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Glossopharyngeal denervation alters responses to nutrients and toxic substances
Physiology & Behavior, Vol. 56, No. 6, pp. 1179-1184, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0031-9384/94 $6.00 + .00
Pergamon 0031-9384(94)00265-7
Glossopharyngeal Denervation Alters Responses to Nutrients and Toxic Substances YUZO
N I N O M I Y A , .1 H I D E A K I K A J I U R A , t " Y U K I O N A I T O , : ~ K A Z U M I C H I HIDEO KATSUKAWA* AND KUNIO TORII§
MOCHIZUKI,
*Department of Oral Physiology, Asahi University School of Dentistry, Hozumi-cho, Motosu-gun, Gifu-Pref 501-02, i'Kirin Brewery Co., Tokyo 103, SNagoya City University, Nagoya 467, and §Ajinomoto, Co., Yokohama 244, Japan NINOMIYA, Y., H. KAJIURA, Y. NAITO, K. MOCHIZUKI, H. KATSUKAWA AND K. TORII. GIossopharyngealdenervation alters responsesto nutrients and toxic substances. PHYSIOL BEHAV 56(6) 1179-1184, 1994.--Functional roles of the glossopharyngeal (GL) nerve on food and fluid intake were studied by examining effects of the GL denervation on two biologically different activities induced by specific diets using mice and rats. First, we examined whether GL section alters the acceptability of a bitter tasting essential amino acid, L-lysine (Lys), by Lys-deficiency in mice. The aversion threshold for Lys, normally about 3 uM in mice, increased to about 300 uM when mice were fed the Lys-deficient diet for 10 days. This increase of the Lys aversion threshold (increase of acceptability for Lys) by Lys-deficiency was also evident in mice with the chorda tympani denervation but was not observed in mice with the GL denervation. Next, we examined whether GL section alters the induction of a salivary protein, cystatin S (a cysteine proteinase inhibitor), by a diet containing papain (a cysteine proteinase) in rats. GL denervation largely inhibited the induction of cystatin S in the rat submandibular glands by papain. These results collectively suggest that chemosensory information conveyed by the GL nerve plays important roles on recognition of both nutrient and toxic compounds in the diet and induction of biological responses that protect the animal from both nutritional deficiency and exogenous toxic compounds. Chemosensory information Glossopharyngeal nerve Lysine-deficient diet Induction of a salivary protein Rat cystatin S Papain containing diet
INTRODUCTION
PREVIOUS electrophysiological studies in several species of mammals (2,3,6,17,18,20,21,25) have shown that bitter tasting substances, such as quinine, phenylthiourea and sucrose octaacetate, elicit greater responses in the glossopharyngeal (GL) nerve than in the chorda tympani (CT) nerve, while the reverse is true for salty (e.g., NaCI) and sweet tasting (e.g., sugars) substances. Hence, it is generally believed that the GL nerve is a dominant taste input for behaviorally aversive substances, while the CT nerve is that for acceptable substances. However, a recent study in C57BL/KsJ strain of mice (19) has shown that the GL nerve produces larger responses than the CT nerve to umami (e.g., monosodium glutamate:MSG) and essential (e.g., L-tryptophan, phenylalanine, histidine, etc.) amino acids, which are nutrients. This suggests the possibility that chemosensory information from the GL nerve plays a major role on recognition of not only behaviorally aversive and toxic but also nutritionally important substances. To investigate further this possibility, in this study using mice and rats, we examined effects of the denervation of the GL nerve on the following two biologically different activities induced by specific diets. The first experiment examined whether GL section alters behavioral acceptability of a bitter-tasting essential amino acid, L-lysine (Lys), when animals are fed a Lys-deficient diet. In rats (12,23), it has been reported that Lys-deficiency changes
To whom requests for reprints should be addressed. 1179
Lysine preference
the taste preference and induces selective behavior to favor ingestion of Lys, which is normally aversive for the animal. Based on these findings, it has been suggested, but not yet proved (12,23), that taste information plays a role on the selection of Lys from other substances available. The second experiment examined whether GL section alters induction of a salivary protein, rat cystatin S (a cysteine proteinase inhibitor), in the submandibular glands of rats fed a diet containing papain (a cysteine proteinase). It is reported that this protein is induced by papain but not by pepsin (an aspartic proteinase) (1,14). This stimulus-specific feature of cystatin S induction might have a neural basis. The results obtained from the present study suggested the importance of chemosensory information conveyed by the GL nerve for the induction of both biologically different activities. METHOD
EXPERIMENT I
Changes in Acceptability for Lys by Lys-deficiency in Mice Subjects. Subjects were male and female mice (C57BL/KsJ strain) weighing 2 0 - 3 5 g ( 8 - 1 6 wk of age). Animals were first divided into 3 groups, intact, C T denervated, and GL denervated groups. Then each group was further divided into two subgroups,
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a control group and a Lys-deficient group. The number of subjects in each subgroup from which data were obtained ranged from 5 to 8 animals. The control group was supplied with the control diet containing Lys and the Lys-deficient group was supplied with the Lys-deficient diet. Four or five animals in each group were housed together and maintained on ad lib food, but only given access to water during the training and testing sessions. The training session started at the 6th day after the presentation of the experimental diets and lasted 5 days. Diet. The contents of the control and the Lys-deficient diets were based on experimental diets determined previously (24). The control diet, in which the main constituents were starch and wheat gluten plus the L-amino acid mixture and Lys, contained more protein than the usual commercially available diet (e.g., laboratory chow)--(Table 1). The Lys-deficient diet, almost the same as the control diet but with Lys omitted, contained a level of Lys too low to maintain appropriate physiological processes and functions, because Lys is an essential amino acid for rats. To make the Lys-deficient diet and control diet isocaloric and isonitrogenic, 1.08% glutamine and 0.27% starch were added to the Lys-deficient diet. Procedures. On the first day of training, each animal was placed in a test box and given free access to distilled water during a 1-h session from a single drinking tube via a circular window (5 mm diameter). The tip of a polyethylene tube (1.5 mm inner diameter) was located 2.0 mm outside the window. This arrangement prevented contact of the tip of the tube with animal's lips. Licks were detected by a lickometer with a photo lick sensor and were recorded on a pen recorder. From the second to the fifth day, training session time was reduced from 1 h to 30 min. During this period, the animal was trained to drink distilled water on a fixed interval schedule, consisting of 10-s periods of presentation of the distilled water alternated with 20-s intertrial intervals, resulting in 3 0 - 5 0 trials during 30-min session. From the sixth to the eighth day, the number of licks for each test solution and distilled water given by each animal was counted during the first 10 s after the animal's first lick. During test sessions, the Lys solutions of different concentrations were given alternatedly with distilled water in a descending order. The mean number of licks across the three test days was obtained for each of the test solutions in each mouse. Solutions. Test solutions used were 0.1, 1.0, 3.0, 10, 30, 100, 300 #M, 1.0, 3.0, 10, 30, 100 mM and 1.0 M Lys and distilled water.
Surgery. The denervation of the CT and GL nerves was made under pentobarbital anesthesia (40-50 mg/kg). Bilateral CT or GL nerves of mice were sectioned at their entry to the bulla or in the neck under the digastric muscle. This surgery was done more than 10 days before the start of the training period. Data analysis. The mean numbers of licks for each distilled water and Lys solution were calculated in each group. The aversion threshold for Lys in each group was obtained as the concentration at which the number of licks per 10 s was significantly lower than that to distilled water (t-test, p < 0.05). EXPERIMENT II
The Induction of Cystatin S by Papain-Containing Diet in Rats Subjects. Subjects were male Wistar rats weighing 180-230 g (8 wk of age). Animals were first divided into two groups, an intact group and a GL denervated group. Each group was further divided into two subgroups: control and papain groups. The number of subjects in each subgroup from which data were obtained ranged from 5 to 8 animals. The control group was supplied with the control diet, whereas the papain group was supplied with the
TABLE 1 COMPOSITION OF DIETS USED IN THE EXPERIMENT
Starch Wheat gluten Cellulose Mineral mixture Vitamin mixture Choline chloride Corn oil Vitamin E L-amino acid mixture Threonine Valine Methionine Isoleucine Leueine Tyrosine Phenylalanine Histidine Arginine Tryptophan Lysine-HC! Glutamine Total
Control*
Lysine Deficient (% of weight)
55.69 24.35 4.00 4.00 1.00 0.20 5.00 0.01 3.72 0.43 0.56 0.50 0.46 0.55 0.18 0.14 0.09 0.69 0.12 1.35 0.68 100.00
55.96 24.35 4.00 4.00 1.00 0.20 5.00 0.01 3.72 0.43 0.56 0.50 0.46 0.55 0.18 0.14 0.09 0.69 0.12 -1.76 100.00
* Control diet and lysine-deficient diet are isocaloric and isonitrogenic.
papain containing diet for one, three or five days. Two or three animals in each group were housed together and maintained on ad lib food and water. In some experiments, rats were used whose unilateral GL nerve was denervated and rats whose oral cavity was treated twice dally with 20% trifluoroacetic acid and 15% hydrogen peroxide for five days. Diet. The contents of diet for control group were same as those used in the previous mouse study, shown in Table 1. The Papain containing diet was made of control diet mixed with 1.0% papain. Procedures. Submandibular glands were removed from rats under pentobarbital anesthesia (50 mg/kg) at one, three or five days after the start of experimental diets. In some experiments, submandibular saliva was collected under light anesthesia with pentobarbital (30 mg/kg). Isoproterenol (IPR: 20 mg/kg) was used as a secretory stimulant for collecting saliva. Quantitation of rat cystatin S. Rat cystatin S was purified from submandibular saliva of rat treated with IPR chronically, as described previously (13). The submandibular glands were sliced into thin sections with a razor blade and homogenized in 10 vol. (w/v) of ice-cooled 20 mM phosphate buffer (pH 7.2) containing 10 mM ethlenediamine-tetraacetic acid, 2 mM L-trans-epoxysuccinyl-leucyamide-(4-guanide)-butane and 2 mM phenylmethylsulfonylfluoride with a glass-Teflon homogenizer. The homogenate was centrifuged at 15,000 g for 30 min at 4°C and the supematant was separated. The concentration of cystatin S in the supernatant and in the saliva was quantified by a single radial immunodiffusion method employing polyclonal antibody against the rat cystatin S as described in a previous report (13). SDS-PAGE of saliva proteins. Electrophoresis with reduction was performed in 15% polyacrylamine gels according to the method of Laemmli (8). One hundred ug of submandibular saliva
GLOSSOPHARYNGEAL DENERVATION
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proteins were electrophoresed and stained by Coomassie Brilliant Blue.
chorda tympani denervated mice. The threshold changed from 3 /zM to I0 mM. These results suggest that the taste information from the glossopharyngeal nerve plays a more important role on the change in behavioral responses of mice to Lys by the Lys deficiency than that from the chorda tympani nerve.
RESULTS
Experiment I Figure 1 shows the concentration-response relationships for Lys in each group. A dotted line in each concentration-response curve indicates the aversion threshold in each group. In intact groups, the aversion threshold for Lys, normally about 3.0 #M, increased to about 300 #M when animals were fed the lysinedeficient diet. In the glossopharyngeal denervated mice, the Lysdeficiency did not change the aversion threshold for Lys. Both control and Lys-deficient mice showed an aversion threshold of about 300 uM. This indicates that the aversion threshold was increased from 3 #M to 300 #M by the GL denervation. This change is equivalent to that which occurred following the Lysdeficiency in the intact group. The greatest increase in the lysine aversion threshold by the lysine-deficiency was found in the
Experiment H As shown in Fig. 2, electrophoretic patterns of rat submandibular saliva proteins on SDS-polyacrylamide gels clearly indicate that cystatin S (an apparent molecular weight = 11,840 (14), the most strongly stained band in SDS-PAGE) was induced in the submandibular saliva of the intact mice fed the papain containing diet for 5 days (Intact), but not in that of control mice fed the control diet (Control). The bilateral or unilateral GL denervation largely inhibited the induction of cystatin S by the papain containing diet (R & L GL denerv and R. or L. GL denerv). This suggests the importance of neural information conveyed by the glossopharyngeal nerve on the induction of rat submandibular
: L-Lysmo deficient 0-0 : Control
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L- Lysine concentration (M) FiG. 1. The number of licks per 10 s (mean _+ SE obtained from 5 to 8 animals) for L-lysine at various concentrations in intact, glossopharyngeal (GL) denervated and chorda tympani (CT) denervated mice supplied with the control (open circles) and the L-lysine deficient diet (filled circles). Each dotted line indicates the aversion threshold for Lys (the concentration of Lys to which the number of licks per l0 s was significantlydifferent from that to distilled water). *: t-test for a difference in the number of licks between L-lysinedeficient and control groups, p < 0.05; **: p < 0.01; ***:p < 0.001.
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Rat submandibular gland proteins
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FIG. 2. SDS-PAGE of the submandibular salivary proteins in rats supplied with the control diet (Control) or the papain containing diet (Papain: 6 lanes) for 5 days, and in rats whose oral cavity was treated with 20% trifluoroaceticacid (TFA 20%) and 15% hydrogenperoxide (H202 15%) twice daily for 5 days. Papain groups consist of intact, unilateral (R or L) and bilateral (R & L) GL denervated animals. Cystatin S (arrow: m.w. 11,840) was strongly induced by papain in intact animals but only slightly in unilateral GL denervated animals and almost not at all in bilateral GL denervated animals.
cystatin S. It is also noted that the cystatin S induction was not observed in rats whose oral cavity was treated with noxious stimulants such as trifluoroacetic acid and hydrogen dioxide solution for 5 days, suggesting that cystatin S might not be induced by nociceptive chemical information from the oral cavity. Figure 3 shows the body weight gain and the amount of cystatin S induced in the submandibular gland of rats after the start of the control and the papain-containingdiet. There was no clear difference in the body weight gain between the GL denervatedcontrol and intact-control [23.2 _ 2.0 g (GL denervated-control) vs. 23.2 _+ 4.1 g (intact-control)for 5 days, t-test, p > 0.05]. This indicates that the denervation of the glossopharyngeal nerve itself did not affect the body weight gain. However, the papain diet group either with or without the GL denervation showed a clear retardation of growth after the start of papain diet as compared with the control diet groups. In the intact-papain group, body weight gain restarted from the third day after the papain diet (13.4 _ 3.1 g for 5 days, significantly smaller than that of control diet groups, t-test, p < 0.001), but the GL denervated-papain group showed only a slight body weight gain for 5 days (3.5 _ 1.9 g, significantly smaller than that of intact-papain and control groups, t-test, p < 0.001). As shown in Fig. 3 (the lower graph), cystatin S was induced in the submandibular glands of the intact-papain group from the third day after the start of the papain diet. The amount of cystatin S of the glands at the 5th day in this group was 4.8 _ 1.1 mg/gland. It
is noted that the amount of cystatin S is parallel with the body weight gain in this group. Comparably, the cystatin S induction was largely inhibited in the GL denervated-papain group showing only a slight body weight gain (0.32 _ 0.2 mg/gland, significantly smaller than that of the intact-papain group, t-test, p < 0.001). DISCUSSION
Changes in Acceptability for Lys by Lys-Deficiency in Mice Previous behavioral studies in rats (23) demonstrated that Lys-deficiency drastically changes the behavioral response to 0.2 M Lys from aversion to preference as compared with water. In the present mouse study, although no such a drastic change was observed, we found a change in acceptability for Lys by Lysdeficiency. The aversion threshold for Lys was increased by Lysdeficiency from 3.0 #M to 300 #M (about 2.0 log units) in intact mice, and from 3.0 #M to 10 mM (about 3.5 log units) in the CT denervated mice, while the aversion threshold for Lys in the GL denervated mice was unaffected by the deficiency (Fig. 1). In further behavioral studies (Kajiura et al., in preparation), we found that Lys-deficiency did not significantly affect drinking responses to other essential amino acids, such as L-phenylalanine and -teucine and a bitter-tasting substance, quinine and various other taste substances, and that the increase of acceptability for Lys by Lys-deficiency lasted for more than l0 days, until animals were supplied with the control diet. This suggests that Lys-defi-
GLOSSOPHARYNGEAL DENERVATION
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m30-
GL denervated- control Intact -control Intact- papain GL denervated- papain
~5- ~ 0
1
2
3
4
5
Time (day) FIG. 3. Body weight gain and the amount of cystatin S of the submandibular glands (mean ___ S.E.) in intact and the GL denervated rats supplied with the control diet (Control) or the papain containing diet (Papain). Data were obtained from 5-8 rats. ciency specifically affects responses to Lys and that repletion for Lys during test sessions was not sufficient for animals to recover from the deficiency. Our previous electrophysiological study in the same strain of mouse (7) demonstrated that the threshold for the neural response to Lys was about 1.0 #M in the GL nerve, and was about 300 #M in the CT nerve. This indicates that the aversion threshold for Lys in control mice was close to the neural threshold for Lys in the GL nerve, and its change by Lys-deficiency was within the concentration range effective in the GL nerve (The CT nerve does not respond to Lys in this concentration range). Taking this together with the fact that the aversion threshold in the GL denervated mice was unaffected by the Lysdeficiency, it is probable that chemosensory information conveyed by the GL nerve plays a major role in changes in acceptability of Lys by Lys-deficiency, while information conveyed by the CT and other taste nerves, such as superior laryngeal nerve innervating taste buds on the larynx, and greater superficial petrosal nerve innervating taste buds in the nasoincisor ducts and on the soft palate, are less important or do not contribute to the deficiency-induced change in aversiveness for Lys at the lower concentration range. In contract to this, the evidence that the greatest increase of the aversion threshold for Lys was found in the CT denervated mice suggests the possibility that information for Lys from the CT nerve may relate to aversive responsiveness. Our preliminary electrophysiological study (Kajiura et al., in preparation) suggests that Lys-deficiency hardly affects the neural thresholds for Lys in mouse CT and GL nerves. Therefore, it is probable that changes in acceptability for the deficient Lys are regulated in the central nervous system with chemosensory information mainly from the GL nerve. Tabuchi et al. (23) reported that in rats, lateral hypothalamic (LHA) neurons specifically responding to Lys were found in animals fed the Lys-deficient diet but not observed in animals fed the control diet. This suggests that preference for deficient amino acids might be mediated, in part at least, in the LHA.
The Induction of Cystatin S by Papain Containing Diet in Rats Previous biochemical studies have shown that some salivary proteins, such as proline-rich proteins and rat cystatin S, normally undetectable, are induced by the treatment with the beta-adrenergic agonist, isoproterenol (IPR), and proteinase or when animals were fed a diet containing tannin (9-11,14). Recently, Naito et al. (14) reported that cystatin S was induced in the rat submandibular saliva by the oral application of papain, but not by that of pepsin. This stimulus-specific feature of cystatin S induction in rats suggests the possibility that chemosensory inputs might participate in the cystatin S induction. The present study confirmed that rat cystatin S induction occurred when papain was given to the animal as a part of the diet, and demonstrated the importance of chemosensory informationfrom the GL nerve for the salivary protein induction (Fig. 1). We did not examine the effect of the chorda tympani denervation because the chorda tympani nerve contains parasympathetic efferents to the submandibulargland. Naito et al. (14) found that metoprolol, a selective beta receptor antagonist, inhibits induction of cystatin S by papain application, suggesting that the induction is primarily mediated by the beta-adrenergic receptors similar to the effect of IPR. Taking this into consideration, it is probable that chemosensory information from the GL nerve leads to the activation of the beta-adrenergic receptors in the submandibular glands. The present study also showed that strong noxious stimulants (trifluoroacetic acid and hydrogen peroxide) applied to the oral cavity twice daily for 5 days did not cause the protein induction in the submandibular glands. This further suggests the possibility of the chemo-specific induction of cystafin S. In this regard, it is reported that in humans (15) and rabbits (4,5), sweet taste, or sweet-tasting substances have a sympathetic-like stimulatory effect and induce higher concentration of amylase than other tastes. It was found that rats fed the papain containing diet showed a retardation of growth compared to control animals, and this growth
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retardation was more prominent in the GL denervated group with no cystatin S in their saliva than in the intact group with cystafin S in the saliva. The induced cystatin S in the saliva of the intact group might act as a cysteine proteinase inhibitor, reduce the activity of papain (a cysteine proteinase) in the diet, and lead to the increased body weight gain, as shown in Fig. 3. Similarly, it is reported that in rats and mice, diets of sorghum, which is high in tannins, induce proline-rich proteins in the parotid and submandibular glands ( 9 11). The initial weight loss experienced by animals on the hightannin sorghum diet was reversed at the 3rd day, at the time when the induction of proline-rich proteins was maximal (10). Therefore, induction of these salivary proteins by the diet containing specific chemical compounds might be a biological response and it is a first line of defense that protects the animal from injury caused by exogenous toxic substances.
Functional Roles of Chemosensory Information From the GL Nerve The results of these two different lines of research using mice and rats suggested that taste neural inputs from the C T and GL nerves plays different roles not only for taste recognition but also for physiological activities maintaining the animal' s homeostatic balance. Previous electrophysiological studies have demonstrated that taste responsiveness of the other two taste nerves, the superior laryngeal nerve and greater superficial petrosal nerve, are also considerably different from those of the CT and GL
nerve (e.g., 22). Chemosensory information conveyed by the GL nerve is very important for recognition of either nutrient or toxic substances in the diet and induction of biological responses for adjustments against the nutritional deficiencies and defenses against exogenous toxic substances. For detection of such chemical compounds, specific receptors and neural coding mechanisms for them may exist in the chemosensory system. In our previous single fiber study using a different strain of mice (SIc:ICR) (16), we found some fibers of the GL nerve which are highly sensitive to an umami amino acid, MSG, but only slightly if at all responsive to the four basic taste stimuli (NaCl, sucrose, HC1 and quinine). Therefore, there may exist a specific receptor and a neural channel for umami amino acids at least on the posterior part of the mouse tongue innervated by the GL nerve. However, no report has demonstrated the existence of single fibers showing selective responses to a single essential amino acid, although as mentioned above (7), the fact that the threshold of the integrated responses of the GL nerve of mice for Lys is much lower than that of the CT nerve, suggests the possibility of existence of the high-affinity receptor type for Lys in taste cells innervated by the GL nerve in this species. Further extensive studies on single fiber recordings in these species may provide the answer to these questions. ACKNOWLEDGEMENTS This research was supported in part by Grant-in-Aid for Scientific Research 05671560 from the Ministry of Education, Science and Culture of Japan, and a grant from Kirin Brewery.
REFERENCES 1. Ershoff, B. H.; Bajwa, G. S. Submandibular gland hypertrophy in rats fed proteolytic enzymes. Proc. Exp. Biol. Med. 113:878-881; 1963. 2. Frank, M. An analysis of hamster afferent taste nerve response function. J. Gen. Physiol. 61:588-613; 1973. 3. Funakoshi, M.; Kawamura, Y. Relations between taste qualities and parotid gland secretion rate. In: Hayashi, T., ed. Olfaction and taste vol. 2. New York: Pergamon Press; 1967:281-287. 4. Gjorstrup, P. Parotid secretion of fluid and amylase in rabbits during feeding. J. Physiol. 309:101-116; 1980. 5. Gjorsa'up, P. Taste and chewing as stimuli for the secretion of amylase from the parotid gland of the rabbit. Acta Physiol. Scand. 110:295-301; 1980. 6. Hanamori, T.; Miller, I. J.; Smith, D. V. Gustatory responsiveness of fibers in the hamster glossopharyngeal nerve. J. Neurophysiol. 60:478-498; 1988. 7. Kajiura, H.; Ninomiya, Y.; Tanimukai, T.; Yoshida, S.; Torii, K. Two different receptor mechanisms for L-lysine in the moose peripheral taste system. Proc. Jpn. Sym. Taste and Smell 26:89-92; 1992. 8. Laemmli, UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685; 1970. 9. Mehansho, H.; Carlson, D. M. Induction of protein and glycoprotein synthesis in rat submandibular glands by isoproterenol. J. Biol. Chem. 258:6616-6620; 1983. 10. Mehansho, H.; Hagerman, A.; Clements, S.; Butler, L.; Rogler, J.; Carlson, D. M. Modulation of proline-rich protein biosynthesis in rat parotid glands by sorghums with high tannin levels. Proc. Natl. Acad. Sci. USA 80:3948-3952; 1983. 11. Mehansho, H.; Ciements, S.; Sheares, B. T.; Smith, S.; Carlson, D. M. Induction of proline-rich glycoprotein synthesis in mouse salivary glands by isoproterenol and by tannins. J. Biol. Chem. 260:4418-4423; 1985. 12. Mori, M.; Kawada, T.; Ono, T.; Torii, K. Taste preference and protein nutrition and L-amino acid homeostasis in male Sprague-Dawley rats. Physiol. Behav. 49:987-996; 1991. 13. Naito, Y. Studies on LM protein appearing in submandibular glands of isoproterenol-treated rats. I. Purification and characteristics of its appearance. Chem. Pharm. Bull. Tokyo. 29:1365-1372; 198 I.
14. Naito, Y.; Suzuki, I.; Hasegawa, S. Induction of cystatin S in rat submandibular glands by papain. Comp. Biochem. Physiol. 102:861-865; 1992. 15. Newbrun, E. Observations on the amylase content and flow rate of human saliva following gustatory stimulation. J. Dent. Res. 41:459465; 1962. 16. Ninomiya, Y.; Funakoshi, M. Qualitative discrimination among umami and the four basic taste substances in mice. In: Kawamura, Y.; Kare, M. R., eds. Umami: A basic taste. New York: Mercel Dekker; 1987:365-385. 17. Ninomiya, Y.; Funakoshi, M. Peripheral neural basis for behavioral discrimination between glutamate and the four basic taste substances in mice. Comp. Biochem. Physiol. 92:371-376; 1989. 18. Ninomiya, Y.; Tanimukai, T.; Yoshida, S.; Funakoshi, M. Gustatory neural responses in preweanling mice. Physiol. Behav. 49:913-918; 1991. 19. Ninomiya, Y.; Kajiura, H.; Mochizuki, K. Differential taste responses of mouse chorda tympani and glossopharyngeal nerves to sugars and amino acids. Neurosci. Let. 163:197- 200; 1993. 20. Ogawa, H. Taste response characteristics in glossopharyngeal nerve of the rat. Kumamoto Med. J. 25: ! 37-147; 1972. 21. Shingai, T.; Beidler, L. M. Response characteristics of three taste nerves in mice. Brain Res. 335:245-249; 1985. 22. Smith, D. V.; Frank, M. Sensory coding by peripheral taste fibers. In: Simon, S. A.; Roper, S. D., eds. Mechanisms of Taste Transduction. Boca Raton: CRC Press; 1993:295-338. 23. Tabuchi, E.; Ono, T.; Nishijo, H.; Torii, K. Amino acid and NaCl appetite, and LHA neuron responses of iysine-deficient rat. Physiol. Behav. 49:951-964; 199 I. 24. Torii, K.; Mimura, T.; Yugari, Y. Biochemical mechanism of umami taste perception and effect of dietary protein on the taste preference for amino acids and sodium chloride in rats. In: Kawamura, Y.; Kate, M. R., eds. Umami: A basic taste. New York: Mercel Dekker; 1987:543-563. 25. Yamamoto, T.; Matsuo, R.; Kiyomitsu, Y.; Kitamura, R. Taste effects of umami substances in hamsters as studied by electrophysiological and conditioned taste aversion techniques. Brain Res. 451:147-162; 1988.
Pergamon 0031-9384(94)00265-7
Glossopharyngeal Denervation Alters Responses to Nutrients and Toxic Substances YUZO
N I N O M I Y A , .1 H I D E A K I K A J I U R A , t " Y U K I O N A I T O , : ~ K A Z U M I C H I HIDEO KATSUKAWA* AND KUNIO TORII§
MOCHIZUKI,
*Department of Oral Physiology, Asahi University School of Dentistry, Hozumi-cho, Motosu-gun, Gifu-Pref 501-02, i'Kirin Brewery Co., Tokyo 103, SNagoya City University, Nagoya 467, and §Ajinomoto, Co., Yokohama 244, Japan NINOMIYA, Y., H. KAJIURA, Y. NAITO, K. MOCHIZUKI, H. KATSUKAWA AND K. TORII. GIossopharyngealdenervation alters responsesto nutrients and toxic substances. PHYSIOL BEHAV 56(6) 1179-1184, 1994.--Functional roles of the glossopharyngeal (GL) nerve on food and fluid intake were studied by examining effects of the GL denervation on two biologically different activities induced by specific diets using mice and rats. First, we examined whether GL section alters the acceptability of a bitter tasting essential amino acid, L-lysine (Lys), by Lys-deficiency in mice. The aversion threshold for Lys, normally about 3 uM in mice, increased to about 300 uM when mice were fed the Lys-deficient diet for 10 days. This increase of the Lys aversion threshold (increase of acceptability for Lys) by Lys-deficiency was also evident in mice with the chorda tympani denervation but was not observed in mice with the GL denervation. Next, we examined whether GL section alters the induction of a salivary protein, cystatin S (a cysteine proteinase inhibitor), by a diet containing papain (a cysteine proteinase) in rats. GL denervation largely inhibited the induction of cystatin S in the rat submandibular glands by papain. These results collectively suggest that chemosensory information conveyed by the GL nerve plays important roles on recognition of both nutrient and toxic compounds in the diet and induction of biological responses that protect the animal from both nutritional deficiency and exogenous toxic compounds. Chemosensory information Glossopharyngeal nerve Lysine-deficient diet Induction of a salivary protein Rat cystatin S Papain containing diet
INTRODUCTION
PREVIOUS electrophysiological studies in several species of mammals (2,3,6,17,18,20,21,25) have shown that bitter tasting substances, such as quinine, phenylthiourea and sucrose octaacetate, elicit greater responses in the glossopharyngeal (GL) nerve than in the chorda tympani (CT) nerve, while the reverse is true for salty (e.g., NaCI) and sweet tasting (e.g., sugars) substances. Hence, it is generally believed that the GL nerve is a dominant taste input for behaviorally aversive substances, while the CT nerve is that for acceptable substances. However, a recent study in C57BL/KsJ strain of mice (19) has shown that the GL nerve produces larger responses than the CT nerve to umami (e.g., monosodium glutamate:MSG) and essential (e.g., L-tryptophan, phenylalanine, histidine, etc.) amino acids, which are nutrients. This suggests the possibility that chemosensory information from the GL nerve plays a major role on recognition of not only behaviorally aversive and toxic but also nutritionally important substances. To investigate further this possibility, in this study using mice and rats, we examined effects of the denervation of the GL nerve on the following two biologically different activities induced by specific diets. The first experiment examined whether GL section alters behavioral acceptability of a bitter-tasting essential amino acid, L-lysine (Lys), when animals are fed a Lys-deficient diet. In rats (12,23), it has been reported that Lys-deficiency changes
To whom requests for reprints should be addressed. 1179
Lysine preference
the taste preference and induces selective behavior to favor ingestion of Lys, which is normally aversive for the animal. Based on these findings, it has been suggested, but not yet proved (12,23), that taste information plays a role on the selection of Lys from other substances available. The second experiment examined whether GL section alters induction of a salivary protein, rat cystatin S (a cysteine proteinase inhibitor), in the submandibular glands of rats fed a diet containing papain (a cysteine proteinase). It is reported that this protein is induced by papain but not by pepsin (an aspartic proteinase) (1,14). This stimulus-specific feature of cystatin S induction might have a neural basis. The results obtained from the present study suggested the importance of chemosensory information conveyed by the GL nerve for the induction of both biologically different activities. METHOD
EXPERIMENT I
Changes in Acceptability for Lys by Lys-deficiency in Mice Subjects. Subjects were male and female mice (C57BL/KsJ strain) weighing 2 0 - 3 5 g ( 8 - 1 6 wk of age). Animals were first divided into 3 groups, intact, C T denervated, and GL denervated groups. Then each group was further divided into two subgroups,
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a control group and a Lys-deficient group. The number of subjects in each subgroup from which data were obtained ranged from 5 to 8 animals. The control group was supplied with the control diet containing Lys and the Lys-deficient group was supplied with the Lys-deficient diet. Four or five animals in each group were housed together and maintained on ad lib food, but only given access to water during the training and testing sessions. The training session started at the 6th day after the presentation of the experimental diets and lasted 5 days. Diet. The contents of the control and the Lys-deficient diets were based on experimental diets determined previously (24). The control diet, in which the main constituents were starch and wheat gluten plus the L-amino acid mixture and Lys, contained more protein than the usual commercially available diet (e.g., laboratory chow)--(Table 1). The Lys-deficient diet, almost the same as the control diet but with Lys omitted, contained a level of Lys too low to maintain appropriate physiological processes and functions, because Lys is an essential amino acid for rats. To make the Lys-deficient diet and control diet isocaloric and isonitrogenic, 1.08% glutamine and 0.27% starch were added to the Lys-deficient diet. Procedures. On the first day of training, each animal was placed in a test box and given free access to distilled water during a 1-h session from a single drinking tube via a circular window (5 mm diameter). The tip of a polyethylene tube (1.5 mm inner diameter) was located 2.0 mm outside the window. This arrangement prevented contact of the tip of the tube with animal's lips. Licks were detected by a lickometer with a photo lick sensor and were recorded on a pen recorder. From the second to the fifth day, training session time was reduced from 1 h to 30 min. During this period, the animal was trained to drink distilled water on a fixed interval schedule, consisting of 10-s periods of presentation of the distilled water alternated with 20-s intertrial intervals, resulting in 3 0 - 5 0 trials during 30-min session. From the sixth to the eighth day, the number of licks for each test solution and distilled water given by each animal was counted during the first 10 s after the animal's first lick. During test sessions, the Lys solutions of different concentrations were given alternatedly with distilled water in a descending order. The mean number of licks across the three test days was obtained for each of the test solutions in each mouse. Solutions. Test solutions used were 0.1, 1.0, 3.0, 10, 30, 100, 300 #M, 1.0, 3.0, 10, 30, 100 mM and 1.0 M Lys and distilled water.
Surgery. The denervation of the CT and GL nerves was made under pentobarbital anesthesia (40-50 mg/kg). Bilateral CT or GL nerves of mice were sectioned at their entry to the bulla or in the neck under the digastric muscle. This surgery was done more than 10 days before the start of the training period. Data analysis. The mean numbers of licks for each distilled water and Lys solution were calculated in each group. The aversion threshold for Lys in each group was obtained as the concentration at which the number of licks per 10 s was significantly lower than that to distilled water (t-test, p < 0.05). EXPERIMENT II
The Induction of Cystatin S by Papain-Containing Diet in Rats Subjects. Subjects were male Wistar rats weighing 180-230 g (8 wk of age). Animals were first divided into two groups, an intact group and a GL denervated group. Each group was further divided into two subgroups: control and papain groups. The number of subjects in each subgroup from which data were obtained ranged from 5 to 8 animals. The control group was supplied with the control diet, whereas the papain group was supplied with the
TABLE 1 COMPOSITION OF DIETS USED IN THE EXPERIMENT
Starch Wheat gluten Cellulose Mineral mixture Vitamin mixture Choline chloride Corn oil Vitamin E L-amino acid mixture Threonine Valine Methionine Isoleucine Leueine Tyrosine Phenylalanine Histidine Arginine Tryptophan Lysine-HC! Glutamine Total
Control*
Lysine Deficient (% of weight)
55.69 24.35 4.00 4.00 1.00 0.20 5.00 0.01 3.72 0.43 0.56 0.50 0.46 0.55 0.18 0.14 0.09 0.69 0.12 1.35 0.68 100.00
55.96 24.35 4.00 4.00 1.00 0.20 5.00 0.01 3.72 0.43 0.56 0.50 0.46 0.55 0.18 0.14 0.09 0.69 0.12 -1.76 100.00
* Control diet and lysine-deficient diet are isocaloric and isonitrogenic.
papain containing diet for one, three or five days. Two or three animals in each group were housed together and maintained on ad lib food and water. In some experiments, rats were used whose unilateral GL nerve was denervated and rats whose oral cavity was treated twice dally with 20% trifluoroacetic acid and 15% hydrogen peroxide for five days. Diet. The contents of diet for control group were same as those used in the previous mouse study, shown in Table 1. The Papain containing diet was made of control diet mixed with 1.0% papain. Procedures. Submandibular glands were removed from rats under pentobarbital anesthesia (50 mg/kg) at one, three or five days after the start of experimental diets. In some experiments, submandibular saliva was collected under light anesthesia with pentobarbital (30 mg/kg). Isoproterenol (IPR: 20 mg/kg) was used as a secretory stimulant for collecting saliva. Quantitation of rat cystatin S. Rat cystatin S was purified from submandibular saliva of rat treated with IPR chronically, as described previously (13). The submandibular glands were sliced into thin sections with a razor blade and homogenized in 10 vol. (w/v) of ice-cooled 20 mM phosphate buffer (pH 7.2) containing 10 mM ethlenediamine-tetraacetic acid, 2 mM L-trans-epoxysuccinyl-leucyamide-(4-guanide)-butane and 2 mM phenylmethylsulfonylfluoride with a glass-Teflon homogenizer. The homogenate was centrifuged at 15,000 g for 30 min at 4°C and the supematant was separated. The concentration of cystatin S in the supernatant and in the saliva was quantified by a single radial immunodiffusion method employing polyclonal antibody against the rat cystatin S as described in a previous report (13). SDS-PAGE of saliva proteins. Electrophoresis with reduction was performed in 15% polyacrylamine gels according to the method of Laemmli (8). One hundred ug of submandibular saliva
GLOSSOPHARYNGEAL DENERVATION
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proteins were electrophoresed and stained by Coomassie Brilliant Blue.
chorda tympani denervated mice. The threshold changed from 3 /zM to I0 mM. These results suggest that the taste information from the glossopharyngeal nerve plays a more important role on the change in behavioral responses of mice to Lys by the Lys deficiency than that from the chorda tympani nerve.
RESULTS
Experiment I Figure 1 shows the concentration-response relationships for Lys in each group. A dotted line in each concentration-response curve indicates the aversion threshold in each group. In intact groups, the aversion threshold for Lys, normally about 3.0 #M, increased to about 300 #M when animals were fed the lysinedeficient diet. In the glossopharyngeal denervated mice, the Lysdeficiency did not change the aversion threshold for Lys. Both control and Lys-deficient mice showed an aversion threshold of about 300 uM. This indicates that the aversion threshold was increased from 3 #M to 300 #M by the GL denervation. This change is equivalent to that which occurred following the Lysdeficiency in the intact group. The greatest increase in the lysine aversion threshold by the lysine-deficiency was found in the
Experiment H As shown in Fig. 2, electrophoretic patterns of rat submandibular saliva proteins on SDS-polyacrylamide gels clearly indicate that cystatin S (an apparent molecular weight = 11,840 (14), the most strongly stained band in SDS-PAGE) was induced in the submandibular saliva of the intact mice fed the papain containing diet for 5 days (Intact), but not in that of control mice fed the control diet (Control). The bilateral or unilateral GL denervation largely inhibited the induction of cystatin S by the papain containing diet (R & L GL denerv and R. or L. GL denerv). This suggests the importance of neural information conveyed by the glossopharyngeal nerve on the induction of rat submandibular
: L-Lysmo deficient 0-0 : Control
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L- Lysine concentration (M) FiG. 1. The number of licks per 10 s (mean _+ SE obtained from 5 to 8 animals) for L-lysine at various concentrations in intact, glossopharyngeal (GL) denervated and chorda tympani (CT) denervated mice supplied with the control (open circles) and the L-lysine deficient diet (filled circles). Each dotted line indicates the aversion threshold for Lys (the concentration of Lys to which the number of licks per l0 s was significantlydifferent from that to distilled water). *: t-test for a difference in the number of licks between L-lysinedeficient and control groups, p < 0.05; **: p < 0.01; ***:p < 0.001.
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NINOMIYA ET AL.
Rat submandibular gland proteins
t/l r-
o
FIG. 2. SDS-PAGE of the submandibular salivary proteins in rats supplied with the control diet (Control) or the papain containing diet (Papain: 6 lanes) for 5 days, and in rats whose oral cavity was treated with 20% trifluoroaceticacid (TFA 20%) and 15% hydrogenperoxide (H202 15%) twice daily for 5 days. Papain groups consist of intact, unilateral (R or L) and bilateral (R & L) GL denervated animals. Cystatin S (arrow: m.w. 11,840) was strongly induced by papain in intact animals but only slightly in unilateral GL denervated animals and almost not at all in bilateral GL denervated animals.
cystatin S. It is also noted that the cystatin S induction was not observed in rats whose oral cavity was treated with noxious stimulants such as trifluoroacetic acid and hydrogen dioxide solution for 5 days, suggesting that cystatin S might not be induced by nociceptive chemical information from the oral cavity. Figure 3 shows the body weight gain and the amount of cystatin S induced in the submandibular gland of rats after the start of the control and the papain-containingdiet. There was no clear difference in the body weight gain between the GL denervatedcontrol and intact-control [23.2 _ 2.0 g (GL denervated-control) vs. 23.2 _+ 4.1 g (intact-control)for 5 days, t-test, p > 0.05]. This indicates that the denervation of the glossopharyngeal nerve itself did not affect the body weight gain. However, the papain diet group either with or without the GL denervation showed a clear retardation of growth after the start of papain diet as compared with the control diet groups. In the intact-papain group, body weight gain restarted from the third day after the papain diet (13.4 _ 3.1 g for 5 days, significantly smaller than that of control diet groups, t-test, p < 0.001), but the GL denervated-papain group showed only a slight body weight gain for 5 days (3.5 _ 1.9 g, significantly smaller than that of intact-papain and control groups, t-test, p < 0.001). As shown in Fig. 3 (the lower graph), cystatin S was induced in the submandibular glands of the intact-papain group from the third day after the start of the papain diet. The amount of cystatin S of the glands at the 5th day in this group was 4.8 _ 1.1 mg/gland. It
is noted that the amount of cystatin S is parallel with the body weight gain in this group. Comparably, the cystatin S induction was largely inhibited in the GL denervated-papain group showing only a slight body weight gain (0.32 _ 0.2 mg/gland, significantly smaller than that of the intact-papain group, t-test, p < 0.001). DISCUSSION
Changes in Acceptability for Lys by Lys-Deficiency in Mice Previous behavioral studies in rats (23) demonstrated that Lys-deficiency drastically changes the behavioral response to 0.2 M Lys from aversion to preference as compared with water. In the present mouse study, although no such a drastic change was observed, we found a change in acceptability for Lys by Lysdeficiency. The aversion threshold for Lys was increased by Lysdeficiency from 3.0 #M to 300 #M (about 2.0 log units) in intact mice, and from 3.0 #M to 10 mM (about 3.5 log units) in the CT denervated mice, while the aversion threshold for Lys in the GL denervated mice was unaffected by the deficiency (Fig. 1). In further behavioral studies (Kajiura et al., in preparation), we found that Lys-deficiency did not significantly affect drinking responses to other essential amino acids, such as L-phenylalanine and -teucine and a bitter-tasting substance, quinine and various other taste substances, and that the increase of acceptability for Lys by Lys-deficiency lasted for more than l0 days, until animals were supplied with the control diet. This suggests that Lys-defi-
GLOSSOPHARYNGEAL DENERVATION
1183
m30-
GL denervated- control Intact -control Intact- papain GL denervated- papain
~5- ~ 0
1
2
3
4
5
Time (day) FIG. 3. Body weight gain and the amount of cystatin S of the submandibular glands (mean ___ S.E.) in intact and the GL denervated rats supplied with the control diet (Control) or the papain containing diet (Papain). Data were obtained from 5-8 rats. ciency specifically affects responses to Lys and that repletion for Lys during test sessions was not sufficient for animals to recover from the deficiency. Our previous electrophysiological study in the same strain of mouse (7) demonstrated that the threshold for the neural response to Lys was about 1.0 #M in the GL nerve, and was about 300 #M in the CT nerve. This indicates that the aversion threshold for Lys in control mice was close to the neural threshold for Lys in the GL nerve, and its change by Lys-deficiency was within the concentration range effective in the GL nerve (The CT nerve does not respond to Lys in this concentration range). Taking this together with the fact that the aversion threshold in the GL denervated mice was unaffected by the Lysdeficiency, it is probable that chemosensory information conveyed by the GL nerve plays a major role in changes in acceptability of Lys by Lys-deficiency, while information conveyed by the CT and other taste nerves, such as superior laryngeal nerve innervating taste buds on the larynx, and greater superficial petrosal nerve innervating taste buds in the nasoincisor ducts and on the soft palate, are less important or do not contribute to the deficiency-induced change in aversiveness for Lys at the lower concentration range. In contract to this, the evidence that the greatest increase of the aversion threshold for Lys was found in the CT denervated mice suggests the possibility that information for Lys from the CT nerve may relate to aversive responsiveness. Our preliminary electrophysiological study (Kajiura et al., in preparation) suggests that Lys-deficiency hardly affects the neural thresholds for Lys in mouse CT and GL nerves. Therefore, it is probable that changes in acceptability for the deficient Lys are regulated in the central nervous system with chemosensory information mainly from the GL nerve. Tabuchi et al. (23) reported that in rats, lateral hypothalamic (LHA) neurons specifically responding to Lys were found in animals fed the Lys-deficient diet but not observed in animals fed the control diet. This suggests that preference for deficient amino acids might be mediated, in part at least, in the LHA.
The Induction of Cystatin S by Papain Containing Diet in Rats Previous biochemical studies have shown that some salivary proteins, such as proline-rich proteins and rat cystatin S, normally undetectable, are induced by the treatment with the beta-adrenergic agonist, isoproterenol (IPR), and proteinase or when animals were fed a diet containing tannin (9-11,14). Recently, Naito et al. (14) reported that cystatin S was induced in the rat submandibular saliva by the oral application of papain, but not by that of pepsin. This stimulus-specific feature of cystatin S induction in rats suggests the possibility that chemosensory inputs might participate in the cystatin S induction. The present study confirmed that rat cystatin S induction occurred when papain was given to the animal as a part of the diet, and demonstrated the importance of chemosensory informationfrom the GL nerve for the salivary protein induction (Fig. 1). We did not examine the effect of the chorda tympani denervation because the chorda tympani nerve contains parasympathetic efferents to the submandibulargland. Naito et al. (14) found that metoprolol, a selective beta receptor antagonist, inhibits induction of cystatin S by papain application, suggesting that the induction is primarily mediated by the beta-adrenergic receptors similar to the effect of IPR. Taking this into consideration, it is probable that chemosensory information from the GL nerve leads to the activation of the beta-adrenergic receptors in the submandibular glands. The present study also showed that strong noxious stimulants (trifluoroacetic acid and hydrogen peroxide) applied to the oral cavity twice daily for 5 days did not cause the protein induction in the submandibular glands. This further suggests the possibility of the chemo-specific induction of cystafin S. In this regard, it is reported that in humans (15) and rabbits (4,5), sweet taste, or sweet-tasting substances have a sympathetic-like stimulatory effect and induce higher concentration of amylase than other tastes. It was found that rats fed the papain containing diet showed a retardation of growth compared to control animals, and this growth
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retardation was more prominent in the GL denervated group with no cystatin S in their saliva than in the intact group with cystafin S in the saliva. The induced cystatin S in the saliva of the intact group might act as a cysteine proteinase inhibitor, reduce the activity of papain (a cysteine proteinase) in the diet, and lead to the increased body weight gain, as shown in Fig. 3. Similarly, it is reported that in rats and mice, diets of sorghum, which is high in tannins, induce proline-rich proteins in the parotid and submandibular glands ( 9 11). The initial weight loss experienced by animals on the hightannin sorghum diet was reversed at the 3rd day, at the time when the induction of proline-rich proteins was maximal (10). Therefore, induction of these salivary proteins by the diet containing specific chemical compounds might be a biological response and it is a first line of defense that protects the animal from injury caused by exogenous toxic substances.
Functional Roles of Chemosensory Information From the GL Nerve The results of these two different lines of research using mice and rats suggested that taste neural inputs from the C T and GL nerves plays different roles not only for taste recognition but also for physiological activities maintaining the animal' s homeostatic balance. Previous electrophysiological studies have demonstrated that taste responsiveness of the other two taste nerves, the superior laryngeal nerve and greater superficial petrosal nerve, are also considerably different from those of the CT and GL
nerve (e.g., 22). Chemosensory information conveyed by the GL nerve is very important for recognition of either nutrient or toxic substances in the diet and induction of biological responses for adjustments against the nutritional deficiencies and defenses against exogenous toxic substances. For detection of such chemical compounds, specific receptors and neural coding mechanisms for them may exist in the chemosensory system. In our previous single fiber study using a different strain of mice (SIc:ICR) (16), we found some fibers of the GL nerve which are highly sensitive to an umami amino acid, MSG, but only slightly if at all responsive to the four basic taste stimuli (NaCl, sucrose, HC1 and quinine). Therefore, there may exist a specific receptor and a neural channel for umami amino acids at least on the posterior part of the mouse tongue innervated by the GL nerve. However, no report has demonstrated the existence of single fibers showing selective responses to a single essential amino acid, although as mentioned above (7), the fact that the threshold of the integrated responses of the GL nerve of mice for Lys is much lower than that of the CT nerve, suggests the possibility of existence of the high-affinity receptor type for Lys in taste cells innervated by the GL nerve in this species. Further extensive studies on single fiber recordings in these species may provide the answer to these questions. ACKNOWLEDGEMENTS This research was supported in part by Grant-in-Aid for Scientific Research 05671560 from the Ministry of Education, Science and Culture of Japan, and a grant from Kirin Brewery.
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14. Naito, Y.; Suzuki, I.; Hasegawa, S. Induction of cystatin S in rat submandibular glands by papain. Comp. Biochem. Physiol. 102:861-865; 1992. 15. Newbrun, E. Observations on the amylase content and flow rate of human saliva following gustatory stimulation. J. Dent. Res. 41:459465; 1962. 16. Ninomiya, Y.; Funakoshi, M. Qualitative discrimination among umami and the four basic taste substances in mice. In: Kawamura, Y.; Kare, M. R., eds. Umami: A basic taste. New York: Mercel Dekker; 1987:365-385. 17. Ninomiya, Y.; Funakoshi, M. Peripheral neural basis for behavioral discrimination between glutamate and the four basic taste substances in mice. Comp. Biochem. Physiol. 92:371-376; 1989. 18. Ninomiya, Y.; Tanimukai, T.; Yoshida, S.; Funakoshi, M. Gustatory neural responses in preweanling mice. Physiol. Behav. 49:913-918; 1991. 19. Ninomiya, Y.; Kajiura, H.; Mochizuki, K. Differential taste responses of mouse chorda tympani and glossopharyngeal nerves to sugars and amino acids. Neurosci. Let. 163:197- 200; 1993. 20. Ogawa, H. Taste response characteristics in glossopharyngeal nerve of the rat. Kumamoto Med. J. 25: ! 37-147; 1972. 21. Shingai, T.; Beidler, L. M. Response characteristics of three taste nerves in mice. Brain Res. 335:245-249; 1985. 22. Smith, D. V.; Frank, M. Sensory coding by peripheral taste fibers. In: Simon, S. A.; Roper, S. D., eds. Mechanisms of Taste Transduction. Boca Raton: CRC Press; 1993:295-338. 23. Tabuchi, E.; Ono, T.; Nishijo, H.; Torii, K. Amino acid and NaCl appetite, and LHA neuron responses of iysine-deficient rat. Physiol. Behav. 49:951-964; 199 I. 24. Torii, K.; Mimura, T.; Yugari, Y. Biochemical mechanism of umami taste perception and effect of dietary protein on the taste preference for amino acids and sodium chloride in rats. In: Kawamura, Y.; Kate, M. R., eds. Umami: A basic taste. New York: Mercel Dekker; 1987:543-563. 25. Yamamoto, T.; Matsuo, R.; Kiyomitsu, Y.; Kitamura, R. Taste effects of umami substances in hamsters as studied by electrophysiological and conditioned taste aversion techniques. Brain Res. 451:147-162; 1988.