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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Chemosens Percept. 2019 Apr 12;12:115–124. doi: 10.1007/s12078-019-09265-9

Development of a Regional Taste Test that uses Edible Circles for Stimulus Delivery

Ray A Abarintos 1, Jayvic C Jimenez 1,3, Robin M Tucker 2, Gregory Smutzer 1
PMCID: PMC6905467  NIHMSID: NIHMS1526936  PMID: 31827664

Abstract

Introduction

Measurements of chemosensory function within specific regions of the tongue can yield important information about the sensitivity of lingual areas to chemosensory stimuli, and may identify possible nerve damage. A novel regional chemosensory test that uses thin edible circles was developed for human testing.

Methods

Edible circles placed at six different regions of the tongue were used to examine regional sensitivity to quinine for bitter taste, NaCl for salt taste, sucralose for sweet taste, and capsaicin for pungency. The six regions included the anterior tip of the tongue, the left and right lateral margins of the tongue (anterior and posterior), and the circumvallate region. Testing was completed with the mouth open, and the mouth closed.

Results

Intensity ratings at all sites were higher in the closed mouth condition for the three taste stimuli. Quinine intensity was highest at the circumvallate region with the mouth closed. NaCl and sucralose intensity were highest at the anterior tip and circumvallate regions. Capsaicin intensity was most highly perceived at the anterior tip of the tongue, but open and closed mouth intensity ratings showed no significant differences.

Conclusions

Regional differences in chemosensory perception were observed on the tongue, and these differences were dependent on the chemosensory stimulus, tongue region, and tasting mode.

Implications

Edible circles show minimal diffusion with saliva, can be used to examine both taste and irritation, and may be used to identify regional papillae counts on the tongue. Finally, edible circles should be invaluable for examining damage to the oral cavity.

Keywords: regional taste test, edible circles, bitter taste, salt taste, sweet taste, capsaicin, psychophysics

Introduction

Taste perception occurs in taste buds that are located on the tongue and oral cavity. Approximately three-fourths of all taste buds are confined to the dorsal surface of the tongue in elevations known as papillae (Norton, 2007). These elevations are classified as circumvallate (CV), foliate, or fungiform papillae, along with a class of papillae that do not contain taste buds (filiform papillae) (Epstein et al., 2016). Fungiform papillae populate the anterior two-thirds of the tongue, and exhibit highest densities at the tongue tip (Eldeghaidy et al., 2018). Foliate papillae occur on the posterolateral tongue, and possess a gill-like appearance (Scully, 2013). Finally, up to twelve CV papillae are arranged in an arc at the base of the tongue (Kobayashi et al., 1994).

Taste buds serve as the anatomic location for taste receptor cells, and these modified epithelial cells form synapses with primary afferent axons from branches of the facial, glossopharyngeal, and vagus nerves (Marlow et al., 1965; Lehman et al., 1995; Mu and Sanders, 2010). The glossopharyngeal nerve innervates the oropharynx and posterior third of the tongue, the middle ear, and Eustachian tube. The chorda tympani branch of the facial nerve originates from taste buds in the anterior two-thirds of the tongue, and carries gustatory information to the brain. Finally, the vagus nerve innervates the root of the tongue, and lower epiglottis.

Taste receptor cells within fungiform papillae on the anterior tongue are innervated exclusively by the chorda tympani branch of the facial nerve. In addition, taste receptor cells of CV papillae are innervated exclusively by the lingual branch of the glossopharyngeal nerve. Finally, taste buds in the epiglottis and esophagus are innervated by the superior laryngeal branch of the vagus nerve. The human tongue and palate also detect chemosensory stimuli that activate Transient Receptor Potential (TRP) channels (Smutzer and Devassy, 2016; Simon and Guttierez, 2017). Vanilloids such as capsaicin activate TRP channels that localize to neurons of the trigeminal nerve, a nerve that supplies sensory input to the anterior two-thirds of the tongue and roof of the mouth. This spatial distribution of sensory input to the CNS has allowed researchers to examine the impact of localized sensory damage to the tongue and oral cavity by regional taste tests (Snyder et al., 2015).

Early regional taste studies by Shore (1892) and Hanig (1901), along with more recent studies by Feeney and Hayes (2014), provided evidence that the greatest sensitivity of bitter-tasting compounds occurred at the back of the tongue. For salt taste and sweet taste, the anterior tongue is markedly more sensitive than the more posterior CV region. For these two stimuli, the tip of the tongue yields greater suprathreshold values when compared to the lateral tongue (Colvin et al., 2018; Doty et al., 2001; Matsuda and Doty, 1995). Other regional taste studies indicate that taste threshold and suprathreshold sensitivities are similar on the left and right anterior tongue for most individuals (McMahon et al., 2001; Coldwell et al., 2011).

Relatively few studies have examined regional differences to capsaicin on the tongue surface. Duner-Engstrom et al. (1986) used cotton swabs to demonstrate that the anterior tongue was highly responsive to capsaicin, a region that was rich in intraepithelial substance P immunoreactive nerves. They further reported no response to capsaicin at the base of the tongue. Lawless and Stevens (1988) used 7-mm filter paper discs to demonstrate that capsaicin elicited relatively more intense responses at the tongue tip when compared to the posterior tongue, or the side of tongue. These authors also reported minimal contribution of sensation from the anterior (hard) palate.

Since an estimated 0.6 % of the US population experience taste disturbances (Hawkes, 2002), regional testing may be useful for identifying taste disturbances. These disturbances are often caused by damage to one of the four nerves that innervate the tongue (Marlow et al., 1965; Lehman et al., 1995; Mu and Sanders, 2010). These taste disturbances may be localized, and may be caused by environmental factors that include nerve damage from radiation therapy, otitis media, or head injury (Bartoshuk et al., 1996; Bartoshuk et al. 2012; Epstein et al., 2016). For example, chorda tympani nerve damage may be caused by ear infections, middle-ear procedures, jaw injuries, or dental procedures (Bartoshuk et al, 1996; Bartoshuk et al., 2005; Ozkurt et al., 2011). Unilateral chorda tympani loss may lead to increased whole-mouth perceived bitterness via increased contralateral taste sensation of the glossypharyngeal nerve (Kveton and Bartoshuk, 1994). These studies suggest that deficits, enhancements, or confusions in taste or irritant perception may occur (Coldwell et al., 2014). Thus, a thorough gustatory assessment should include regional testing for examining possible nerve damage as an underlying cause of chemosensory disturbances.

Regional taste tests are conducted by applying chemosensory stimuli to the tongue by wire loops (Nilsson, 1979), aqueous solutions (Delwiche et al., 1999), glass stimulators (Hebhardt et al., 1999), moistened cotton swabs (Feeney and Hayes, 2014; Colvin et al., 2018), impregnated filter paper discs (Satoh-Kuriwada et al. 2014; Manzi and Hummel, 2014), or by electrogustometry (Doty et al. 2016). In most cases, these measurements are recorded while the mouth remains open. A regional test that examines chemosensory perception with both the mouth open and closed allows the possibility of examining chemosensory responses under different conditions. For example, a mouth closed condition may more closely mimic taste perception when food is consumed (Colvin et al., 2018). In this pilot study, a novel method for spatial testing is described that utilizes thin, rapidly dissolving edible circles as vehicles for delivering chemosensory stimuli to the tongue. Regional studies with three primary taste stimuli (quinine HCl, NaCl, and sucralose) and a representative trigeminal stimulus (capsaicin) are conducted at six different regions of the human tongue. In this study, intensity measurements were recorded under both open and closed mouth conditions.

Materials and Methods

Preparation of Edible Circles for Regional Studies

Pullulan-based edible films were prepared as previously described (Smutzer et al., 2008). Briefly, pullulan (α−1,4-; α−1,6-glucan; NutriScience Innovations, LLC, Trumbull, CT) was combined with the polymer hydroxypropyl-methylcellulose (Dow Chemical Co., Midland, MI) at a weight ratio of 11.5:1, and a final polymer concentration of 3.0% (w/v). Blue food coloring (McCormick & Co., Hunt Valley, MD) was obtained from a local supermarket, and was added to aid in visualizing edible circles (0.02% v/v). Water was obtained from Deer Park (Stamford, CT). Stock solutions of capsaicin (Pfaltz and Bauer, Waterbury, CT) were prepared in 95% ethanol, and directly used or stored at −80 °C before use. Quinine HCl (anhydrous) was obtained from Sigma-Aldrich (St. Louis, MO), NaCl was obtained from Fisher Scientific (Hampton, NH), and sucralose was obtained from Tate & Lyle (McIntosh, AL). The three taste stimuli were added in powder form to the polymer solution, and fully dissolved before the solution was poured.

For production of edible films, polymer solutions that contained capsaicin were cast onto 7.6 × 8.3 cm surfaces while quinine HCl, NaCl, sucralose, and control films were cast onto 8.3 × 10.1 cm surfaces, and allowed to dry for 12 to 18 hours at RT in the dark (Smutzer et al., 2008). This drying procedure caused the evaporation of 95% ethanol that was previously used to dissolve capsaicin. After drying, the clear films were removed with tweezers. Edible circles were then prepared by placing aluminum foil on both sides of the film, and detaching 5/16 inch (0.79 cm) diameter circles from the film with a McGill hole punch (Advantus Corp., Jacksonville, FL). The area of each edible circle was 0.50 cm2 (0.077 square inches), and each circle corresponded to a volume of 41 μliters of the polymer solution that was used to pour the film. After removal, edible circles were placed in 35 mm cell culture dishes that were lined with filter paper, and stored in the dark at 4° C or at −10° C in a plastic sealable bag for no more than one month. Control (blank) circles were prepared as described above except that no chemosensory stimulus was added.

The stimulus content for quinine HCl was 125.0 nmoles, NaCl was 4.10 μmoles, sucralose was 500.0 nmoles, and capsaicin was 0.54 nmoles. The amount of NaCl in edible circles represented the maximal amount of NaCl that could be incorporated into edible films that were used to prepare circles. Along with controls, these edible circles were used to examine the perceived intensities of each stimulus at six different regions on the tongue.

Participants

A total of 88 healthy adults (39 female and 49 male, mean age was 22.9 ±0.8 years) participated in this regional study with oral stimuli. This population included 51.7% Asian subjects, 31.8% Caucasian subjects, 14.2% Black/African-American subjects, and 2.3% Hispanic subjects. One test subject reported zero intensity values at all data points, and was not included in the data analysis. An additional four subjects participated in the study involving edible circle diffusion. All 88 participants were pre-screened by asking a series of questions to determine whether the subject had normal taste function. All participants refrained from eating or drinking for 30 minutes prior to the start of the study. For the capsaicin substudy, subjects refrained from consuming capsaicin-containing foods for 24 hours before the test date. Eleven of the participants completed two studies, and three participants completed three chemosensory studies. For participants in multiple sub-studies, only one chemosensory stimulus was examined at a testing session, and only one testing session occurred on a single day. Each testing session lasted from 25 to 30 minutes.

Study participants were recruited through flyers and by word of mouth. The study protocol was approved by our university Institutional Review Board, and all study participants provided written informed consent. Participants were reimbursed for their time.

Chemosensory Studies

The general Labeled Magnitude Scale (gLMS) was used for all intensity measurements (Bartoshuk et al., 2004). This higher order polynomial scale contains intensity labels for no sensation (0.0), barely detectable (1.4), weak (6.0), moderate (17.0), strong (34.7), very strong (52.5), and strongest imaginable sensation of any kind (100.0). All subjects were trained in the use of the gLMS by asking them to rate the perceived intensities of actual, imagined, or remembered sensations that included both gustatory and non-gustatory stimuli (Smutzer et al., 2018).

The spacing of the four lateral regions of the tongue were carried out according to Doty and coworkers (2001), and the medial CV region was examined according to Green and Hayes (2004). For chemosensory studies, edible circles were placed on the surface of the tongue in one of six locations with sterile forceps (Fisher Scientific) by the test administrator (Figure 1). These six regions included the tongue tip, right and left lateral margins of the tongue 1.7 cm from the tongue tip, the left and right lateral margins 3.4 cm from the tongue tip, and the medial CV region at the back of the tongue. Each trial consisted of two edible circles. One edible circle contained a stimulus, and one edible circle contained no stimulus (control circle). The presentation of the two circles for each trial was randomized.

Figure 1. Regional testing locations and properties of edible circles.

Figure 1.

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A. This figure shows the six regions on the tongue surface that were examined (filled circles). Short black arrow represents a length of 1.7 cm, and longer black arrow represents a length of 3.4 cm. Small open circles at base of tongue represent CV papillae. (This figure is not to scale). B. This image represents a 5/16 inch diameter control circle that contained 0.02% blue food color. Each mark on the upper edge of the ruler represents 1/16 inch.

Edible circles were presented to subjects in a pseudorandom order at the different regions of the tongue (see Figure 1A). However, we found that it was more convenient to test the CV region last because this region was at the base of the tongue. A water rinse was used following the presentation of each taste circle. The interval between trials was approximately two minutes, or until no residual taste remained in the oral cavity.

Intensities at the six tongue sites were measured by two different sampling modes. These modes were similar to the approach used by Colvin et al. (2018). One mode occurred with the mouth continuously open for 15 seconds (passive, or open condition). A second mode occurred with the mouth open for 7 seconds, and then closed for the remaining 8 seconds (closed condition, or active pressing mode). For the closed mouth condition, each participant was instructed to touch the roof of his or her mouth without moving their tongue laterally. After 15 seconds, participants were asked to report a gLMS intensity for each edible circle. The open mouth condition permitted intensity measurements with little or no nasal airflow, and the closed mouth condition allowed measurements in the presence of nasal airflow.

The dissolving time for edible circles occurred by direct observation of the time for the circle to completely dissolve on the tongue. For spreading analysis (diffusion), one edible circle contained 0.1% blue food coloring. A second circle (5/16” diameter Whatman No. 1 filter paper disc (Fisher Scientific)) served as a control. The two discs were placed flatly on the tongue adjacent to each other. The tongue was photographed after 15 seconds, and after 1 minute with the mouth remaining open. ImageJ (Version 1.52, NIH) was used for size estimations. For imaging studies, a yellow dot was visually placed in the center of each image of a blue or white circle, and four diameter lines were drawn through the center dot. The four diameter lengths were then averaged for each circle to directly compare the mean diameters of the circles at the two time points.

Statistical Analysis

SPSS (version 24, IBM Corporation, Armonk, NY) and Microsoft Excel (version 15.32, Microsoft, Redmond, WA) were used to analyze data that was obtained from psychophysical studies. Intensity ratings are presented as means ± standard deviations (SD). Since edible circles may cause a mild tactile response, data were standardized by subtracting control strip intensity ratings from the stimulus ratings. Repeated measures analysis of variance with Bonferroni correction for multiple comparisons was used to examine differences in intensity ratings between the six tongue sites. Paired t-tests with Holm-Bonferroni correction for multiple comparisons examined differences between open versus closed mouth conditions.

Results

Pullulan-based edible circles that contained quinine HCl, NaCl, sucralose, capsaicin, or no stimulus were successfully prepared with a 5/16” (0.79 cm) in diameter single hole punch from 0.030 mm-thick films. These circles were used for subsequent chemosensory studies. Figure 1B is a gray scale image of a control (blank) circle that identifies the size and shape of these edible circles.

The mean gLMS intensity (on a scale of 0 – 100) for control (blank) circles used in all four substudies was in the “barely detectable” range (gLMS = 1.32 ±0.32, n = 930 circles). Supplemental Figure 1 is a color image of an edible circle that was placed near the anterior tip of the tongue for dissolution studies.

The dissolving time for 3.0% edible circles with no taste stimulus was 3.5 ±0.5 seconds (n = 4). Image analysis of edible circles on the tongue surface exhibited almost no diffusion across the surface of the tongue. After 15 seconds, the ratio of the diameter of edible circles to filter paper was 1.04 ±0.02 (open mouth condition). After 60 seconds, the ratio of the two diameters was 0.99 ±0.04 (n = 4). These results indicate that edible discs showed almost no spreading when placed on the tongue surface during the time that stimulus intensities were recorded.

Table 1 summarizes the mean intensities and SD for all four chemosensory stimuli at six different tongue locations for both the open mouth and closed mouth conditions. For taste stimuli, quinine HCl showed the highest mean intensity in the CV region with the mouth closed, and highest intensity in the anterior tip with the mouth open. NaCl and sucralose also yielded the greatest gLMS intensity values at the tip of the tongue for both mouth conditions. In contrast, capsaicin yielded highest intensity scores at the tip of the tongue for both mouth conditions, with lowest intensity scores in the CV region. As opposed to the three taste stimuli, mouth condition (open vs. closed) did not significantly affect intensity ratings for capsaicin.

Table 1.

Mean gLMS intensity ratings by stimulus and condition (open mouth vs. closed mouth)

Quinine NaCl Sucralose Capsaicin
Open Closed Open Closed Open Closed Open Closed
Anterior 11.4 ± 9.7 17.6 ± 10.4 8.9 ± 8.3 12.9 ± 9.0 6.3 ± 9.4 18.7 ± 14.2 16.7 ± 10.2 17.3 ± 12.1
Left anterior 7.6 ± 9.2 13.1 ± 8.9 4.1 ± 5.4 7.8 ± 5.2 2.2 ± 3.0 10.9 ± 10.5 5.1 ± 6.2 5.0 ± 7.6
Left posterior 6.6 ± 6.3 14.3 ± 12.7 2.8 ± 3.4 6.4 ± 6.5 2.8 ± 4.2 8.0 ± 7.3 2.2 ± 3.4 3.6 ± 5.4
Right anterior 6.9 ± 7.2 12.2 ± 11.0 5.9 ± 6.6 8.6 ± 5.4 2.3 ± 3.3 11.8 ± 10.7 4.3 ± 4.3 3.5 ± 4.5
Right posterior 8.2 ± 9.0 14.6 ± 16.0 4.2 ± 6.6 9.1 ± 10.4 1.4 ± 3.2 9.2 ± 8.9 3.2 ± 6.0 2.5 ± 3.3
Circumvallate 9.9 ± 10.6 20.6 ± 19.2 7.8 ± 8.4 11.6 ± 9.3 2.3 ± 5.9 12.9 ± 10.9 1.0 ± 3.4 2.9 ± 4.8
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Regional Taste Studies with the Bitter Taste Stimulus Quinine

For our study population, mean gLMS intensity scores for quinine were in the moderate range for all six tongue regions for both the mouth open and mouth closed conditions. As shown in Figure 2, the four lateral regions of the tongue yielded mean intensities that were slightly lower than those observed in both the CV region and tip of the tongue. However, none of these six regions of the tongue were statistically significant.

Figure 2.

Figure 2.

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Mean gLMS intensity values for quinine by mouth condition (mouth open or mouth closed) at six different locations on the tongue (n = 30). Gray columns represent open mouth condition, and black columns represent closed mouth condition. Asterisks identify regions where quinine intensity was rated as significantly more intense in the closed mouth condition (p ≤ 0.025 for all regions). Error bars represent standard deviation.

When intensity ratings at each of the six regions were compared for the open mouth and closed mouth conditions, the intensity of quinine was rated more intense in the closed mouth condition at the tongue tip (p = 0.018), left anterior (p = 0.005), and right anterior (p = 0.025) regions (Figure 2). Although mean intensities were higher in the mouth closed condition for all six regions, no statistical differences were observed between the left and right posterior sites when intensity values for the two mouth conditions were compared.

As a reliability check, intensity ratings from the left and right sides of the tongue for the two mouth conditions for quinine were compared by Pearson correlation coefficient analysis. All four correlations were positive, but the closed mouth condition yielded stronger correlations between the left and right sides of the tongue (see Table II). Similar results were observed for salt taste, sweet taste, and the pungent sensation of capsaicin.

Table 2.

Correlations between ratings by location and condition

Stimulus Anterior Open P Anterior Closed P Posterior Open P Posterior Closed P
Quinine 0.326 NS 0.658 <0.001 0.564 0.001 0.762 <0.001
NaCl 0.734 <0.001 0.498 0.008 0.143 NS 0.494 0.009
Sucralose 0.136 NS 0.797 <0.001 0.628 0.007 0.655 0.004
Capsaicin −0.084 NS −0.011 NS 0.125 NS 0.406 0.026
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Correlation coefficients and p-values for right and left side ratings for anterior and posterior locations under open and closed mouth conditions. Correlations between right and left side ratings were more consistent under the closed mouth condition.

Regional Taste Studies with the Salt Taste Stimulus Sodium Chloride

For our study population, mean gLMS intensity ratings were in the weak to moderate range, with maximal intensity at the tongue tip in both the mouth open and mouth closed conditions (Figure 3). The four lateral tongue regions yielded NaCl ratings as weak for both mouth conditions. NaCl intensity was rated as significantly more intense at the tongue tip when compared to both the left anterior (p = 0.045) and left posterior (p = 0.026) regions in the open mouth condition. In the closed mouth condition, NaCl was rated as significantly more intense at the tongue tip when compared to the left anterior (p = 0.033) and left posterior (p = 0.019) regions. Participants rated this stimulus as more intense at the CV region when compared to the left posterior tongue region in the closed mouth condition (p = 0.029). None of the other sites differed statistically from each other.

Figure 3.

Figure 3.

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Mean gLMS intensity values for NaCl by mouth condition at six locations on the tongue (n = 27). Gray columns represent open mouth condition, and black columns represent closed mouth condition. Asterisks identify regions where NaCl intensity was rated as significantly more intense in the closed mouth condition (p ≤ 0.025 for all regions). Error bars represent standard deviation.

NaCl was rated as significantly more intense at the tongue tip when compared to the left anterior and left posterior sites in both the open mouth (p ≤ 0.045 for both) and closed mouth conditions (p ≤ 0.033 for both). Participants rated the stimulus as more intense at the CV region when compared to the left posterior region in the closed condition (p = 0.029). For all six regions of the tongue, a higher mean intensity rating for NaCl was observed with the mouth closed condition, when the tongue came in contact with the hard palate (p ≤ 0.049 for all regions).

Regional Taste Studies with the Sweet Taste Stimulus Sucralose

Mean gLMS intensity ratings were in the weak to moderate range for all six tongue regions. Maximal intensity was identified at the tongue tip for both mouth conditions. No significant differences in intensity by region were observed in the open mouth condition (Figure 4, gray columns). For the closed mouth condition, intensity was rated higher in the tongue tip when compared to the left posterior region (p = 0.035) (Figure 4, black columns). None of the other sites differed statistically from each other. When comparing across mouth conditions, differences were observed in all six regions. Intensity was rated lower at each site in the open condition compared to the closed (p ≤ 0.008 for all regions) (Figure 4).

Figure 4.

Figure 4.

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Mean gLMS intensity values for sucralose by mouth condition at six locations on the tongue (n = 17). Gray columns represent open mouth condition, and black columns represent closed mouth condition. Asterisks identify regions where sucralose intensity was rated as significantly more intense in the closed mouth condition (p ≤ 0.025 for all regions). Error bars represent standard deviation.

Regional Studies with the Trigeminal Stimulus Capsaicin

The regional study with capsaicin yielded a different sensitivity profile when compared to the three taste stimuli. As shown in Figure 5, mean intensity ratings for capsaicin were rated as significantly more intense at the anterior tip of the tongue when compared to the other five regions. This maximal intensity was observed both the open mouth (p ≤ 0.002 for all regions), and closed mouth condition (p ≤ 0.001 for all regions). Mean intensities at the anterior tip of the tongue fell into the “moderate” range of the gLMS while mean intensities at the lateral regions were primarily reported as “weak”. Finally, the CV region yielded almost no response to capsaicin (barely detectable) with the mouth open. However, mean intensities for capsaicin slightly increased in the CV region in the closed mouth position. As opposed to results with the three taste stimuli, none of the six tongue regions statistically differed from each other in either of the two mouth conditions. Notably, mean intensity ratings at the tip of the tongue for the open and closed mouth conditions were nearly identical (see Figure 5).

Figure 5.

Figure 5.

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Mean gLMS intensity values for capsaicin by mouth condition at six locations on the tongue surface. Gray columns represent open mouth condition, and black columns represent closed mouth condition (n = 30). No statistical differences in mean intensity values were observed between the open mouth and closed mouth condition at the six locations (p ≤ 0.025). Error bars represent standard deviation.

Evaluation of Results for Mouth Condition and the Two Classes of Chemosensory Stimuli

For the three primary taste qualities, mean gLMS intensities were greatest at the tip of the tongue for salt and sweet taste (both mouth conditions). However, maximal quinine intensity was dependent on mouth condition where only the open mouth condition showed maximal intensities at the tongue tip. In contrast, capsaicin showed highest intensities at the tip of the tongue, with minimal intensity values at the CV region. Finally, taste-intensity ratings on the left and right side of the tongue were correlated for the three taste qualities.

The regional study involved two different classes of chemosensory stimuli (taste stimuli and a trigeminal stimulus), and allowed a direct comparison of intensity ratings for closed and open mouth condition for all four stimuli. Figure 6 identifies the gLMS intensity ratios for each of the four chemosensory stimuli when closed (active pressing) and open (passive) mouth positions were compared for all six regions. The mean intensities in the closed mouth condition at all six regions were higher for all stimuli, with intensity ratios greater than one. Of the three taste stimuli, mean intensity ratios were greatest for sucralose, and lowest for capsaicin.

Figure 6.

Figure 6.

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Combined mean intensity ratios for all six tongue regions for NaCl, quinine HCl, sucralose, and capsaicin. The gLMS intensity ratio (mean closed mouth intensity for all six regions divided by mean open mouth intensity for all six regions) is shown for each of the four stimuli.

Discussion

Since the first studies by Shore in 1892, a variety of methods have delivered chemosensory stimuli to different regions of the tongue. This study describes a new tool for regional testing of chemosensory stimuli in the oral cavity. In this report, rapidly dissolving edible circles were successfully used as a vehicle for administering precise amounts of chemosensory stimuli to specific regions of the tongue surface. By using circles that contained food coloring, this study confirmed that edible circles remained localized to the placement area on the tongue, and underwent essentially no spreading from this location.

The amount of quinine in edible circles was similar to the amount that was used in previous suprathreshold studies with one-inch square strips (125 nmoles for circles vs. 148 nmoles for one-inch square strips) (Smutzer et al., 2013). Although edible circles covered one-thirteenth the surface area of the tongue when compared to edible strips, both circles and strips yielded roughly similar intensity ratings (moderate intensity range). Experiments that vary the surface area of edible films but maintain the same stimulus amount for examining specific regions of the tongue may prove useful for examining receptor responses and taste coding at the peripheral level (Roper and Chaudhari, 2017).

Minimal intensity responses were observed for capsaicin in the CV region of the tongue. The absence of trigeminal innervation in this region of the tongue is the most likely explanation for these results. Although controversial (reviewed by Roper, 2014), immunostaining studies suggest that mammalian taste receptor cells in CV papillae contain TRPV1 receptors (Moon et al., 2010). Our psychophysical study argues against a significant activation of TRP receptors in the CV region. Nonetheless, these circles may not have contained sufficient amounts of capsaicin to stimulate a noticeable chemosensory response in CV papillae (Green and Hayes, 2003).

Although intensities were low, capsaicin showed a small increase in intensity in the CV region with the mouth closed. This increase could be due to a swallowing reflex in some individuals after mouth closure. This reflex would allow capsaicin to come in contact with the soft palate, a region that is innervated by both the glossopharyngeal and vagus nerves (Rentmeister-Bryant and Green, 1997). This higher sensitivity of the pharyngeal mucosa to capsaicin could in turn explain the small increase in perception of capsaicin in this region (Rentmeister-Bryant and Green, 1997) with the mouth closed.

When averaged over all six regions of the tongue, the closed mouth condition increased mean intensities for all four stimuli. Although the open mouth condition was tested first, the variability in closed to open mouth ratios for the four different stimuli argues against increased familiarity with the stimulus in the mouth closed condition. Possible explanations for increased intensity of the three taste stimuli in the mouth closed condition might include the transfer of some stimulus from the tongue to the hard palate. This transfer could increase the area of chemosensory stimulation in the oral cavity, and increase perceived intensity due to spatial summation (Smith 1971; Delwiche et al. 2001; Colvin et al., 2018). In contrast, the small increase in closed mouth to open mouth ratio for capsaicin may relate to decreased volatility of capsaicin when compared to taste stimuli (Rozin, 1982), or lower TRP receptor densities (or lower activation of TRP receptors) on the palate.

In addition to examining possible nerve damage to the tongue, edible circles should be valuable tools for identifying peripheral and CNS suppression of chemosensory stimuli in the oral cavity. By including mixtures in edible circles, this approach may identify partition ratios of central to peripheral nervous system suppression. In particular, split-tongue studies could be useful for examining capsaicin suppression by sweet taste stimuli in order to determine if this suppression is primarily peripheral, central, on both central and peripheral in origin (Kroeze and Bartoshuk, 1985; Smutzer et al., 2018).

Edible circles may also be useful for identifying secondary responses to trigeminal stimuli in the CV region. For example, capsaicin can both stimulate and desensitize bitter sensation in some individuals when this irritant is applied to CV papillae (Green and Hayes, 2003). Other evidence suggests that capsaicin elicits a salt taste (Lyall et al., 2004). The potential to selectively examine individual CV papillae with edible circles may help to extend these results.

Several limitations of this study should be considered. Edible circles may cause a small tactile response in some individuals, which could slightly increase gLMS intensity scores. For this study, ratings for control circles were subtracted in order to account for this possibility. Only one rating was obtained for each stimulus at each location. However, Feeney and Hayes (2014) reported stability for regional intensity ratings over several days. Another limitation of our study is that diet could affect capsaicin perception in the human oral cavity (Naswari and Pangborn, 1990). Currently, no evidence suggests that a diet rich in capsaicin could show regional differences on the tongue surface. However, diet could affect overall intensity ratings for individual subjects.

Our results indicate that edible circles represent a promising delivery method for examining both taste and trigeminal stimuli on a regional basis in the oral cavity. These edible circles are easy to prepare, and are easy to administer to participants. These edible circles can target a precise location on the tongue with minimal spreading of stimulus. The inclusion of food dye in edible circles allows the potential to count papillae in specific regions of the tongue during regional chemosensory studies. In addition, these circles can be prepared in a variety of sizes, shapes, and thicknesses for detecting a chemosensory stimulus in the oral cavity. Notably, these circles have the potential to examine CV papillae responses without inducing a gag reflex that may occur with liquid stimuli (Green and Hayes, 2004). Also, this approach should be useful for fMRI studies involving irritant and taste stimuli (Kawakami et al., 2016). Finally, this approach shows promise for examining regional taste differences in pediatric populations and aged individuals where the fear of choking is an important concern.

In summary, our results identify regional differences in chemosensory responsiveness between different regions of the tongue surface, and that regional responsiveness is dependent on both the stimulus and the mouth condition during chemosensory testing. These results indicate that edible circles represent a promising delivery method for examining chemosensory responses at defined regions of the tongue surface. The amount of stimulus in these circles can be modified for both threshold and suprathreshold studies, and should be useful for clinical studies of chemosensory function. Future studies will include regional analysis of sour and savory stimuli in order to further extend the versatility of edible circles in psychophysical research.

Supplementary Material

12078_2019_9265_MOESM1_ESM

Acknowledgements

This work was supported by NIDCD R44 DC007291, and funded in part by the URP program at Temple University. The authors thank Dow Chemical Co. for the hydroxypropyl methylcellulose, and Jacqueline Tanaka, Edward Gruberg, and Craig Brumwell for valuable discussions. An earlier version of this work was presented as an abstract at the 17th International Symposium of Olfaction and Taste (ISOT 2012).

Footnotes

Declaration of Conflict of Interests

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Compliance with Ethical Standards.

All protocols performed in this study were in accordance with ethical standards of the sponsoring university’s institutional review board. Informed consent was obtained from all subjects who participated in this study.

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