigated with Tyrode solution. The recording electrode was applied to the cell with a micromanipulator from basolateral side, and electrical signals were obtained under the extracellular recording configuration. B: photograph of the tip of the stimulating pipette. Fast Green (1 mg/ml) was applied inside the stimulating pipette for visualization of the separation between bath and stimulating solution.

Tyrode solution contained (in mM) 140 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose, and 10 sodium pyruvate. The pH was adjusted to 7.40 with NaOH. Taste stimuli were the following (mM): 10300 NaCl; 130 sodium saccharin (Sac); 0.310 HCl; 120 quinine-HCl (quinine or QHCl); 300 sucrose; 100 D-phenylalanine (D-phe). As the standard taste stimuli, we used the following four taste stimuli, 0.3 M NaCl, 0.01 M HCl, 0.02 M Sac, and 0.02 M QHCl. We use Sac as sweet stimuli because viscosity of the sugar solutions such as sucrose (especially at high concentration) prohibits the smooth exchange of stimulating solution in our experimental setup. We recorded responses of each cell to the four stimuli with at least two repeated trials. The receptor membrane was rinsed with distilled water (DW) 30 s before and after taste stimulation. If the cell responded both to 0.3 M NaCl and 0.02 M Sac, we also examined responses to 0.02 M NaCl to study possible contribution of the sodium component to the Sac response. Chemicals were dissolved in distilled water (DW) and used at room temperature (25°C). All chemicals were purchased from Wako (Tokyo, Japan) and Nacalai Tesque (Kyoto, Japan).

The electrical responses of taste receptor cells in isolated taste buds were recorded from the basolateral side at room temperature (25°C). Recording electrodes were made from borosilicate glass capillaries (B150-86-10; Shutter Instrument, Novato, CA) pulled with a vertical puller and fire-polished on a microforge (MF-830; Narishige) to a resistance of 1.57 ± 0.79 (SD) M when filled with extracellular solution (Tyrode). Inner diameters of the tip of recording electrodes were 1–3 µm, which were smaller than predicted maximum diameters of single taste bud cells. Seal resistances were typically 310 times the pipette resistances (9.10 ± 5.71 M). Electrical responses of taste receptor cells were recorded by a high-impedance patch-clamp amplifier (Axopatch 200B; Axon Instruments, Foster City, CA) interfaced to a computer (Windows 98 or XP) by an A/D board (Digidata 1320A; Axon Instruments). Signals were filtered at 1 kHz, sampled at 5 kHz, and stored on the hard-disk drive on a computer by using pCLAMP software (Gap-Free mode; Axon Instruments) for later analysis.

Single-fiber recordings

Data for mouse CT nerve fibers were sampled from previous (Ninomiya 1996; Ninomiya et al. 1984, 1999) (n = 48 from 17 animals) and additional experiments (n = 57 from 16 animals) using the C57BL strain. The procedures of dissection and recording of responses of fibers were as used previously (Ninomiya 1996; Ninomiya et al. 1984, 1999). Briefly, each mouse was anesthetized with sodium pentobarbital (40–50 mg/kg ip; Abbott Laboratories, Abbott Park, IL). Under anesthesia, the trachea of each animal was cannulated, and the mouse was then fixed in the supine position with a head holder to allow dissection of the CT nerve. The hypoglossal nerve was transected bilaterally to prevent tongue movements. The right CT nerve was exposed at its exit from the lingual nerve by removal of medial pterygoid muscle. The CT nerve was then dissected free from surrounding tissues and cut at the point of its entry to the bulla. For single-fiber recording, a single fiber or a few fibers of the nerve were teased apart with a pair of needles and lifted on the electrode. An indifferent electrode was placed in nearby tissue. Neural responses resulting from taste stimulation of the tongue were fed into an amplifier (K-1; Iyodenshikogaku, Nagoya, Japan), monitored on an oscilloscope and audiomonitor, recorded on a recorder (WS-641G; Nihon-kohden, Tokyo), and stored on magnetic tape or a hard disk drive on a computer by using PowerLab (AD Instruments, Australia) for later analysis. For taste stimulation, the anterior half of the tongue was enclosed in a flow chamber made of silicone rubber (Ninomiya and Funakoshi 1981). Solutions were delivered into the chamber by gravity flow and flowed over the tongue for a controlled period (510 s). Solutions used as taste stimuli were the same as those used for recordings of taste cell responses. The tongue was rinsed for >1 min between successive stimulations. In the analysis of single fiber responses, single fibers were identified by their uniform spike height, singular wave form and intervals between contiguous spikes (Kawai et al. 2000; Ninomiya 1998).

Data analysis

For recordings from multiple taste cells, responses of single cells were segregated with the help of spike wave form analysis (Clampex 9.2; Axon Instruments). We used waveform shape parameters (time of peak-peak, peak amplitude/antipeak amplitude ratio, antipeak amplitude, peak amplitude) to segregate each single unit. Figure 2 shows one example of segregation of single units of taste cell responses. In this case, the spike size in each unit was distinct (Fig. 2B, unit 1: –24.1–86.4 pA, unit 2: –22.3–49.4 pA), but the waveform shape was similar in each unit (Fig. 2B inset). The sizes of action currents in each cell were not necessarily constant because it was reported that the amplitude of action potentials was affected by the depolarization level of taste cells (Furue and Yoshii 1997).

View larger version (23K): [in this window] [in a new window]   FIG. 2. Single-unit analysis of taste cell responses. A: sample recording of taste cell response to 0.02 M Sac. Upper part shows the detected spikes of each unit. Unit 1 responded to 0.02 M Sac. B: spike waveform analysis of sample recording in A. Each spike was plotted in this graph (X: time of peak-peak, Y: antipeak amplitude). Unit 1 () and unit 2 () were discriminated by time of peak-peak. Inset: expanded waveform of spikes.

Breadth of responsiveness of the taste cells was quantified using the following entropy equation (Smith and Travers 1979; Travers and Smith 1979)

where H is the breadth of responsiveness, K is a scaling constant (1.661 for 4 stimuli), p_i is the proportional response to each of the 4 taste stimuli, and logarithms of p_i are taken to the base 10. This entropy value (H) varies continuously from 0.0 for a cell that responds exclusively to one stimulus to 1.0 for a cell that responds equivalently to all four of the taste stimuli (Smith and Travers 1979).

Hierarchical cluster analysis was used to determine the extent to which the various response profiles fall into meaningful clusters (Ninomiya et al. 1982). The clustering program (cluster97.xls for Microsoft Excel) processed the cell and fiber profiles based on a matrix of the Pearson correlation coefficients between all possible pairs of profiles and amalgamated the cell and fiber sequentially into the cluster solution using the Ward method.

	RESULTS

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Experimental setup and spike activities of taste cells

We used isolated mouse fungiform taste buds in this study. As shown in Fig. 1B, the structure of a taste bud was identified when an isolated taste bud was held to the stimulating pipette. On holding an isolated taste bud to the stimulating pipette, the taste cells could be accessed by the recording electrode without major difficulties. By flowing dye solution (1 mg/ml fast green) in the stimulating pipette, we visually confirmed that the stimulating solution actually reached the mucosal membrane and the exchange of the solution was done immediately after the delivery of the solution to the tip of the polyethylene tube inside the stimulating pipette. In addition, this visual observation confirmed that the solution did not leak into the bath solution. In the extracellular recording, we found that some taste bud cells generated spontaneous spikes. As previously shown in our study (Yoshida et al. 2005), spike activities of taste bud cells tested were TTX-sensitive action potentials.

Response of receptor cells generating action currents

A total of 267 cells were tested for responses to at least four taste stimuli. Of these, 219 (82%) showed spontaneous activity, and 72 (27%) showed responses to taste stimuli. Sample recordings of responses of cells sensitive to single or multiple taste qualities are shown in Fig. 3. Figure 3A shows a taste cell responding only to 0.02 M Sac but not to 0.3 M NaCl, 0.01 M HCl, and 0.02 M quinine-HCl. This cell also responded to other sweet substances, 0.3 M sucrose and 0.1 M D-phe. Figure 3, B and C, shows taste cells responding to multiple taste stimuli, such as 0.3 M NaCl, 0.02 M Sac, and 0.3 M sucrose but not to 0.01 M HCl and 0.02 M quinine-HCl (Fig. 3B), and responding to 0.3 M NaCl and 0.01 M HCl but not to 0.02 M Sac and 0.02 M quinine-HCl (Fig. 3C).

View larger version (22K): [in this window] [in a new window]   FIG. 3. Sample recordings from taste receptor cells responding to apical taste stimuli. These recordings were obtained from single or multiple units in different preparations. Arrowheads indicate the onset of stimulation. Dots in B indicate spikes obtained from the same unit.

We recorded responses to the four taste stimuli from 72 mouse fungiform taste cells and compared them with those recorded from 105 CT nerve fibers. Under the criterion for occurrence of response employed (>2 SDs greater than spontaneous discharge or 3 spikes), 48 (67%) of 72 taste cells responded to one of four taste stimuli, 22 (30%) responded to two stimuli, and the remaining 2 cells (3%) responded to three stimuli. Comparably, out of 105 CT fibers, 66 fibers (63%) responded to one, 24 (23%) responded to two, 11 (10%) responded to three, and the rest (4 fibers, 4%) responded to four taste stimuli. One example of single fiber recordings from CT fiber is shown in Fig. 4. To examine the range of responsiveness more precisely, the entropy of the breadth of responsiveness of each taste cell and fibers was calculated by using the equation described in METHODS. The relative distribution of the breadth of responsiveness (H) for mouse fungiform taste receptor cells and CT nerve fibers is shown in Fig. 5. The mean entropy of the breadth of responsiveness of taste cells in mouse fungiform papillae is 0.158 ± 0.234 (average ± SD, n = 72). This value is not significantly different from that of mouse CT fibers (0.183 ± 0.262, n = 105, t-test P > 0.05). Also, the mean entropy value for fungiform taste cells categorized by best stimulus (Sac-best: 0.133 ± 0.232, n = 39, NaCl-best: 0.2 ± 0.246, n = 17, HCl-best: 0.153 ± 0.227, n = 12, QHCl-best: 0.24 ± 0.277, n = 4) is not significantly different from that for CT fibers (Sac-best: 0.09 ± 0.202, n = 44, NaCl-best: 0.183 ± 0.262, n = 27, HCl-best: 0.294 ± 0.293, n = 24, QHCl-best: 0.333 ± 0.274, n = 10, t-test P > 0.05).

View larger version (28K): [in this window] [in a new window]   FIG. 4. An example of single-fiber recordings from the chorda tympani (CT) nerve. , onset of stimulation.

View larger version (41K): [in this window] [in a new window]   FIG. 5. Relative distribution of the breadth of responsiveness of mouse fungiform taste receptor cells () and CT nerve fibers (). The breadth of responsiveness was calculated using the equation for entropy described in METHODS. The mean entropy of the breadth of responsiveness for taste cell and nerve fiber is 0.158 ± 0.234 (SD) and 0.183 ± 0.262, respectively.

View larger version (28K): [in this window] [in a new window]   FIG. 6. Cluster dendrograms showing the relationships among response profiles of mouse fungiform taste receptor cells (A) and CT fibers (B). The response profile of each cell and fiber is indicated on the left. Capital letters indicate the stimulus producing the maximum response (shown first) and all others with responses 50% of maximum. Lowercase letters indicate responses <50% of the maximum. The order of the letters indicates the relative magnitude of the response to each stimulus (S or s: Sac; N or n: NaCl; H or h: HCl, Q or q: quinine). The 4 clusters are labeled NaCl, quinine, HCl, and Sac, according to which stimulus was common to all members of the group (but not necessarily the best stimulus for every member of the group).

	DISCUSSION

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Our results demonstrated that by our response criterion (>2 SD of spontaneous impulse discharges in two repeated trials) about 6070% of taste cells responded to one of the four basic taste stimuli. This proportion of cells responding to only one of four taste stimuli in mouse fungiform taste bud cells is much larger than that obtained from intracellular recording experiments (Tonosaki and Funakoshi 1984) but is very similar to that in mouse circumvallate taste bud cells measured by a Ca²⁺ imaging (Caicedo et al. 2002). As suggested by previous reports (Avenet and Lindemann 1989; Herness and Gilbertson 1999; Kinnamon 1988; Lindemann 1996), intracellular recordings require invasive procedures that may cause cell damage. However, experimental methods employed in the present study and a Ca²⁺ -imaging study using slice preparation of tissue containing circumvallate taste papilla in the previous study (Caicedo et al. 2002) may produce cell damage to lesser extent and allow more restricted stimulus application to the apical membrane of taste cells. It is possible that our recording configuration did not necessarily record responses from only one single cell. However, even if this would be the case, the presumed incidence of single cells responding to only one stimulus should not decrease but rather increase. The relatively high proportion of taste cells responding to a limited number of taste qualities, consistently obtained from different taste cell populations (anterior fungiform vs. posterior circumvallate taste buds) and by different experimental methods (extracellular recording vs. Ca²⁺ imaging), may reveal common characteristics of taste cells in mice; although a substantial population of mouse taste cells (40%) possesses multiple taste sensitivities, and the population could increase with increasing number of stimuli employed.

Recent molecular studies using in situ hybridization analysis demonstrated that most taste bud cells expressing bitter receptors (T2Rs) do not co-express a component of sweet receptors, T1R3 (Adler et al. 2000; Nelson et al. 2001), suggesting the possibility that taste receptor cells sensitive to bitter might be different from those sensitive to sweet stimuli. In the present study, however, although the incidence is small, 2 of 39 fungiform taste bud cells that responded best to Sac also responded to quinine. Because saccharin possesses a lingering bitter aftertaste in humans and was recently shown to activate two members of human bitter receptor T2R family (hT2R43 and T2R44) (Kuhn et al. 2004), one might suppose that in mice saccharin could also activate not only sweet receptor T1R2/T1R3 heterodimers but bitter T2R receptors as well. However, it has been shown that in T1R3-KO mice responses of the CT nerve to 0.02 M Sac reduced to be 10% of control in wild type, which is lower than is the case for 0.3 M sucrose (20%) (Damak et al. 2003). This indicates that almost all Sac responses in the fungiform taste cells at this concentration may be elicited by activation of T1R3 sweet receptor component. In contrast, in the same T1R3-KO mice, Sac responses of the glossopharyngeal nerve were not largely reduced as compared with those in wild-type mice (Yasumatsu et al., unpublished observation), suggesting that a considerable amount of the Sac responses from the posterior tongue may occur by activation of receptors other than the T1R3 sweet receptor component. The previous Ca²⁺ imaging study (Caicedo et al. 2002), more reliably, revealed that 14% (22 of 160 cells) of mouse circumvallate taste cells were sensitive to both sweet (sucrose) and bitter (cycloheximide) stimuli. Therefore segregation of taste cells based on bitter versus sweet sensitivity may not necessarily be evident in the analysis of physiological responses of taste cells.

In the present study, response patterns to the four basic taste stimuli in fungiform taste bud cells were compared with those of their information-transmissible CT nerve fibers, and many similarities were found between them. For example, by examining response selectively among four taste stimuli evaluated by the entropy value of the breadth of responsiveness of taste cells and fibers (Smith and Travers 1979; Travers and Smith 1979), we found that the mean values of taste cells and fibers were not significantly different (taste cells: 0.158 ± 0.234, n = 72; CT fibers: 0.183 ± 0.262, n = 105, t-test, P > 0.05). In rats, the mean entropy value of response of fungiform taste cells recorded by the whole cell patch clamp method was 0.429 ± 0.321 (n = 43) (Gilbertson et al. 2001), which is significantly lower than that for CT fibers [0.588 ± 0.187, n = 50 (Yamamoto et al. 1984), t-test P < 0.01]. However, in the same report, they also calculated the entropy value based on the data of 13 taste cells of the fungiform papillae or the palate that possessed both Na⁺ and K⁺ currents required for generation of action potentials. The mean entropy value obtained from these cells was 0.660 ± 0.281, which is not significantly different from that for CT fibers (t-test, P > 0.05). This indicates that the range of responsiveness of taste cells generating action potentials may be close to that of innervating axons in both mice and rats. The dendrogram for both taste cells and fibers forms four major clusters labeled as NaCl, HCl, quinine, and Sac (Fig. 6). The four cell groups segregated by the cluster analysis were mostly consistent with those categorized by their best stimulus. The occurrence of each class of taste cells with different taste responsiveness to four taste stimuli was comparable with that of nerve fiber (Table 1). Only the class which responded to three tastants (NaCl, HCl, and quinine) was significantly lower in taste cells than in the fibers (Table 1). These results suggest that there may be corresponding groupings of taste cells generating action potentials and nerve fibers except classes with broad taste responsiveness.

The functional significance of action potentials in taste cells may be to affect voltage-dependent components for transmitter release such as voltage-gated Ca²⁺ channels (Béhé et al. 1990). It has been shown that in the rat fungiform taste buds a subset of receptor cells possess voltage-gated Na⁺ and Ca²⁺ currents and fire action potentials in response to sweet stimulation. A Na⁺ action potential is proposed to be required to fully activate Ca²⁺ inward currents (Béhé et al. 1990). In contrast, in the rat and mouse circumvallate taste bud cells, it has been shown that inflow of extracellular Ca²⁺ may not be absolutely necessary for transmitter release in response to bitter and sweet substances (Akabas et al. 1988; Bernhardt et al. 1996; Huang et al. 2005; Ogura and Kinnamon 1999). Moreover, in the mouse circumvallate taste cells, a subset of taste cells that contains alpha-gustducin, a G protein involved in bitter transduction, has been shown to possess only small voltage-gated inward Na⁺ and outward K⁺ currents but no voltage-gated Ca²⁺ currents (Medler et al. 2003). This evidence together with the fact that in mutant mice lacking alpha-gustducin the incidence of cells responding to bitter stimuli was reduced by 70% (which leads to decreased taste nerve and behavioral bitter responses) (Caicedo et al. 2003) suggests that there exists a component of receptor cells that may lack the ability to generate action potentials but that can nevertheless transmit taste information to the taste nerve fiber. Therefore it is probable that mouse fungiform taste bud cells showing action potentials in the present study may not contain this component of taste receptor cells if it exists in the fungiform papillae. The observation that the incidence of classes of cells that respond to multiple taste stimuli (including bitter stimuli) is lower than for CT nerve fibers (Table 1) may be partially due to the lack of this taste cell component.

The impulse frequency of mouse taste cells evoked by taste stimuli was about 5–10 times lower than that of CT nerve fibers (Table 2). This may be because single nerve fibers innervate multiple taste receptor cells. Previous anatomical studies demonstrated that each CT fiber innervates one to four taste buds in rats (Miller 1971) and an average of three taste buds in cats (Oakley 1975), and within a single taste bud, each fiber innervated an average of 1.65 taste cells for 34 nerve fibers in the mouse circumvallate papilla (Kinnamon et al. 1988). Therefore the observed lower impulse frequency of taste cells to taste stimuli than that of CT nerve fiber appears to be reasonable.

In summary, we examined response profiles of mouse fungiform taste cells that responded to taste stimuli with generating action potentials by using a newly developed extracellular recording technique and compared them with those of CT nerve fibers. We found that taste cells and CT fibers share similar characteristics in the breadth of responsiveness (response selectively) to the four taste stimuli, the grouping based on hierarchical cluster analysis, and the occurrence of each class of taste cells with different taste responsiveness to four taste stimuli (except for classes with broad taste sensitivities). These results suggest that information derived from receptor cells generating action potentials may form a major component of taste information that is transmitted to gustatory nerve fibers. In the present study, we could not examine molecular mechanisms for information transmission and possible contribution of receptor cells with no action potentials. Thus to wholly elucidate transmission of taste information from all types of taste cells to nerve fibers, further studies are needed.

	GRANTS

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This work was supported by Grant-in-Aids 18109013 and 18077004 to Y. Ninomiya and 17791325 to R. Yoshida for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

	ACKNOWLEDGMENTS

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We thank Dr. Robert F. Margolskee for valuable suggestions on the manuscript and Drs. Stephen D. Roper and David V. Smith for valuable comments on the first version of the manuscript, and Dr. Bernd Lindemann for technical advice.

	FOOTNOTES

 
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.

Address for reprint requests and other correspondence: Y. Ninomiya, Section of Oral Neuroscience, Graduate School of Dental Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (E-mail: nino{at}dent.kyushu-u.ac.jp)

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