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Elucidating coding of taste qualities with the taste modifier miraculin in the common marmoset
Brain Research Bulletin 68 (2006) 315–321
Elucidating coding of taste qualities with the taste modifier miraculin in the common marmoset Vicktoria Danilova, G¨oran Hellekant ∗ Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1655 Linden Drive, Madison, WI 53706, USA Received 2 June 2005; received in revised form 2 September 2005; accepted 10 September 2005 Available online 18 October 2005
Abstract To investigate the relationships between the activity in different types of taste fibers and the gustatory behavior in marmosets, we used the taste modifier miraculin, which in humans adds a sweet taste quality to sour stimuli. In behavioral experiments, we measured marmosets’ consumption of acids before and after tongue application of miraculin. In electrophysiological experiments responses of single taste fibers in chorda tympani and glossopharyngeal nerves were recorded before and after tongue application of miraculin. We found that after miraculin marmosets consumed acids more readily. Taste nerve recordings showed that after miraculin taste fibers which usually respond only to sweeteners, S fibers, became responsive to acids. These results further support our hypothesis that the activity in S fibers is translated into a hedonically positive behavioral response. © 2005 Elsevier Inc. All rights reserved. Keywords: Taste coding; Sweet taste; Marmoset; Non-human primates; Taste modifier; Miraculin
1. Introduction Humans as well as other primates like sweet and have no problems distinguishing sweet from bitter. The difference is evident already to newborns as shown by the striking facial expressions of neonatal babies when tasting sweet or bitter [24]. This leads to the question: what is the code in the taste nerves that never fails to give us correct information? To answer this question we have studied over the last 20 years the relationship between taste fibers and taste qualities in several non-human primates, including the common marmoset, Callithrix jacchus jacchus. It is a New World primate belonging to a family of four known genera and 15 species, found mainly in the Amazon region. Although common marmosets and humans belong to the same primate suborder, Anthropoidea (also called Simii), they represent different infraorders. Consequently common marmoset, belonging to Platyrrhina infraorder, is less related to humans than for example, macaques. C. jacchus is a small diurnal monkey, weighing less than 500 g (e.g. ∗ Corresponding author. Present address: Department of Physiology & Pharmacology, Medical School, University of Minnesota-Duluth, 1035 University Dr. Duluth, MN 55812, USA. Tel.: +1 608 262 1056; fax: +1 608 262 7420. E-mail address: [email protected] (G. Hellekant).
0361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.09.008
[17,23]). From the point of view of diet, it can be characterized as an omnivore, because it eats insects, small vertebrates, eggs, fruits and tree exudates [21]. This study is the third in a series exploring the sense of taste in C. jacchus. In the first study, we recorded from its chorda tympani nerve (CT) and glossopharyngeal (NG) nerve while we stimulated the tongue with 31 sweeteners, 5 bitter, 4 sour and 3 salty stimuli [5]. Hierarchial cluster analysis showed that the taste fibers in both nerves cluster in groups according to the human taste qualities. In agreement with other investigators, we call the fibers responding to sweet compounds, S fibers, and to bitter compounds, Q fibers. We also identified a third cluster of taste fibers responding to acids, H fibers, but found few fibers responding to NaCl, N fibers. This is noteworthy, because N fibers form a large well-defined group in most mammalian species. In our second study, we used two-bottle preference (TBP) and conditioned taste aversion (CTA) tests to investigate the relationship between behavior and single fiber activity [6]. We found that activity in the S fibers was linked to liking of the compounds and Q fiber activity to aversion. These findings agree with our previous work in other primates, chimpanzee and rhesus monkey, and suggest that in all species S fiber activity triggers intake and Q fiber activity triggers rejection.
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However, there is one further way to test this conclusion. This is offered by the taste modifier miraculin. It is a glycoprotein (Mw 22,000) from ripe berries of West African shrub Synsepalum dulcificum that was introduced to the Western World in mid-19th century. The people of native tribes have used these berries for ages to improve taste of acidic beverages and home brewed beer. Miraculin has no taste in itself. However, its presence on the tongue can be verified by tasting sour compounds, because after miraculin is applied on the human tongue, a sweet taste is added to the sourness of the acids, e.g. citric acid [18]. This change in perception is accompanied by an enhancement of the whole CT response to citric acid [9]. More than 20 years ago we recorded a change of behavior from rejection to preference and an increase of whole CT response to citric acid after miraculin in C. jacchus [13]. This suggests that miraculin exerts similar effects in C. jacchus as in humans. In this final study of taste in C. jacchus we combine intake behavior and recordings of single taste fibers before and after miraculin application. We address two questions: (i) what kind of taste fibers miraculin effects and (ii) whether a change of activity in a particular type of taste fibers is correlated with a particular intake behavior. As will be shown in the following, our results support the idea that S fibers trigger intake, or expressed in a somewhat different way, code for hedonically positive responses. 2. Methods All animals were housed in the Wisconsin Primate Research Center. The University of Wisconsin’s Animal Care and Use Committee approved all experiments performed with these marmosets.
2.1. Behavior experiments Nine naive adult marmosets of both sexes, housed as pairs, were used as subjects. They were separated only during the experiments (about 1 h). The animals were first trained to drink water from a lickometer, a device that measures consumption as a number of licks from bottles mounted in a carousel. Each lick broke an infrared beam positioned between the animal and the bottle in use and triggered one count by the computer. The first lick started a timer that limited the presentation of each stimulus to 30 s. Upon completion of training the animals drank only from the lickometer. To expose marmosets’ tongue to miraculin the animals were approached with 1 ml of 2 mg/ml miraculin in a pipette. When the animal tried to bite the pipette, small quantities of the miraculin solution were squeezed into its mouth. This allowed the delivery of 1 ml over 1 min and assured that the tongue was exposed to miraculin. The marmosets were offered water and four acids: 40 mM citric acid, 50 mM ascorbic acid, 50 mM aspartic acid and 10 mM HCl. One set of data was obtained without application of miraculin and another within 30 min after the animals were given miraculin. The two data sets were obtained during different days. Each stimulus was presented three times in random order. The consumption of stimuli was expressed as percent of water consumption. To compare the results we calculated relative differences in consumption before and after miraculin: (After − Before)/(After + Before). Thus, equal consumptions before and after miraculin would result in relative difference of 0. Two-tailed t-test for correlated data was used to assess differences in consumptions before and after miraculin. A p < 0.01 value was considered a significant difference in the statistical analysis. We also conducted TBP tests with five of these animals. Two bottles, one with water and another with 20 mM citric acid, were presented for 15 min, beginning
from the moment when the animals had tasted solutions. In these experiments the preference ratios were calculated as the amount of a test solution consumed divided by the total amount of liquid consumed.
2.2. Nerve recordings In six male common marmosets responses of single taste fibers were recorded. Anesthesia was initiated with i.m. saffan 0.9 ml/animal and maintained with 10 mg/ml sodium pentobarbital i.v. as needed. Surgery and recording Table 1 List of compounds used in electrophysiological experiments Compound
Concentrations
Salts NaCl LiCl KCl
0.1 M 0.1 M 0.1 M
Acids Citric acid Ascorbic acid Aspartic acid Hydrochloric acid
40 mM 50 mM 50 mM 10 mM
Bitter compounds QHCl Caffeine Denatonium benzoate SOA Aristolochic acid Tannic acid
5 mM 0.1 M 1 mM 1 mM 0.01 mM 2 mM
Sweeteners Sucrose Acesulfame-K Alitame Ampame Aspartame CAM CAMPA CCGA CGA Cyanosuosan Cyclamate d-Phenylalanine d-Tryptophan Dulcin Fructose MAGAP NC-00174 NC-00351 NHDHC Saccharin SC-45647 Stevioside Suosan Super-aspartame TGC Sucralose Xylitol
0.3 M 13.8 mM 0.3 mM 11.9/17.8 mM 5 mM 0.18 mM 0.028 mM 0.21 mM 0.77 mM 2.5 mM 9.9 mM 0.1 M 19.5 mM 1.59 mM 0.3 M 0.055 mM 0.23 mM 0.022 mM 0.49 mM 1.6 mM 0.12 mM 0.62 mM 1.1 mM 0.23 mM 0.17 mM 0.5 mM 0.82 M
Different concentrations of ampame we used for stimulation of the chorda tympani (CT) and glossopharyngeal (NG) nerves. QHCl, quinine hydrochloride; SOA, sucrose octaacetate; CAM, N-4-cyanophenylcarbamoyl-l-aspartyl-(R)␣-methylbenzylamine; CAMPA, N-4-cyanophenylcarbamoyl-(R,S)-3-amino3-(3,4-methylenedioxyphenyl) propionic acid; CCGA, N-4-cyanophenyl-N cyanoguanidineacetate; CGA, N-4-cyanophenyl-guanidineacetate; MAGAP, N-(S)-2-methylhexanoyl-l-glutamyl-5-amino-2-pyridinecarbonitrile; TGC, Ntrifluoroacetyl-l-glutamyl-4-aminophenylcarbonitrile.
V. Danilova, G. Hellekant / Brain Research Bulletin 68 (2006) 315–321 technique were described elsewhere [4,16]. Body temperature, heart and respiratory rates were continuously monitored. Fluid was replenished with i.v. lactated Ringer’s solution. Table 1 presents the stimuli and their concentrations. All compounds except QHCl, which for solubility reasons was dissolved in distilled water, were dissolved in artificial saliva [12]. Stimulation time was 5 s. Between stimulations the tongue was rinsed for 55 s with the artificial saliva. Responses of single fibers to all stimuli we first recorded before and then after 3 min application of 2 ml miraculin (1 mg/ml) to the receptive field of the fiber. Because miraculin’s effect lasts at least 1 h, the treatment, miraculin application, was done at the end of experiments. Although this constraint limited the number of fibers we were able to record from, it assured that we began every single fiber recording with unaffected taste buds. Recordings were obtained in both CT and NG taste fibers. Single taste fibers’ responses were recorded with a custom-made amplifier and fed into an impulse–amplitude analyzer, which had a window with adjustable upper and lower levels. A pulse to a computer was triggered when a nerve impulse exceeded the lower but not the upper level. Custom-made software controlled stimulus delivery and stored information on the presented stimulus [16].
2.3. Data analyses The measure of response was the total number of impulses during 5 s of stimulation minus the spontaneous impulses recorded during 5 s of the prestimulus period. A fiber was considered to respond to a stimulus if the nerve impulse rate was larger than two times the S.D. of the spontaneous activity of the fiber. The fibers presented here are a subpopulation of previously described fibers, which have been classified with hierarchical cluster analysis [5]. Differences between clusters’ responses before and after miraculin were assessed using ttests. Probability less than 0.05 was considered significant. To visualize similarities between different compounds we used multidimensional scaling (MDS) analysis (SYSTAT for Macintosh). MDS computes coordinates of points in a multidimensional space where each point represents a particular stimulus. As a result of MDS we have a map where the closeness between points reflects similarities between stimuli.
3. Results Fig. 1 shows the mean intake of the acids before and after mirculin application. For citric acid and ascorbic acid the rela-
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tive differences in consumption were significantly larger than 0, indicating that the animals drunk these acids more readily after they were exposed to miraculin. The animals consumed more of the aspartic and hydrochloric acids after miraculin, although the increase was not significant. Because of the limited supply of miraculin, we did not conduct TBP test for miraculin solution itself. However, we observed that marmosets had no interest in miraculin itself. Furthermore, it has no taste to humans, and rhesus monkeys show no behavioral reaction to the solution in itself. Fig. 2 illustrates the responses in one of the S fibers before and after stimulation with the four acids, NaCl, QHCl, sucrose and the high intensity sweetener NC-00174. It is evident that after miraculin all acids elicited nerve activity, whereas the response to the non-sour stimuli was unchanged. In the following we present the average responses of the S, Q and H clusters to 3 salts, 4 acids, 6 bitter and 28 sweet compounds before and after miraculin. In our previous marmoset study we found no difference between the clusters in the CT and NG. Therefore, we combined the results of the two nerves. 3.1. S clusters Fig. 3A presents the average response profile of seven S fibers before (white columns) and after miraculin (black columns). It shows that before miraculin these fibers responded only to sweeteners; salts, acids and bitter compounds did not elicit any response in these fibers. The absence of a response to CAMPA, NHDHC, cyclamate and aspartame can be explained by our earlier finding that these stimuli have no taste to marmosets; they do not stimulate their S fibers and are neither preferred in TBP tests nor rejected in CTA tests [5]. After miraculin the response profile of the S fibers was changed. The black columns in the S cluster of Fig. 3A show that citric, ascorbic and aspartic acids elicited nerve responses that were increased by at least 10 times. 3.2. Q clusters Fig. 3B presents the responses in two Q fibers to the same array of stimuli. As has been reported earlier, Q fibers respond to bitter taste. However, if a sweet compound has an additional bitter taste, as for example saccharin, then it elicits a response in Q fibers in addition to its S fiber response. Responses of these Q fibers after application of miraculin did not differ from their responses before miraculin. 3.3. H clusters
Fig. 1. Results of behavioral tests before and after miraculin in nine marmosets. Relative difference in consumption was calculated as (After − Before)/(After + Before). A value of 0 would indicate no change. The error bars show the S.E.; (*) a significant change.
Fig. 3C presents the results from two H fibers. As shown in our previous study, H fibers respond predominantly to acids. Thus, citric acid elicited responses in all H fibers, whereas aspartic and ascorbic acid stimulated fewer fibers and HCl the least. In general H fibers did not discharge during stimulation with other taste qualities. It is important that miraculin did not significantly
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Fig. 2. Recordings from the CT fiber MA97F18E belonging to the S cluster. Top panels show responses before miraculin and bottom panels show responses after miraculin. Stimuli are noted above each set of two panels. The 5 s stimulation period is between the two dashed vertical lines. After miraculin the fiber became responsive to acids, whereas the responses to the other taste qualities did not change.
change the H fibers’ responses to the compounds, especially the acids. 3.4. Multidimensional scaling of stimulus relationship Fig. 4 compares the result of MDS before and after miraculin. The distribution was calculated with the use of Pearson correlation coefficients between the stimuli across 11 fibers, 7 CT and 4 NG fibers. The left plot shows result of MDS based on data before miraculin application. A large group of sweeteners (dark circles) are positioned separately from the rest of stimuli. These sweeteners stimulated only S fibers and were preferred by marmosets. A small group of sweeteners are positioned between this group and stimuli of the other taste qualities. Some of these compounds stimulated both the Q and S fibers (saccharin, stevioside and NC-00351) or did not stimulate any fibers at all (aspartame and cyclamate). The acids (open triangles) are positioned further away from the sweeteners and closer to the salts (hatched circles) and bitter compounds (grey circles). The right plot is based on data acquired after miraculin. It shows that the acids (black triangles) have moved so that they are now positioned closer to the sweeteners than in previous plot. At the same time the positions of the salts and the bitter compounds remain far from sweeteners. 4. Discussion These data in marmosets complement and corroborate our previous primate studies in marmoset, rhesus monkey and chimpanzee [3,5–8,14,20]. Behaviorally, in all three primates we
observed that acids, which were not preferred before application of miraculin to the taste buds, were preferred after its application. Furthermore, single fiber recordings showed that after miraculin the S fibers responded to acids. Such a response was not present before miraculin. Miraculin had no effect on the responses in Q and H fibers. We have earlier suggested that S fiber activity triggers intake and Q fibers rejection. As a test of this conclusion we explored the relationship between intake and nerve responses in the Q and S fibers for bitter or/and sweet compounds. As measure of intake we used the preference ratios from the TBP tests and as measure of nerve response, the Net responses from the S and Q fibers. We define the Net response for each stimulus as: Net response = (SCT + SNG ) − (QCT + QNG ) where SCT and SNG are the average responses of the S fibers, and QCT and QNT are the average responses of Q fibers to the same stimulus. The values for bitter and sweet compounds were taken from our previous study [5] and for citric acid as described under Section 2. Fig. 5 suggests a strong correlation between preference ratios and Net nerve response. The Pearson correlation coefficient between these parameters was 0.85 (p < 0.01). Thus, intake was high for compounds that stimulate only the S fibers and low for compounds that stimulated only the Q fibers. Intake of compounds with complex taste depends on the balance between the S and Q fibers’ responses. The white triangle shows the result of citric acid before miraculin application and the black triangle after miraculin. Fig. 5 shows that after miraculin citric acid was moved toward the sweet compounds. Our interpretation is that citric acid after miraculin tasted more like a sweet compound
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Fig. 3. Effect of miraculin on average responses of the S, Q and H fibers. Data for CT and NG fibers belonging to similar clusters were combined. Only S-cluster responses were changed. The error bars show the S.E.; (*) a significant change.
than before. That is, miraculin added a sweet taste quality to the sour. We suggest that sweet taste is coded the same way in humans as it is in non-human primates. This conclusion is based on psychophysical and electrophysiological data, which we summarize in the following. Our conclusion is based on comparisons of human and non-human primate results, not only with miraculin, but also with a second taste modifier, gymnemic acid from a shrub Gymnema sylvestre [10]. It is a compound that abolishes sweet taste in humans. First, in humans it is well documented that miraculin adds sweet taste to sour compounds [1,2,19]. Monkeys and chimpanzees increase their intake of sour compounds after miraculin revealing the addition of a hedonically positive taste. A similar increase of intake of acids has been recorded with addition of sucrose to acids in monkeys [11]. Second, miraculin increased the human CT summated response to citric acid [9]. In chimpanzees and monkeys, a similar increase of summated CT responses to acids after miraculin was observed. Single fiber recordings showed that the increase originated from S fibers [14].
Third, in humans the sour taste of acids remains after miraculin [2,19]. This agrees with CT recordings in chimpanzee and marmoset, which show that miraculin did not change the H fiber response to acids [14]. Fourth, in humans gymnemic acid suppresses perceived sweetness [10] as well as the CT nerve responses to sweeteners [22,25]. This parallels the findings in chimpanzee of decreased liking of sweeteners and disappearance of the S fiber response to sweeteners after gymnemic acid [14,15]. Fifth, in humans the sweetness caused by miraculin could be removed by gymnemic acid in both psychophysical and electrophysiological experiments [1,9]. In rhesus and chimpanzee, gymnemic acid abolished both the increased CT response to acids and the increased intake of acids by miraculin [11,14]. Although no single taste fiber recordings have been published from human CT, we interpret the sweetness to acids after miraculin in humans as the result of increased activity in one type of taste fibers, the S fibers. In conclusion, we suggest that in primates attractive taste (in human sweet taste quality) is coded by S fibers.
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Fig. 4. Distribution of taste stimuli in a two-dimensional space resulting from multidimensional scaling before and after miraculin. The distribution was calculated using Pearson correlation coefficients between stimuli across 11 CT and NG fibers. Hatched symbols depict salts, grey—bitter compounds, black—sweeteners and open triangles—acids, as determined by human taste. After miraculin the acids (black triangles) were positioned closer to the sweeteners.
extend our gratitude to Dr. D. Abbott and his group at Wisconsin Regional Primate Center. This is NIH RR00167/WRPRC Publication No. 41-007. References
Fig. 5. Relationship between preference ratios in behavioral experiments and Net responses in electrophysiological experiments. The Net response was calculated as (SCT + SNG ) − (QCT + QNG ). A linear relationship exists between preference and the Net response. The Spearman correlation coefficient for these two parameters was 0.85 (p < 0.01). Hatched squares indicate bitter stimuli (they stimulated only Q fibers); grey circles—sweeteners, which stimulated both Q and S fibers, black circles—sweeteners which stimulated only S fibers, white circles—sweeteners which did not stimulate any fibers. The open triangle depicts citric acid before miraculin and the inverted black triangle—citric acid after the treatment.
Acknowledgements This research was supported in part by NIH grants R01DC6016 (GH) and R03DC005336 (VD). We would like to
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Elucidating coding of taste qualities with the taste modifier miraculin in the common marmoset Vicktoria Danilova, G¨oran Hellekant ∗ Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1655 Linden Drive, Madison, WI 53706, USA Received 2 June 2005; received in revised form 2 September 2005; accepted 10 September 2005 Available online 18 October 2005
Abstract To investigate the relationships between the activity in different types of taste fibers and the gustatory behavior in marmosets, we used the taste modifier miraculin, which in humans adds a sweet taste quality to sour stimuli. In behavioral experiments, we measured marmosets’ consumption of acids before and after tongue application of miraculin. In electrophysiological experiments responses of single taste fibers in chorda tympani and glossopharyngeal nerves were recorded before and after tongue application of miraculin. We found that after miraculin marmosets consumed acids more readily. Taste nerve recordings showed that after miraculin taste fibers which usually respond only to sweeteners, S fibers, became responsive to acids. These results further support our hypothesis that the activity in S fibers is translated into a hedonically positive behavioral response. © 2005 Elsevier Inc. All rights reserved. Keywords: Taste coding; Sweet taste; Marmoset; Non-human primates; Taste modifier; Miraculin
1. Introduction Humans as well as other primates like sweet and have no problems distinguishing sweet from bitter. The difference is evident already to newborns as shown by the striking facial expressions of neonatal babies when tasting sweet or bitter [24]. This leads to the question: what is the code in the taste nerves that never fails to give us correct information? To answer this question we have studied over the last 20 years the relationship between taste fibers and taste qualities in several non-human primates, including the common marmoset, Callithrix jacchus jacchus. It is a New World primate belonging to a family of four known genera and 15 species, found mainly in the Amazon region. Although common marmosets and humans belong to the same primate suborder, Anthropoidea (also called Simii), they represent different infraorders. Consequently common marmoset, belonging to Platyrrhina infraorder, is less related to humans than for example, macaques. C. jacchus is a small diurnal monkey, weighing less than 500 g (e.g. ∗ Corresponding author. Present address: Department of Physiology & Pharmacology, Medical School, University of Minnesota-Duluth, 1035 University Dr. Duluth, MN 55812, USA. Tel.: +1 608 262 1056; fax: +1 608 262 7420. E-mail address: [email protected] (G. Hellekant).
0361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.09.008
[17,23]). From the point of view of diet, it can be characterized as an omnivore, because it eats insects, small vertebrates, eggs, fruits and tree exudates [21]. This study is the third in a series exploring the sense of taste in C. jacchus. In the first study, we recorded from its chorda tympani nerve (CT) and glossopharyngeal (NG) nerve while we stimulated the tongue with 31 sweeteners, 5 bitter, 4 sour and 3 salty stimuli [5]. Hierarchial cluster analysis showed that the taste fibers in both nerves cluster in groups according to the human taste qualities. In agreement with other investigators, we call the fibers responding to sweet compounds, S fibers, and to bitter compounds, Q fibers. We also identified a third cluster of taste fibers responding to acids, H fibers, but found few fibers responding to NaCl, N fibers. This is noteworthy, because N fibers form a large well-defined group in most mammalian species. In our second study, we used two-bottle preference (TBP) and conditioned taste aversion (CTA) tests to investigate the relationship between behavior and single fiber activity [6]. We found that activity in the S fibers was linked to liking of the compounds and Q fiber activity to aversion. These findings agree with our previous work in other primates, chimpanzee and rhesus monkey, and suggest that in all species S fiber activity triggers intake and Q fiber activity triggers rejection.
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However, there is one further way to test this conclusion. This is offered by the taste modifier miraculin. It is a glycoprotein (Mw 22,000) from ripe berries of West African shrub Synsepalum dulcificum that was introduced to the Western World in mid-19th century. The people of native tribes have used these berries for ages to improve taste of acidic beverages and home brewed beer. Miraculin has no taste in itself. However, its presence on the tongue can be verified by tasting sour compounds, because after miraculin is applied on the human tongue, a sweet taste is added to the sourness of the acids, e.g. citric acid [18]. This change in perception is accompanied by an enhancement of the whole CT response to citric acid [9]. More than 20 years ago we recorded a change of behavior from rejection to preference and an increase of whole CT response to citric acid after miraculin in C. jacchus [13]. This suggests that miraculin exerts similar effects in C. jacchus as in humans. In this final study of taste in C. jacchus we combine intake behavior and recordings of single taste fibers before and after miraculin application. We address two questions: (i) what kind of taste fibers miraculin effects and (ii) whether a change of activity in a particular type of taste fibers is correlated with a particular intake behavior. As will be shown in the following, our results support the idea that S fibers trigger intake, or expressed in a somewhat different way, code for hedonically positive responses. 2. Methods All animals were housed in the Wisconsin Primate Research Center. The University of Wisconsin’s Animal Care and Use Committee approved all experiments performed with these marmosets.
2.1. Behavior experiments Nine naive adult marmosets of both sexes, housed as pairs, were used as subjects. They were separated only during the experiments (about 1 h). The animals were first trained to drink water from a lickometer, a device that measures consumption as a number of licks from bottles mounted in a carousel. Each lick broke an infrared beam positioned between the animal and the bottle in use and triggered one count by the computer. The first lick started a timer that limited the presentation of each stimulus to 30 s. Upon completion of training the animals drank only from the lickometer. To expose marmosets’ tongue to miraculin the animals were approached with 1 ml of 2 mg/ml miraculin in a pipette. When the animal tried to bite the pipette, small quantities of the miraculin solution were squeezed into its mouth. This allowed the delivery of 1 ml over 1 min and assured that the tongue was exposed to miraculin. The marmosets were offered water and four acids: 40 mM citric acid, 50 mM ascorbic acid, 50 mM aspartic acid and 10 mM HCl. One set of data was obtained without application of miraculin and another within 30 min after the animals were given miraculin. The two data sets were obtained during different days. Each stimulus was presented three times in random order. The consumption of stimuli was expressed as percent of water consumption. To compare the results we calculated relative differences in consumption before and after miraculin: (After − Before)/(After + Before). Thus, equal consumptions before and after miraculin would result in relative difference of 0. Two-tailed t-test for correlated data was used to assess differences in consumptions before and after miraculin. A p < 0.01 value was considered a significant difference in the statistical analysis. We also conducted TBP tests with five of these animals. Two bottles, one with water and another with 20 mM citric acid, were presented for 15 min, beginning
from the moment when the animals had tasted solutions. In these experiments the preference ratios were calculated as the amount of a test solution consumed divided by the total amount of liquid consumed.
2.2. Nerve recordings In six male common marmosets responses of single taste fibers were recorded. Anesthesia was initiated with i.m. saffan 0.9 ml/animal and maintained with 10 mg/ml sodium pentobarbital i.v. as needed. Surgery and recording Table 1 List of compounds used in electrophysiological experiments Compound
Concentrations
Salts NaCl LiCl KCl
0.1 M 0.1 M 0.1 M
Acids Citric acid Ascorbic acid Aspartic acid Hydrochloric acid
40 mM 50 mM 50 mM 10 mM
Bitter compounds QHCl Caffeine Denatonium benzoate SOA Aristolochic acid Tannic acid
5 mM 0.1 M 1 mM 1 mM 0.01 mM 2 mM
Sweeteners Sucrose Acesulfame-K Alitame Ampame Aspartame CAM CAMPA CCGA CGA Cyanosuosan Cyclamate d-Phenylalanine d-Tryptophan Dulcin Fructose MAGAP NC-00174 NC-00351 NHDHC Saccharin SC-45647 Stevioside Suosan Super-aspartame TGC Sucralose Xylitol
0.3 M 13.8 mM 0.3 mM 11.9/17.8 mM 5 mM 0.18 mM 0.028 mM 0.21 mM 0.77 mM 2.5 mM 9.9 mM 0.1 M 19.5 mM 1.59 mM 0.3 M 0.055 mM 0.23 mM 0.022 mM 0.49 mM 1.6 mM 0.12 mM 0.62 mM 1.1 mM 0.23 mM 0.17 mM 0.5 mM 0.82 M
Different concentrations of ampame we used for stimulation of the chorda tympani (CT) and glossopharyngeal (NG) nerves. QHCl, quinine hydrochloride; SOA, sucrose octaacetate; CAM, N-4-cyanophenylcarbamoyl-l-aspartyl-(R)␣-methylbenzylamine; CAMPA, N-4-cyanophenylcarbamoyl-(R,S)-3-amino3-(3,4-methylenedioxyphenyl) propionic acid; CCGA, N-4-cyanophenyl-N cyanoguanidineacetate; CGA, N-4-cyanophenyl-guanidineacetate; MAGAP, N-(S)-2-methylhexanoyl-l-glutamyl-5-amino-2-pyridinecarbonitrile; TGC, Ntrifluoroacetyl-l-glutamyl-4-aminophenylcarbonitrile.
V. Danilova, G. Hellekant / Brain Research Bulletin 68 (2006) 315–321 technique were described elsewhere [4,16]. Body temperature, heart and respiratory rates were continuously monitored. Fluid was replenished with i.v. lactated Ringer’s solution. Table 1 presents the stimuli and their concentrations. All compounds except QHCl, which for solubility reasons was dissolved in distilled water, were dissolved in artificial saliva [12]. Stimulation time was 5 s. Between stimulations the tongue was rinsed for 55 s with the artificial saliva. Responses of single fibers to all stimuli we first recorded before and then after 3 min application of 2 ml miraculin (1 mg/ml) to the receptive field of the fiber. Because miraculin’s effect lasts at least 1 h, the treatment, miraculin application, was done at the end of experiments. Although this constraint limited the number of fibers we were able to record from, it assured that we began every single fiber recording with unaffected taste buds. Recordings were obtained in both CT and NG taste fibers. Single taste fibers’ responses were recorded with a custom-made amplifier and fed into an impulse–amplitude analyzer, which had a window with adjustable upper and lower levels. A pulse to a computer was triggered when a nerve impulse exceeded the lower but not the upper level. Custom-made software controlled stimulus delivery and stored information on the presented stimulus [16].
2.3. Data analyses The measure of response was the total number of impulses during 5 s of stimulation minus the spontaneous impulses recorded during 5 s of the prestimulus period. A fiber was considered to respond to a stimulus if the nerve impulse rate was larger than two times the S.D. of the spontaneous activity of the fiber. The fibers presented here are a subpopulation of previously described fibers, which have been classified with hierarchical cluster analysis [5]. Differences between clusters’ responses before and after miraculin were assessed using ttests. Probability less than 0.05 was considered significant. To visualize similarities between different compounds we used multidimensional scaling (MDS) analysis (SYSTAT for Macintosh). MDS computes coordinates of points in a multidimensional space where each point represents a particular stimulus. As a result of MDS we have a map where the closeness between points reflects similarities between stimuli.
3. Results Fig. 1 shows the mean intake of the acids before and after mirculin application. For citric acid and ascorbic acid the rela-
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tive differences in consumption were significantly larger than 0, indicating that the animals drunk these acids more readily after they were exposed to miraculin. The animals consumed more of the aspartic and hydrochloric acids after miraculin, although the increase was not significant. Because of the limited supply of miraculin, we did not conduct TBP test for miraculin solution itself. However, we observed that marmosets had no interest in miraculin itself. Furthermore, it has no taste to humans, and rhesus monkeys show no behavioral reaction to the solution in itself. Fig. 2 illustrates the responses in one of the S fibers before and after stimulation with the four acids, NaCl, QHCl, sucrose and the high intensity sweetener NC-00174. It is evident that after miraculin all acids elicited nerve activity, whereas the response to the non-sour stimuli was unchanged. In the following we present the average responses of the S, Q and H clusters to 3 salts, 4 acids, 6 bitter and 28 sweet compounds before and after miraculin. In our previous marmoset study we found no difference between the clusters in the CT and NG. Therefore, we combined the results of the two nerves. 3.1. S clusters Fig. 3A presents the average response profile of seven S fibers before (white columns) and after miraculin (black columns). It shows that before miraculin these fibers responded only to sweeteners; salts, acids and bitter compounds did not elicit any response in these fibers. The absence of a response to CAMPA, NHDHC, cyclamate and aspartame can be explained by our earlier finding that these stimuli have no taste to marmosets; they do not stimulate their S fibers and are neither preferred in TBP tests nor rejected in CTA tests [5]. After miraculin the response profile of the S fibers was changed. The black columns in the S cluster of Fig. 3A show that citric, ascorbic and aspartic acids elicited nerve responses that were increased by at least 10 times. 3.2. Q clusters Fig. 3B presents the responses in two Q fibers to the same array of stimuli. As has been reported earlier, Q fibers respond to bitter taste. However, if a sweet compound has an additional bitter taste, as for example saccharin, then it elicits a response in Q fibers in addition to its S fiber response. Responses of these Q fibers after application of miraculin did not differ from their responses before miraculin. 3.3. H clusters
Fig. 1. Results of behavioral tests before and after miraculin in nine marmosets. Relative difference in consumption was calculated as (After − Before)/(After + Before). A value of 0 would indicate no change. The error bars show the S.E.; (*) a significant change.
Fig. 3C presents the results from two H fibers. As shown in our previous study, H fibers respond predominantly to acids. Thus, citric acid elicited responses in all H fibers, whereas aspartic and ascorbic acid stimulated fewer fibers and HCl the least. In general H fibers did not discharge during stimulation with other taste qualities. It is important that miraculin did not significantly
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Fig. 2. Recordings from the CT fiber MA97F18E belonging to the S cluster. Top panels show responses before miraculin and bottom panels show responses after miraculin. Stimuli are noted above each set of two panels. The 5 s stimulation period is between the two dashed vertical lines. After miraculin the fiber became responsive to acids, whereas the responses to the other taste qualities did not change.
change the H fibers’ responses to the compounds, especially the acids. 3.4. Multidimensional scaling of stimulus relationship Fig. 4 compares the result of MDS before and after miraculin. The distribution was calculated with the use of Pearson correlation coefficients between the stimuli across 11 fibers, 7 CT and 4 NG fibers. The left plot shows result of MDS based on data before miraculin application. A large group of sweeteners (dark circles) are positioned separately from the rest of stimuli. These sweeteners stimulated only S fibers and were preferred by marmosets. A small group of sweeteners are positioned between this group and stimuli of the other taste qualities. Some of these compounds stimulated both the Q and S fibers (saccharin, stevioside and NC-00351) or did not stimulate any fibers at all (aspartame and cyclamate). The acids (open triangles) are positioned further away from the sweeteners and closer to the salts (hatched circles) and bitter compounds (grey circles). The right plot is based on data acquired after miraculin. It shows that the acids (black triangles) have moved so that they are now positioned closer to the sweeteners than in previous plot. At the same time the positions of the salts and the bitter compounds remain far from sweeteners. 4. Discussion These data in marmosets complement and corroborate our previous primate studies in marmoset, rhesus monkey and chimpanzee [3,5–8,14,20]. Behaviorally, in all three primates we
observed that acids, which were not preferred before application of miraculin to the taste buds, were preferred after its application. Furthermore, single fiber recordings showed that after miraculin the S fibers responded to acids. Such a response was not present before miraculin. Miraculin had no effect on the responses in Q and H fibers. We have earlier suggested that S fiber activity triggers intake and Q fibers rejection. As a test of this conclusion we explored the relationship between intake and nerve responses in the Q and S fibers for bitter or/and sweet compounds. As measure of intake we used the preference ratios from the TBP tests and as measure of nerve response, the Net responses from the S and Q fibers. We define the Net response for each stimulus as: Net response = (SCT + SNG ) − (QCT + QNG ) where SCT and SNG are the average responses of the S fibers, and QCT and QNT are the average responses of Q fibers to the same stimulus. The values for bitter and sweet compounds were taken from our previous study [5] and for citric acid as described under Section 2. Fig. 5 suggests a strong correlation between preference ratios and Net nerve response. The Pearson correlation coefficient between these parameters was 0.85 (p < 0.01). Thus, intake was high for compounds that stimulate only the S fibers and low for compounds that stimulated only the Q fibers. Intake of compounds with complex taste depends on the balance between the S and Q fibers’ responses. The white triangle shows the result of citric acid before miraculin application and the black triangle after miraculin. Fig. 5 shows that after miraculin citric acid was moved toward the sweet compounds. Our interpretation is that citric acid after miraculin tasted more like a sweet compound
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Fig. 3. Effect of miraculin on average responses of the S, Q and H fibers. Data for CT and NG fibers belonging to similar clusters were combined. Only S-cluster responses were changed. The error bars show the S.E.; (*) a significant change.
than before. That is, miraculin added a sweet taste quality to the sour. We suggest that sweet taste is coded the same way in humans as it is in non-human primates. This conclusion is based on psychophysical and electrophysiological data, which we summarize in the following. Our conclusion is based on comparisons of human and non-human primate results, not only with miraculin, but also with a second taste modifier, gymnemic acid from a shrub Gymnema sylvestre [10]. It is a compound that abolishes sweet taste in humans. First, in humans it is well documented that miraculin adds sweet taste to sour compounds [1,2,19]. Monkeys and chimpanzees increase their intake of sour compounds after miraculin revealing the addition of a hedonically positive taste. A similar increase of intake of acids has been recorded with addition of sucrose to acids in monkeys [11]. Second, miraculin increased the human CT summated response to citric acid [9]. In chimpanzees and monkeys, a similar increase of summated CT responses to acids after miraculin was observed. Single fiber recordings showed that the increase originated from S fibers [14].
Third, in humans the sour taste of acids remains after miraculin [2,19]. This agrees with CT recordings in chimpanzee and marmoset, which show that miraculin did not change the H fiber response to acids [14]. Fourth, in humans gymnemic acid suppresses perceived sweetness [10] as well as the CT nerve responses to sweeteners [22,25]. This parallels the findings in chimpanzee of decreased liking of sweeteners and disappearance of the S fiber response to sweeteners after gymnemic acid [14,15]. Fifth, in humans the sweetness caused by miraculin could be removed by gymnemic acid in both psychophysical and electrophysiological experiments [1,9]. In rhesus and chimpanzee, gymnemic acid abolished both the increased CT response to acids and the increased intake of acids by miraculin [11,14]. Although no single taste fiber recordings have been published from human CT, we interpret the sweetness to acids after miraculin in humans as the result of increased activity in one type of taste fibers, the S fibers. In conclusion, we suggest that in primates attractive taste (in human sweet taste quality) is coded by S fibers.
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Fig. 4. Distribution of taste stimuli in a two-dimensional space resulting from multidimensional scaling before and after miraculin. The distribution was calculated using Pearson correlation coefficients between stimuli across 11 CT and NG fibers. Hatched symbols depict salts, grey—bitter compounds, black—sweeteners and open triangles—acids, as determined by human taste. After miraculin the acids (black triangles) were positioned closer to the sweeteners.
extend our gratitude to Dr. D. Abbott and his group at Wisconsin Regional Primate Center. This is NIH RR00167/WRPRC Publication No. 41-007. References
Fig. 5. Relationship between preference ratios in behavioral experiments and Net responses in electrophysiological experiments. The Net response was calculated as (SCT + SNG ) − (QCT + QNG ). A linear relationship exists between preference and the Net response. The Spearman correlation coefficient for these two parameters was 0.85 (p < 0.01). Hatched squares indicate bitter stimuli (they stimulated only Q fibers); grey circles—sweeteners, which stimulated both Q and S fibers, black circles—sweeteners which stimulated only S fibers, white circles—sweeteners which did not stimulate any fibers. The open triangle depicts citric acid before miraculin and the inverted black triangle—citric acid after the treatment.
Acknowledgements This research was supported in part by NIH grants R01DC6016 (GH) and R03DC005336 (VD). We would like to
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