Bitter taste in single chorda tympani taste fibers from chimpanzee

Physiology & Behavior, Vol. 56, No. 6, pp. 1185-1188, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0031-9384/94 $6.00 + .00 Pergamon 0031-9384(94)00266-5 Bitter Taste in Single Chorda Tympani Taste Fibers From Chimpanzee GORAN HELLEKANT' AND YUZO NINOMIYA University of Wisconsin and Wisconsin Regional Primate Center, Madison, WI USA, and Asahi University, Gifu Prefecture, Japan HELLEKANT, G. AND Y. NINOMIYA. Bitter taste in single chorda tympani tastefibersfrom chimpanzee. PHYSIOL BEHAV 56(6) 1185-! 188, 1994.--We have found earlier that chimpanzee chorda tympani taste fibers fall into groups that conform with the human taste qualities. This study focuses on bitter taste and its relation to sweet taste. Eight fibers were classified as bitter fibers according to their responses to 31 stimuli. The stimuli included the bitter compounds quinine, denatonium benzoate and caffeine. The results indicate a clear dichotomy between the bitter and sweet fibers. Sweet fibers never responded to the bitter compounds. However, in addition to their responses to the above compounds, some of the bitter fibers were stimulated by other compounds. Most prominent were responses to NaCl-amiloride mixture, KC! and xylitol. In most cases the cause could be assumed to be a bitter taste in the compound. These results suggest that the bitter and sweet tastes are conveyed in specific and separate groups of nerve fibers in the chimpanzee. Because of the closeness between chimpanzee and human, this finding has implications on the question of taste coding in human and the concept of taste qualities. Chimpanzee Primate Taste Bitter Sweet Chorda tympani INTRODUCTION Coding Surgery RECENT studies indicate that the chimpanzee (Pan troglodytes) is more closely related to h u m a n s than any other species (6). This is also reflected in the peripheral sense of taste, which we have not found to be different from that of the h u m a n (2,3,5,8). For questions related to h u m a n taste this makes the chimpanzee a superior animal model. Earlier studies show (3,5,8) that taste fibers in the chimpanzee group distinctly in categories congruent with the h u m a n taste qualities. For bitter taste, this infers that taste fibers responding to bitter compounds carry the quality o f bitter taste. As a consequence, fibers classified in the bitter taste quality should mainly respond to bitter compounds. Their response spectrum should not overlap with that o f fibers responding to other taste qualities, especially the sweet quality. This study will present data obtained in the chimpanzee aimed at this question. W e present single fiber recordings from the chorda tympani proper nerve on bitter taste together with representative excerpts o f our data for the sweet and salty qualities. The term proper is used to distinguish the chorda that supplies the mandibular and sublingual salivary glands. A more extensive analysis o f the sweet and salty fiber data will be published elsewhere. The chimpanzees were intubated after an i.m. injection o f ketamine, 400 mg/animal, and atropine, 0.5 mg/animal. They were then maintained on a mixture of oxygen, halothane and NzO2. Fluid losses were replenished with IV 5% dextrose and lactated Ringer's solution. The right chorda tympani proper nerve, CT, was exposed through an incision along the mandibular angle between the rostral lobes of the parotid gland and the mandibular bone. First the tissue attached to the mandibular angle was sectioned. Then blunt dissection was used to follow the caudo-medial side of the pterygoid muscle in the direction of its origin at the pterygoid plate of the skull that contains taste fibers from the one to the C T nerve. The nerve forms generally one bundle although there were exceptions. The nerve is surrounded by small veins. After the experiment, deeper layer was sutured with adsorbable suture while the skin was closed with nylon. The nylon sutures were removed after 7 - 1 0 days. Apparatus The nerve impulse activity was recorded with a P A R 113 amplifier, monitored over a loudspeaker and an oscilloscope, and fed into a recorder (Gould ES 1000) and an I B M P C - A T computer via a DAS-Keithley interface. Our o w n software controlled both stimulating and recording equipment. METHODS Animals The data were obtained from 8 adult chimpanzees lodytes) weighing 35 to 75 kg Single fiber Stimulation (Pan trog- A taste stimulation system was used that delivers 32 different solutions at given intervals and under conditions of constant flow ' Requests for reprints should be addressed to G. Hellekant, 1655 Linden Dr., Madison, WI 53706 USA. 1185 1186 HELLEKANT AND NINOMIYA Chimp 90/91, Main stimuli, All bitter and selected salt. sweet fibers .,,la" FIG. 1. An overview of the results of the single fiber recording in the chimpanzee as a 3-dimensional graph. Each column illustrates the response to one stimulus in one fiber with the spontaneous activity before each stimulation deducted. The stimuli were arranged in the order of salt, bitter and sweet along the x axis, while the fibers were arranged along the z axis in groups, starting with the Na-best group, followed by bitter and finally the sweet group. To facilitate the overview, the fibers within each group have been arranged so the fibers with the highest activity are furthest away from the observer. and temperature (33°(2) on the tongue. Each stimulation lasted for 8 - 1 0 s. Between the stimulations, the tongue was rinsed for 3 0 - 4 5 s with artificial saliva. ulation was deducted. In each single fiber the average response to each quality was calculated. These values were then used to express breath of tuning in that particular fibers. Finally the average breath of tuning was calculated for all fibers. Test Substances and Procedure The sweet stimuli were: 3.5 m M acesulfame-K, 0.3 mM alitame, 3.4 m M aspartame, 10 mM Na-cyclamate, 0.3 M fructose, 1.6 m M Na-saccharine, 0.87 m M stevioside, 0.3 M sucrose, 114 # M of "super-aspartame" (an arylurea-dipeptide which incorporates elements of dipeptide and arylurea structures into a single molecule; its potency is 7,800 × sucrose), and 0.75 M xylitol. The sour stimuli were: 50 m M aspartic acid, 40 m M citric acid, and 10 m M HC1. The salty stimuli were: 70 m M NaCI with and without 0.5 m M amiloride. The bitter stimuli were: 5 m M quinine hydrochloride, 1/~M denatonium benzoate, and 84 m M caffeine. In addition, 0.1 M KC1, 10 m M M S G and 0.3 m M GMP as well as a mixture of the same concentrations of M S G and G M P were used. To human approximately equi-intensive concentrations were used. However, these values were adjusted up or down depending on the magnitude of the summated response they evoke. All compounds except quinine, which for solubility reasons was dissolved in distilled water, were dissolved in artificial saliva (2). Data Analysis The breadth of tuning was calculated (cf. 11). To be able to compare the breadth of tuning with data in other species, we used the response to NaC1, citric acid, quinine, sucrose. The responses are expressed as total number of nerve impulses recorded during the first 5 s of stimulation. The activity during 5 s before stim- RESULTS The taste fibers were grouped according to their responses to the different stimuli in each taste quality. Fig. 1 presents an overview of the results in all bitter fibers and 5 salt and sweet fibers as a 3-dimensional graph. Each column illustrates a response to one stimulus in one fiber. The spontaneous activity before each stimulation was deducted from the responses. The response magnitude is illustrated by the height of the columns. A mark instead of a column indicates that the stimulus did not elicit a response. The stimuli were arranged along the x axis in the order of salt, sour, bitter and sweet, while the fibers along the z axis were arranged in groups, Na-best group, bitter and the sweet group. To facilitate the overview, the fibers within each group have been arranged so the fibers with the highest activity are furthest away from the observer. Figure 1 does not contain all sweet and salt fibers recorded. Forty percent fell within the sweet category, and 28% within the salty. The sour fibers were the smallest category. Figure 1 shows a representative sample of our total population of sweet and salt fibers. Calculated from all fibers, the shady area emphasizes the bitter stimuli and the bitter fibers. Figure 1 indicates that chimpanzee taste fibers are quite specific. Based on 49 fibers, the breadth of tuning is 0.30, (SE 0.39) which indicates a higher specificity than reported in any other mammalian species. BITTER T A S T E IN C H I M P A N Z E E 1187 Several conclusions can be drawn from Fig. 1. First, a comparison between the bitter fibers and the response profiles of the other fibers shows that the specificity of the bitter fibers was less than that of both the salt and sweet fibers. Second, none of the fibers in the sweet group responded to the bitter stimuli. Third, with the exception of one salt fiber none of them responded to the bitter compounds. Fourth, the amount of suppression by amiloride on the NaC1 response seems to be inversely related to the response intensity to KCI, so that the fibers which were most suppressed by amiloride responded least to KCI. The response spectrum of the bitter fibers is shown at a higher resolution in Fig. 2. It can be concluded that the 5 m M quinine concentration gave the largest responses, followed by 84 m M caffeine solution. With regard to the response to the other stimuli, all bitter fibers responded to the amiloride-NaCl mixture, while NaCI in itself gave no response. This suggests that the taste of amiloride was the cause. Further, KC1, as expected from its bitter taste, elicited a response in 5 fibers. Less expected is the response to xylitol in 4 fibers. This can be explained by the mixed taste of xylitol. The response to sucrose and fructose will be discussed in the following. DISCUSSION The terms sweet, salty, sour and bitter, are used in the sense these qualities are tasted and understood by humans. The described single fiber data show good correlation of fiber grouping with the human taste qualities. We found a strict dichotomy between bitter and sweet fibers. Some of the conclusions that can be drawn from these results will be discussed in the following. The bitter and sweet taste qualities can be regarded as representing the end points on a hedonic scale going from dislike to like. It is also well known that the sweet and bitter taste qualities can be separated also when they exist in a mixture. If this ability is based on the existence of specific fibers, i.e. the identity of the taste fibers carries the information on taste quality, a strict di- chotomy between bitter and sweet fibers is necessary. It is important that there is no overlap between the response spectrum of the sweet and bitter fibers. Some of the bitter fibers in Fig. 1 show a response to the sweet compounds, but mostly to the sweeteners whose taste include a bitter component. Thus, xylitol has a nonsweet taste component, saccharine and Na cyclamate are not purely sweet, especially saccharine has a bitter quality at higher concentrations (10), amiloride tastes bitter and KCI has a prominent bitter taste. This explanation can not be applied to sucrose and fructose, they do not taste bitter. However, many nonsweet fibers, both bitter and salt, respond to some extent also to carbohydrates. It is possible that carbohydrates are less stringent ligands than other tastants. Another explanation is that they, due to the high concentration at which they are used, exert nonspecific osmotic effects on the sensory cells as has been suggested by DuBois et al. (1). It may be more important that none of the sweet fibers responded to the bitter compounds. It is not shown here, but our total single fiber material in chimpanzee comprises more than 20 sweet fibers. They all display a very high specificity to sweet compounds, and, as with the sweet fibers in Fig. 1, they never show even a trace of a response to a bitter compound. Thus it seems that the difference in taste sensitivity between sweet and bitter fibers is enough to satisfy the criteria for fiber specificity as a means of coding in taste. Though our findings do not exclude the possibility that taste qualities perceptions are the result of central information processing, they provide an explanation based on a division of stimuli into qualities or quality categories already established in the periphery (cf.9). Such a mechanism is no exception among sensory systems. The coding of colors by the retinal cones is a parallel from another sensory system; light stimuli are divided into three "qualities" by the receptor cells (cones) of the retina. A related mechanism in taste would provide a means to handle a large number of taste stimuli by sorting their taste effects into a finite number of taste classes (qualities). 160 140 120 Loealt=. 9ID • t ¢tt][/11 ~tll"~lLIICl, m FIG. 2. An overview of the single fiber responses from bitter fibers in a similar manner. dlP4t. L , m ~ *JOlly" Fiber ° F IDl'l,~all ,77,77,:'.7-,: c 1188 HELLEKANT AND NINOMIYA If these conclusions are correct, recordings from taste nerves can be used to develop new tastants or isolate potent flavor compounds from existing plants, fruits or food. All tastes have temporal and qualitative characteristics. Recordings of the over-all taste nerve activity can give information on the temporal profile and dose/response relation of the tastant (cf.4). Then single taste fiber recordings can be used to determine its taste quality(ies) and supplemental informarion on dose/response relations. Since the difference between the bitter and sweet fiber groups is very clear and the presence of one or more qualities in a tastant is shown by the response spectrum of the taste fibers, the single taste fiber technique can be used as a means to develop and assess new tastants. ACKNOWLEDGEMENTS The data was collected with financial support from the Umami Manufacturers Association of Japan, NutraSweet R&D, Mt. Prospect, IL, USA. We thank Dis. J. Moor-Jankowski, E. Muchmore and W. W. Socha for permission to study the chimpanzees at the Laboratory for Experimental Medicine and Surgery in Primates (LEMSIP), New York Medical Center, Tuxedo, N.Y., USA. REFERENCES 1. DuBois, G. E.; Walters, D. E.; Schiffman, S. S.; Warwick, Z. S.; Booth, B. J.; Pecore, S. D.; Gibes, K.; Carr, B. T.; Brands, L. M. Concentration-response relationships of sweeteners. In: Waiters, E.; Orthoefer, F. T.; DuBois, G. E., eds. Sweeteners: Discovery, molecular design, and chemoreception. ACS Symposium series 450. Am. Chem. Soc. 1991:261-276. 2. Hellekant, G.; H~d a Segerstad, C.; Roberts, T.; van der Wel, H.; Brouwer, J. N.; Glaser, D.; Hayes, R. J.; Eichberg, J. W. 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