Gustatory neural responses in preweanling mice

Physiology&Behavior,Vol. 49, pp. 913-918. ©Pergamon Press plc, 1991. Printed in the U.S.A.

0031-9384/91 $3.00 + .00

Gustatory Neural Responses in Preweanling Mice YUZO NINOMIYA, TSUTOMU TANIMUKAI,* SADAHIRO YOSHIDA* AND MASAYA FUNAKOSHI

Department of Oral Physiology and *Pediatric Dentistry Asahi University School of Dentistry, Hozumi, Motosu, Gifu 501-02, Japan

NINOMIYA, Y., T. TANIMUKAI, S. YOSHIDA AND M. FUNAKOSHI. Gustatory neural responses in preweanling mice. PHYSIOL BEHAV 49(5) 913-918, 1991.--Taste sensitivity of preweanling mice was studied by examining responses of the chorda tympani (CT) and glossopharyngeal (GL) nerves to various taste stimuli, and was compared to that of adult mice. In mice of 7-10 days of age, comparing to that of the CT nerve, threshold of the GL nerve for monosodium l-glutamate (MSG) was low, but those for sucrose and NaCI were high. Sensitivities to HCI and quinine-HC1 were similar between the CT and GL nerves, although that to quinine-HC1 was larger in the GL nerve than in the CT nerve in adult mice. Enhancement of MSG responses by addition of GMP was observed in the CT nerve but not in the GL nerve in this age group. In mice of 8-16 weeks of age, threshold of the GL nerve for MSG became higher but that for NaC1 became lower. Enhancement of MSG responses by addition of GMP appeared also in the GL nerve. Inhibition of NaCI responses by amiloride was observed in the CT nerve. These results suggest that, in mice, the GL nerve is important taste input for umami substances especially during the preweanling period, whereas the CT nerve is for sweet and salty substances. Properties of umami and salt receptor systems change during the postweanling period. Preweanling mice

Taste sensitivity

Chorda tympani nerve

Glossopharyngeal nerve

Umami

METHOD

DIFFERENCES in responsiveness to taste stimuli between the chorda tympani (CT) and glossopharyngeal (GL) nerves have been reported in several mammalian species (6-8, 15, 18-20, 26, 27, 29). A common response property throughout mammalian species may be that quinine, a bitter substance, elicits a greater response in the GL nerve than in the CT nerve, and the reverse is true for NaC1. Recent behavioral and neurophysiological studies (14,15) have demonstrated that, in mice, the GL nerve is very important for behavioral discrimination between monosodium l-glutamate (MSG), a typical umami substance, and the four basic taste stimuli (NaC1, HC1, quinine-HC1 and sucrose), and there are several fibers in the GL nerve which responded to MSG, but only slightly if any at all responded to the four basic taste stimuli. These differential responses of the CT and GL nerves suggest possible differences in physiological function between the two taste inputs. During preweanling period, taste receptors on the GL nerve region (posterior one-third of the tongue) can be constantly stimulated by milk when the infant sucks on the mammary nipple, whereas receptors on the CT nerve region (anterior two-thirds of the tongue) would be rarely so stimulated. This suggests the possibility that differences in responsiveness between the GL and CT nerves are much more evident during this period. In the present study, therefore, taste sensitivity of the preweanling mice was examined by comparing responsiveness of the CT and GL nerves to the four basic taste and umami substances. Then, it was compared to that of the adult mice to investigate possible developmental changes in taste receptor systems.

Subjects and Stimuli Mice of C57BL/KsJ strain (male and female) were used in this experiment. Animals were divided into two age groups, one is a 7-10 days of age group weighing 5-7 g and the other is a 8-16 weeks of age group weighing 25--40 g. Taste stimuli used were: 1.0 m M - 1 . 0 M NaC1, 0.1 raM-1.0 M MSG, 1.0 m M - 1 . 0 M sucrose, 0.03 raM-0.01 M HCI, 0.03 mM--0.02 M quinine-HC1 (QHC1), 0.5 mM disodium 5'-guanylate (GMP), 0.1 M NH4C1, 0.1 M KC1, mixture of 0.1 m M - 1 . 0 M M S G and 0.5 mM GMP, and mixture of 0.1 M - 1 . 0 M NaC1 and 0.1 mM amiloride. These solutions were made in distilled water at about 20°C, and were flowed over the tongue (about 0.5 ml/s of flow rate) for 20-30 s for taste stimulation. To examine amiloride inhibition of NaC1 response, the tongue was treated with 0.1 mM amiloride for 5 min by running it over the tongue before the stimulation with the mixture of NaC1 and amiloride.

Procedure Each mouse was anesthetized with an intraperitoneal injection of sodium pentobarbital (40-50 mg/kg) and the trachea was cannulated. The CT nerve was exposed, freed from surrounding tissues, and cut at the point of its entry to the bulla. The GL nerve was dissected free and cut near its entry to the posterior lacerated foramen. For whole nerve recording, the entire nerve was placed on a silver wire electrode. An indifferent electrode was positioned nearby in the wound. Neural responses resulting from gustatory stimulation of the tongue were fed into an amplifier and displayed

913

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0.1M

0.1M

NH4C!

0.1M

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0.3M Suc

0.01M HCI

O.02M QHCI

20 sec

FIG. 1. Integrated responses of the chorda tympani (CT) and glossopharyngeal (GL) nerves in mice of 7 days and 8 weeks of age to 0.1 M NH4CI, 0.1 M NaCI, 0.1 M MSG, 0.3 M sucrose (Suc). 0.01 M HC1 and 0.02 M quinine-HCl (QHCI). on an oscilloscope screen. Whole nerve responses were integrated and displayed on a strip chart recorder. The time constant of the integrator used was 300 ms. For data analysis, the magnitude of the integrated response at 10 s after stimulus onset was measured. The relative response to each stimulus was calculated by taking the magnitude of response to 0.1 M NH4C1 as unity (1.0). RESULTS

Differences in Taste Sensitivity Between the CT and GL Nerve in the Preweanling and Adult Mice Integrated responses of the CT and GL nerves of mice of 7 TABLE 1 RELATIVE MAGNITUDES OF THE CHORDA TYMPANI AND GLOSSOPHARYNGEAL NERVE RESPONSES TO THE SIX TASTE STIMULI IN PREWEANLING (7-10 DAYS) AND ADULT MICE (8-16 WEEKS)

7-10 Days of Age Stimuli

CT Nerve

GL Nerve

8-16 Weeks of Age CT Nerve

GL Nerve

0.1 M NHaCI 1.00

1.00

1.00

1.00

0.1 MNaCI 0.1 MMSG 0.3 M sucrose 0.01 M HC1 0.02 M QHCI

0.55---0.16 0.85-+0.14" 0.20-+0.09:~ 0.94-+0.12 0.75+-0.22

0.94+-0.18 * 0.50+-0.09 0.79-+0.14 1.04-+0.14 0.56-+0.09

0.50+0.16+ 0.68-+0.12 * 0.21 -+0.09~: 1.02-+0.13 0.94-0.18"~

0.68-+0.10 0.62+-0.11 0.81-+0.09 1.01-+0.16 0.52-+0.10

Numerals are the mean - S.D., obtained from 4-6 experiments. *ttest, p<0.05; tp<0.01; :~p<0.001.

days and 8 weeks of age to 6 taste stimuli are shown in Fig. 1. The CT and GL nerves of mice responded well to taste stimuli even at 7 days of age. The magnitude of response to 0.1 M NHaC1 was fairly constant throughout different taste nerves and age groups. Responses of both CT and GL nerves of two age groups showed the initial dynamic phase followed by the steady phase. Magnitudes of dynamic responses of the GL nerve to the 6 stimuli were slightly greater than those of the CT nerve. This was probably because the GL nerve has higher sensitivity to mechanical stimulation than the CT nerve. Therefore, response magnitudes of steady phase (10 s after stimulus onset) were measured and the response magnitude to 0.1 M NH4CI was used as the standard for the comparison. The GL nerve tended to show longer " o f f responses" (after rinsing the tongue with distilled water) especially to MSG and QHC1 than the CT nerve in both age groups. Relative magnitudes of the CT and GL nerve responses to the 6 stimuli in the two age groups are presented in Table 1. In mice of 7-10 days of age, the response to 0.1 M MSG was significantly greater in the GL nerve than in the CT nerve (t-test, p < 0 . 0 5 ) , whereas the response to 0.3 M sucrose was four times greater in the CT nerve than in the GL nerve (t-test, p < 0 . 0 0 1 ) . In mice of 8-16 weeks of age, responses in 0.1 M NaC1 and 0.3 M sucrose were greater in the CT nerve than in the GL nerve (0.1 M NaCI: t-test, p < 0 . 0 1 ; 0.3 M sucrose: p < 0 . 0 0 1 ) , whereas the reverse was true for the response to 0.1 M MSG (t-test, p < 0 . 0 5 ) and 0.02 M QHC1 (t-test, p < 0 . 0 1 ) . A significant increment along with the development was observed only in the response of the CT nerve to NaC1 (t-test, p < 0 . 0 5 ) . Relations between concentrations of the five stimuli and the magnitudes of responses are shown in Fig. 2, In mice of 7-10 days of age, the threshold for MSG, roughly estimated from Fig. 2, is lower in the GL nerve

DEVELOPMENT OF TASTE RESPONSES

915

TABLE 2 ENHANCEMENT OF MSG RESPONSES BY ADDITION OF GMP IN THE CHORDA TYMPANI AND GLOSSOPHARYNGEAL NERVES OF PREWEANLING (7-10 DAYS) AND ADULT MICE (8-16 WEEKS) Stimuli 0.1 M MSG 0.5 mM GMP

7-10 Days of Age

8-16 Weeks of Age

CT Nerve

GL Nerve

CT Nerve

GL Nerve

Sum of of responses Response to mixture t-Test

0.72__-0.18 1.24---0.29 p<0.05

0.89_-0.16 0.91 --+0.16 n,s.

0.60__-0.14 0.84±0.10 p<0.05

0.58+__0.12 1.06__-0.20 p<0.01

Numerals are the mean _+ S.D., obtained from 4-6 experiments.

(0.1 mM) than in the CT nerve (1.0 mM), whereas thresholds for NaC1 and sucrose are lower in the CT nerve (1.0 mM and 10 mM) than in the GL nerve (10 mM and 0.1 M). Thresholds for HC1 and QHC1 are 0.03 mM or less in both nerves, showing no clear difference between two nerves. Difference in the threshold between the two age groups is observed only in that for MSG, which is about 1.0 log unit higher in the adult mice (from 0.1 mM to 1.0 mM). These results suggest that as a whole the GL nerve shows higher sensitivities to QHC1 and MSG but lower to sucrose and NaC1 than the CT nerve, and that NaCI responses of the CT nerve and QHC1 responses of the GL nerve slightly increase during development. Difference in sensitivity between MSG and NaC1 is the largest in the GL nerve of preweanling mice.

7 - 10

Developmental Changes in MSG Receptor Sensitivity Several electrophysiological studies in cats (1), rats (23,24) and mice (15) have demonstrated the occurrence of marked enhancement of the CT and GL nerve responses to M S G by the addition of G M P or disodium 5'-inosinate (IMP). Table 2 shows response magnitudes of the CT and GL nerves to the mixture of 0.1 M MSG with 0.5 mM G M P and the sum of the response magnitudes to either of the nerves alone. In the CT nerve, responses to the mixture of MSG with G M P are about 1.7 and 1.4 times greater than the sum of responses to each component stimulus in mice of 7-10 days and 8-16 weeks of age, respectively (t-test, p < 0 . 0 5 ) . This shows the typical enhancement of MSG responses by the addition of GMP. However, in the GL nerve,

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FIG. 2. Relationships of the stimulus concentration to magnitudes of responses for the five kinds of stimuli in the chorda tympani (CT) and glossopharyngeal (GL) nerves of mice of 7-10 days and 8-16 weeks of age groups. Each response magnitude was expressed relative to the response magnitude for 0.1 M NH4C1, and is the mean value obtained from 4-6 mice.

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FIG. 3. Relationships of the stimulus concentration to response magnitudes for MSG and the mixture of MSG and 0.5 mM GMP in the chorda tympani (CT) and glossopharyngeal (GL) nerves of mice of 7-10 days and 8-16 weeks of age groups. Each response magnitude was expressed relative to the response magnitude for 0.1 M NH4C1, and is the mean value obtained from 4-6 mice. Differences between responses to the mixture of MSG and 0.5 mM GMP and the sum of responses to each component were statistically examined by t-test. *p<0.05; **p<0.01.

such enhancement was observed only in the adult mice (1.8 times greater; t-test, p < 0 . 0 1 ) , and in the preweanling mice no significant difference was observed between responses to the mixture and the sum of response to each component. Figure 3 presents concentration-response relationships for MSG, the mixture of MSG with GMP and the sum of the responses to each component stimulus in the CT and GL nerves of the two age groups. In the CT nerve, concentrations of MSG showing the enhancement (ttest, p < 0 . 0 5 ) range from 3 mM to 0.3 M for mice of 7-10 days of age, and from 30 mM to 0.1 M for mice of 8-16 weeks of age, whereas in the GL nerve, they are from 10 rnM to 0.3 M for mice of 8-16 weeks of age, but no significant enhancement is observed in the concentration range of MSG from 0.1 mM to 1.0 M for mice of 7-10 days of age. These results suggest that taste receptors on the GL nerve region of the tongue, responsible for such enhancement, develop during postweanling period, instead of the postweanling reduction of sensitivity to MSG of lower concentrations.

Developmental Changes in NaCl Receptor Sensitivi~ Figure 4 shows responses of the CT and GL nerves of the two age groups to salts. The order of response magnitudes of the GL nerve to 0.1 M salts is NH4C1 > KC1 > NaC1 in both age groups, whereas that of the CT nerve is NH4C1 > KCI > NaC1 in 7-10 days group but is NH4C1 > NaC1 > KC1 in 8-16 weeks group. Responses of the CT nerve to 0,1 and 1.0 M NaC1 significantly increase during development. The lingual treatment with the epithelial sodium transport blocker amiloride suppressed 0.1 and 1.0 M NaC1 responses of the CT nerve by about 65% of the con-

trol in 8-16 weeks group, whereas no such suppression was observed in NaCI responses of the CT nerve in 7-10 days group and those of the GL nerve in both age groups. This suggests that, similar to the previous study in rats (11), the CT nerve responses of mice (C57BL/KsJ) to NaC1 increase during development with the addition of amiloride-sensitive components, whereas such components are not added to the GL nerve responses to NaCI. DISCUSSION

Differences in Taste Sensitivity Between the CT and GL Nerve in the Preweanling and Adult Mice The present study demonstrated that the sensitivity of the CT nerve to taste stimuli is different from that of the GL nerve in both preweanling and adult mice. In general the GL nerve shows higher sensitivities to MSG and QHC1 but lower sensitivities to sucrose and NaCl than the CT nerve. Differential responsiveness of the CT and GL nerves to QHCI, NaC1 and sucrose corresponds well with the data previously shown in rats (20,27), hamsters (6, 8, 18, 29), dogs (7) and mice (15,26). However, the higher sensitivity to MSG in the GL nerve is rather species-specific, which is observed only in mice [C57BL/KsJ strain in this study and Slc:ICR strain in the previous study (15)]. In rats, Ogawa (19) reported that the GL nerve shows the lowest sensitivity to MSG among several sodium salts. In hamsters, the GL nerve shows good responses to 0.3 M NaCI (8,18) but no clear response to 0.3 M MSG (29). In mice, the importance of the taste information from the GL nerve for taste discrimination between M S G and

DEVELOPMENT OF TASTE RESPONSES

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D-phenylalanine and D-tryptophan, and sugars, such as, sucrose and fructose. Therefore, it is probable that taste information of the GL nerve is very important for preweanling animals to perceive the taste of milk, and that in other interpretations, during this neonatal period repeated stimulations with milk determine the taste sensitivity of the GL nerve of mice throughout the animal's whole life.

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FIG. 4. Relative response magnitudesof the chorda tympani (CT) and glossopharyngeal (GL) nerves of mice of 7-10 days (open columns)and 8-16 weeks of age (shaded columns)to 0.1 M NH4C1 (NH4, as a unity: 1.0), 0.1 M KCI (K), 0.1 and 1.0 M NaC1 (Na) and the mixture of 0.1 and 1.0 M NaC1 and 0.1 mM amiloride (Na + Ami). Each value indicates the mean---S.E., obtainedfrom 4--6 mice. *t-test, p<0.05.

NaC1 was clearly demonstrated in the behavioral study using a conditioned taste aversion paradigm (14), in which behavioral responses to MSG were different from those to any of the four basic stimuli, but the denervation of bilateral GL nerve reduced behavioral discrimination between MSG and NaC1, although it was not the case after destruction of the CT nerve. Unlike mice, rats and hamsters, which have a lower sensitivity of the GL nerve to MSG, could not behaviorally discriminate between MSG and NaC1 because of a possible strong salty component taste common to both MSG and NaC1 for which information could be conveyed mainly by the CT nerve (28,29). The present study showed that differences in sensitivities to MSG and NaC1 are the most evident in the GL nerve of the preweanling mice. The GL nerve of the preweanling mice shows about two log units lower threshold for MSG than that for NaC1. At the considerably wide concentration range from 0.3 mM to 10 mM, the GL nerve responded to MSG without responding to NaC1. This high sensitivity to MSG at the physiological range of concentration suggests the importance of such amino acid for preweanling animals. Rassin et al. (21) reported that glutamate and glutamine are the most abundant free amino acids in human, chimpanzee and monkey milk, and are the third most abundant amino acids in mouse (C57BL/6J) milk, suggesting that they are one of most important components of milk for mammals. Our recent studies (Ninomiya et al., in preparation) suggest that in both preweanling and adult mice the GL nerve shows very high sensitivities to components of the milk, including MSG and other L-form essential amino acids, such as, L-leucine, L-histidine, L-phenylalanine and L-tryptophan, and sugars, such as, galactose and lactose, whereas the CT nerve strongly responded to sweettasting D-form amino acids, such as D-leucine, D-histidine,

In the adult mice, the threshold for MSG of the GL nerve, roughly estimated, was 1.0 mM, which was higher than that in the preweanling mice. This indicates a slight reduction of sensitivity to MSG during postweanling period. Instead of this, enhancing effects of GMP on MSG responses, which are not seen in the GL nerve of preweanling mice, became evident in responses of the GL nerve of the adult mice, although such enhancement was constantly observed in responses of the CT nerve of both preweanling and adult mice. This suggests that properties of MSG receptors responsible for the enhancement on the GL nerve region of the tongue are newly acquired during postweanling development. During the suckling period, taste receptors on the GL nerve region of the tongue would rarely be so stimulated by 5'-ribonucleotides, such as GMP and IMP, because they are not components of milk, whereas during the postweanling period taste receptors are stimulated by chemical compounds of foods, such as meats and plants, containing 5'-ribonucleotides. Under such condition, the postweanling foods, if available, possibly trigger the alternation of the properties of the MSG receptors on that region. The other possible factor to cause the developmental changes in the MSG receptors is the component of the saliva. Redman (22) described that, although mouse sublingual glands immediately after birth can function similarly to those of the adulthood, the functional maturation of the submandibular glands need at least 2 weeks after birth. Neonatal development of parotid glands is slowest among the three major glands. Significant secretion of the enzymes of parotid glands does not begin until the weanling process. Therefore, it is possible that developmental changes in the component of the saliva would induce changes in properties of the MSG receptors on the GL nerve region of the tongue. The amount of glutamate in the saliva of the adult rats increases during eating (K. Torii, personal communication). This may also affect the MSG sensitivity, possibly accounting for the observed slight reduction of the MSG sensitivity of the GL nerve during postweanling development.

Developmental Changes in NaCI Receptor Sensitivity The present study demonstrated that response magnitudes to 0.1 M salts were in the order of NHaC1 > KC1 > NaC1 for the GL nerve in the preweanling and adult mice and for the CT nerve in the preweanling mice, whereas they were in the order of NHaC1 > NaC1 > KC1 for the CT nerve in the adult mice, and that NaCI responses of the CT nerve increased during the postweanling development. In the adult animals, the order of sensitivities to salts for both CT and GL nerves corresponds quite well to that reported in the previous studies in rats (2,17), sheeps (4) and mice (24). The observed developmental increment of the NaC1 responses of the CT nerve in mice is also consistent with that reported in rats (5,10) and sheeps (13). Amiloride is known to be an inhibitor of passive sodium transport of various epithelial cells (3). Recent human psychophysical (25) and rat electrophysiological (9) studies demonstrated that this drug suppresses taste responses to NaC1. Hill and Bour (11) found that amiloride did not suppress responses of the CT nerve to 0.5 M NaC1 in rats at 12-13 days of age, but the drug suppressed the

918

N I N O M I Y A E'I AL.

NaC1 responses by about 50% o f the control in rats at 29-31 days or more of age. In the present study in mice, similar developmental changes in amiloride inhibition o f NaCI responses o f the CT nerve were observed, although NaC1 responses of the GL nerve were not inhibited by amiloride even in the adult mice. Ninomiya et al. (17) examined amiloride inhibition of NaC1 responses o f the CT nerve in four inbred strains (C57BL/6CrSlc, DBA/2CrSlc, BALB/cCrSlc and C3H/HeSlc), and found that the larger the NaC1/KC1 response ratio in the mouse strain, the greater the magnitude o f amiloride inhibition of NaC1 responses. This is also true in this study on the CT and GL nerve responses of mice (C57BL/ KsJ strain) of two age groups and the CT nerve responses o f rats (11), suggesting that the relation between NaCI/KC1 response ratio and the magnitude of amiloride inhibition of NaCI responses is fairly constant throughout species, ages and the taste nerves. Therefore, it is probable that in this strain o f mice during maturation the amiloride-sensitive components add the NaC1 receptors

on the CT nerve region of the tongue, but did not on the (iL nerve region, and that KCl receptors on both CT and GL nerve regions show no clear developmental change. Induction o f the amiloride-sensitive NaCI receptor components in rats was suppressed by deprivation of dietary sodimn (12) and was enhanced by desalivation of the sublingual glands ~16) during the preweanling period. Taking these facts together with the present result that the GL nerve sensitivity fits well Io perceive the taste of milk, it is suggested that functional maturation o f the taste receptor system considerably depends on environmental stimulation during the preweanling period. ACKNOWLEDGEMENTS We are grateful to Drs. Sako and Nomura for their technical assistance. This study is supported in part by Grants-in-Aid for Scientific Research (No.63304063, 63480418 and 01304045) from the Minist~ of Education, Science and Culture of Japan.

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