Taste effects of ‘umami’ substances in hamsters as studied by electrophysiological and conditioned taste aversion techniques

Brain Research, 451 (1988) 147-162

147

Elsevier BRE 13598

Taste effects of 'umami' substances in hamsters as studied by electrophysiological and conditioned taste aversion techniques Takashi Yamamoto 1, Ryuji Matsuo l, Yoshitaka Kiyomitsu 2 and Ryuji Kitamura 3 Departments of 1OralPhysiology, 2DentalAnesthesiology and 3OralSurgery, Facultyof Dentistry, Osaka University, Osaka (Japan) (Accepted 1 December 1987)

Key words: Umami; Taste quality; Conditioned taste aversion; Chorda tympani; Glossopharyngeal nerve; Single fiber analysis

Behavioral and electrophysiological experiments were performed to examine whether or not the taste of 'umami' substances such as monosodium glutamate (MSG), disodium 5'-inosinate (IMP), and disodium 5'-guanilate (GMP) is really unique in hamsters. When the animals were conditioned to avoid ingestion.of MSG (or IMP) or their mixture by pairing its ingestion with an i.p. injection of LiCI, suppression of drinking generalized to IMP (or MSG), GMP, NaCI, and other sodium salts. Suppression of drinking after conditioning to NaCI generalized to MSG, IMP, GMP, and inorganic sodium salts. These learned aversions to umami substances and sodium salts were abolished by bilateral deafferentation of the chorda tympani, but were not affected by destruction of the bilateral glossopharyngeal nerves. The integrated whole-nerve responses of the chorda tympani to MSG, IMP, and NaCI were similar to each other, consisting of the initial dynamic phase and the following tonic phase. Synergism of chorda tympani responses to a mixture of MSG and IMP was not observed. Across-fiber response patterns of the chorda tympani for MSG, IMP, or their mixture were very similar to that for NaCi. Even the high concentrations of umami substances (0.3 M MSG, 0.3 M IMP, and the mixture) did not elicit any detectable responses in the glossopharyngeal nerve. These results suggest that the taste of umami substances is not unique in the hamster, but is similar to that of sodium salts, and is mediated exclusively via the chorda tympani. INTRODUCTION Sea tangle has been used as a natural seasoning in the Japanese cuisine for a long time. In 1909, Ikeda 25 first extracted monosodium L-glutamate (MSG) from sea tangle (Laminaria japonica), and identified it as an essential element for the flavor enhancing effect of sea tangle. Besides its seasoning effect, M S G possesses a unique taste named ' u m a m i ' in Japanese by Ikeda, which means 'delicious taste' or 'savory taste'. Later in 1913, K o d a m a 29 identified 5'-inosinate from bonito flakes, which is now considered to be another key ingredient in umami. According to the results of subsequent studies (see refs. 34, 74 for reviews) following these pioneering works, the most typical umami substances are now divided into two groups: a group of L-amino acids represented by MSG, and another of 5'-ribonucleotides and their derivatives represented by disodium 5'-inosinate (or inosine 5'-

monophosphate, IMP), and disodium 5'-guanylate (or guanosine 5'-monophosphate, GMP). These substances elicit hedonically preferable taste effects both in humans 65,76 and animals 36'46-48'7°, although the hamster 66 showed much less preference for M S G than mouse and rat 66'69. Several investigators 15'59'75,76's6, using the multidimensional scaling method in humans, have pointed out that umami is a unique taste which does not belong to any of the classical 4 basic taste qualities, i.e. sweet, salty, sour and bitter. Yamaguchi 75 insists that umami is the 5th fundamental taste which necessitates construction of another dimension independent of the 4 basic tastes. In animal experiments, using mice of the SIc:ICR strain, Ninomiya and Funakoshi 39 also suggested that the taste of umami substances was independent of the 4 basic tastes. In favor of these suggestions, single fiber analyses of the taste nerve fibers through electrophysiological ex-

Correspondence: T. Yamamoto, Department of Oral Physiology, Faculty of Dentistry, Osaka University, 1-8 Yamadaoka, Suita, Osaka 565, Japan. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

148 periments showed that some fibers in the chorda tympani of the cat ~ and in the glossopharyngeal nerve of the Slc:ICR mouse 39 were especially more sensitive to umami substances than the conventional 4 basic taste stimuli. However, the chemical structure of MSG suggests that this substance has a potency of eliciting any of the 4 basic taste qualities 9. Actually, human judgement of the taste of umami substances, especially in Western people, does not always indicate that this taste is unique, but may be described by single or combinations of words conventionally used for expressing the taste qualities 6'~7'5°. Although the lack of a codable term for the taste of umami substances in the West may reflect cultural differences with different cuisines between Oriental and Western people 5°, there seems to be some evidence for the reports 19' 33.63 that the taste of MSG is related to salty and/or sweet components. For example, Kurihara 32 showed that the taste of MSG changed to salty taste after treatment of the tongue with gymnemic acid which is known to abolish only sweet taste. In animal experiments, single unit analyses of rat taste fibers showed that umami substances induced good responses in fibers sensitive to sucrose ~5,57 or NaC11. Responses to umami substances were highly correlated with those to NaCI in cortical taste-responsive neurons in the rat 84 and hamster 82, indicating that these animals are unable to discriminate the taste of umami substances from that of NaC1. Moreover, Boudreau 1° could not find any taste units in the peripheral taste nerves t h a t responded specifically to umami substances in the rat, cat, dog and goat. There are thus some inconsistencies in defining the properties of the taste of umami substances among researchers and among species of animals. When the two groups of umami substances are mixed, umami of the mixture becomes stronger than the sum of umami of the individual components in the mixture. Such a synergistic effect was first reported by Kuninaka 3°,31 in the 1960s in human subjects using a mixture of MSG and IMP or GMP. This phenomenon has been studied extensively in human psychophysical studies 31'73,77-79, and in electrophysiological and biochemical studies using a variety of animals including the carp 87, mouse 39, hamsters6, rat 52-55'57's5, cat 2-4'27, and bovine 12,68. According to the results of animal experiments, the synergistic taste responses

occur at the receptor level rather than in the central nervous system. Sato et al. 56, however, showed that the synergistic mixture effect was smaller in hamsters than in rats, suggesting a possible existence of species differences in synergism as well. To examine whether or not umami really elicits a unique taste independent of the 4 basic tastes in animals, we have tried to evaluate this taste in terms of two major issues, i.e. its taste quality, and its synergism in mixture. The hamster was used in the present study, since this animal is widely used in the field of taste physiology, and is known to have similar taste sensitivity to the four basic tastes as in humans 43, but is suggested to have somewhat different sensitivity to umami substances from other animals as described above. We carried out electrophysiological experiments to analyze taste nerve responses to umami substances, and behavioral experiments by means of a conditioned taste aversion technique. The taste aversion methodology, where animals conditioned to avoid a stimulus also avoid other stimuli with similar tastes, has been proved to be useful to determine a behavioral categorization of taste qualities 14'16'38'41'51'64'67'84.Preliminary results of this study were reported elsewhere 35,83. BEHAVIORAL EXPERIMENT

Materials and methods Male golden hamsters, weighing 80-100 g at the beginning of the experiments, were used. Animals ate solid pellets (MF, Oriental Yeast, Osaka) ad lib throughout the experiment, but they were given access to water during training sessions and water plus taste solutions during testing sessions. The animals were trained and tested in a 25 x 25 x 32-cm test box, which had a single drinking tube attached to an outer wall of the box. The glass spout (3 m m i.d.) of this tube was fixed 2 mm outside a round opening (7 mm diameter) at one end of the box. The center of the opening was 5 cm above the wire-mesh floor and 12.5 cm from the sides. The taste solutions, made with distilled water, were (in M): sucrose 0.1, NaCI 0.03, HC1 0.003, quinine hydrochloride 0.001, sodium saccharin 0.005 and 0.03, CH3COONa 0.03, Na2SO 4 0.03, N a N O 3 0.03, KCI 0.03, CaCI 2 0.03, NH4C1 0.03, MSG 0.03, monopotassium L-glutamate (MPG) 0.03, monoam-

149 monium L-glutamate (MAG) 0.03, monocalcium diL-glutamate (MCG) 0.03, IMP 0.03, GMP 0.03, glycine (Gly) 0.5, L-proline (Pro) 0.5, L-alanine (Ala) 0.5, L-aspartic acid (Asp) 0.01, L-glutamic acid (Glu) 0.01, L-histidine (His) 0.05, L-glutamine (Gin), Lphenylalanine (Phe) 0.1, L-tryptophan (Trp) 0.05, and L-arginine (Arg) 0.05. Quinine hydrochloride was Japanese pharmaceutical grade; MSG, MPG, MAG, MCG, IMP and GMP (purity, 99%) were supplied from Ajinomoto company (Tokyo), and the rest of the chemicals were reagent grade. The concentration of each chemical was fixed throughout the experiments unless otherwise noted. All stimuli were presented at room temperature. The animals were deprived of water for 23 h, and were trained to drink for 1 h only in the test box during the first 5 days (training session). On the 6th day, conditioned taste aversion was induced; each animal was given an intraperitoneal injection of 0.15 M LiCI (2% of body weight) soon after a sufficient intake of one of the taste stimuli listed above. The 7th day was a recovery period, during which the animal ate ad lib in its home cage, and drank water for 1 h in the test box. On the 8th, 9th and 10th days the generalization of the conditioned taste aversion was tested using the taste solutions. Each test stimulus was licked for 20 s, and the number of licks was counted by a capacitive sensor 37. The intertrial interval was at least 3 min. The mean number of licks across the 3 test days was obtained for each of the test solutions in each hamster. The behavioral data were quantified by expressing the aversion as a ratio of the standard and postconditioning licking rates to each taste stimulus 43. The hamsters presented with distilled water as a conditioning stimulus provided the standard for the number of licks to each test stimulus. Suppression of drinking of a test stimulus was expressed as a suppression ratio according to the formula suppression ratio = 1-(no. of licks/20 s in E)/(no. of licks/20 s in C) where E is for the experimental group, which received sapid solutions as conditioning stimuli, and C is for the control group in which distilled water was a conditioning stimulus. Suppression ratio multiplied by 100 gives percent suppression ratio.

Generalization of aversion among amino acids Procedure. The animals were randomly divided into 17 groups of 5 to 6 hamsters each. The taste solutions were 4 basic taste solutions (sucrose, NaCI, HC1 and quinine hydrochloride), 2 umami substances (MSG and IMP), and 10 amino acids (Gly, Pro, Ala, Asp, Glu, His, Gin, Phe, Trp and Arg). Hamsters in each group were conditioned to avoid one of these 16 taste stimuli and distilled water, forming 16 experimental groups (n = 5 in each group) and one control group (n = 6), and were tested for generalization of aversion to each of these stimuli. When Asp, His, and Arg were used as conditioned stimuli, some animals could not avoid these chemicals within 10 s after the first lick by single paring of the ingestion with LiCI injection. In this case, a repeated conditioning was performed until they acquired a firm conditioned taste aversion learning. Only single pairing was used throughout the subsequent experiments. Results and discussion. The mean suppression ratios calculated in each group are summarized in Fig 1. The dotted columns indicate that the number of licks after conditioning was statistically significantly (P < 0.05, Mann-Whitney U-test) smaller than in the control animals. The generalization pattern of suppression of licking was very similar, i.e. only intakes of IMP, MSG, and NaC1 were greatly suppressed, when the aversions were conditioned to either IMP, MSG or NaCI, except that intakes of Gly and Ala were slightly suppressed after learned aversion to IMP. Besides the large reduction in intake of IMP by aversions to the three taste stimuli, a small, but statistically significant, decrease (about 40% of the control) in intake of IMP was also induced by conditioning to Pro and Gly, while conditioning to other taste stimuli did not influence the licking rate to IMP. Similar resuits were obtained for MSG except that learned aversions to Ala and Gln, but not Pro and Gly, generalized to MSG. Intake of NaCI was reduced only when the animals were conditioned to avoid NaCI itself, MSG or IMP. We calculated Pearson's product-moment correlation coefficients between pairs of all the 16 taste stimuli. These correlations were used for a multidimensional scaling analysis, and production of a dendrogram. Fig. 2A shows a dendrogram. When a correlation at 0.62 (1% significance level) is taken as the critical level, several stimuli were clustered into

150

Suc

suc Pro

Gly Ala IMP MSG NaCI Gin His Asp HCI Glu Arg Trp Phe Qui

I

Pro

Gly

Ala

IMP

MSG

NaCI

Gin

I I

m EE

0 50 100

0 50 100

0 SO 100

0 50 100

0 50 100

0 SO 100

0 50 100

Suc

0 S0 100

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Pro Gly Ala IMP MSG NaCI Gin His Asp HCI Glu Arg Trp Phe Qui

0 50 100

0 50 100

0 50 100

0 50 100 0 50 100

0 50 100

0 50 100

0 50 100

Suppression Ratio (%) Fig. 1. Mean suppression ratios across 16 taste stimuli after aversionswere conditionedto each of the stimuli. The conditionedstimulus is indicated on the top of each graph. Suc, 0.1 M sucrose; Pro, 0.5 M proline; Gly, 0.5 M glycine; Ala, 0.5 M alanine; IMP, 0.05 M IMP; MSG, 0.05 M MSG; NaC1, 0.03 M NaCI; Gin, 0.2 M glutamine; His, 0.05 M histidine; Asp, 0.0t M aspartic acid; HC1,0.003 M HCI; Glu, 0.01 M glutamic acid; Arg, 0.05 M arginine; Trp, 0.05 M tryptophan;Phe, 0.1 M phenylalanine; Qui, 0.001 M quinine hydrochlodde. Dotted columnsindicate that the licking rate of a test solution was suppressed by a significant (P < 0.05) percentage.

groups: sucrose-Pro-Gly group, NaC1-MSG-IMP group, HC1-GIu-Asp group, and quinine-Phe-Trp group. Some of these groupings are known to show similar taste in human psychophysical studies 28'6°. Ala, His, Gin and Arg are more independent and do not belong to any group. However, when the correlation level is set at 0.3, the taste stimuli were clustered into 4 groups corresponding to sweet, salty, sour and bitter tastes in human sensation. At the level of 0, the taste stimuli were divided in two groups, probably reflecting hedonics of taste, i.e. pleasant and un-

pleasant, or acceptable and rejective. Fig, 2B shows a taste space indicating the spatial representation of the similarities among the 16 stimuli, which was obtained through multidimensional scaling. The locations of the 4 basic taste stimuli (sucrose, NaCl, HCt and quinine) are spread widely in the space, and the taste tetrahedron can be roughly formed (dotted lines). The peak of the tetrahedron is taken at the center of each of the 4 dusters depicted from the dendrogram. The clusters are indicated by circles in the taste space.

151

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generalization of aversion to sucrose, sodium saccharin, CH3COONa , Na2SO4, NaNO3, NaCl, IMP, MSG, KCI, CaCl2, NH4CI and a mixture containing 0.024 M (0.45%) MSG and 0.0026 M (0.11%) IMP. Results and discussion. As shown in Fig. 3, intakes of all the 8 sodium salt solutions were suppressed statistically significantly. However, the rate of licking for the low concentration of sodium saccharin, sucrose, KCI, CaC12 and NH4CI did not deviate from the comparable range of control licking rates. In order to dissociate the taste effects produced by cations (Na ions) and anions of umami substances, the following several behavioral experiments were performed. Conditioning to mixture of MSG and IMP If the mixture of MSG and IMP enhances umami responses in hamsters as has been reported in humans3°'31'74, conditioning to the mixture is expected

Glu

t

( O.03M MSG )

SUC 0.005

Sacch

0.03 Sacch CH3COONa Na2S04 NAN03

Fig. 2. Behavioral similarity of taste stimuli as expressed lay a dendrogram (A) and a 3-dimensional taste space obtained through multidimensional scaling (B). Value of stress for the space was 0.0300. Abbreviations are the same as those used in Fig. I except S, N, H, Q, M, and I for sucrose, NaCl, HCI, quinine, MSG and IMP, respectively.

NaCI

IMP i !iii ililiii iilii~~ ii i~i~ilii~ i ~iii!iiii!~iii!i|

MSG M+I

KCI " ~ CaCI 2

The results described above suggest that behavioral similarity of the tastes of MSG, I M P and NaC1 is attributed to taste effectiveness of Na ions c o m m o n l y contained in these three chemicals. In the next experiment, therefore, we have e x a m i n e d generalization of aversions among different salts including a variety of sodium salts after conditioning to MSG.

Generalization of aversion among salts Procedure. Ten hamsters were divided into two groups of 5 each, and were conditioned to avoid either 0.03 M MSG or distilled water, and tested for

NH4Cl ---~ f

-20

0

2'o 4'0

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Suppreasion Ratio

%

Fig. 3. Pattern of suppression of licking across the 13 taste stimuli after aversion to 0.03 M MSG was conditioned. Each column with a bar represents mean suppression ratio + S.E.M. (n = 5). Suc, 0.1 M sucrose; 0.005 Sacch, 0.005 M Na saccharin; 0.03 Sacch, 0.03 M sodium saccharin; CH3COONa, 0.03 M CH3COONa; Na2SO4, 0.03 M Na~SO4; NaNO 3, 0.03 M NaNO3; NaCI, 0.03 M NaCl; IMP, 0.03 M IMP; MSG, 0.03 M (0.56%) MSG; M + I, 0.024 M (0.45%) MSG + 0.0026 M (0.11%) IMP; KCI, 0.03 M KCI; NH4CI, 0.03 M NH4CI. Dotted columns indicate that the suppression of licking was statistically significant.

152 to generalize to other umami substances as well as, or rather than the sodium salts. Procedure. Eight hamsters were divided into 2 groups of 6 each, and were conditioned to avoid either distilled water or a mixture containing 0.024 M (0.45%) MSG and 0.0026 M (0.11%) IMP, and tested for generalization of aversion to NaCI, MSG, IMP, the mixture, MPG, M A G and MCG. Results and discu/ssion. After acquisition of learned aversion to the mixture of MSG and IMP, suppression of licking to 0.03 M NaC1 was similarly predominant (76 + 7, mean + S.E.M., n = 6) as to 0.03 M IMP (90 + 6), 0.03 M MSG (87 + 4), or the mixture (conditioned stimulus) (79 + 9). Licking rates to other umami substances without Na ions such as MPG, MAG, and MCG were not affected at all, with the suppression scores of 0 + 8, - 2 + 7 and - 8 + 9, respectively. These results again indicate that the hamsters cannot detect differences in taste between NaC1 and the umami substances, and that the mixture of MSG and IMP does not enhance umami responses in this species.

Conditioning after preexposure to MS G or NaCl Acquisition of conditioned taste aversion learning is most effective when conditioned stimulus is the first experience to the animals. If the conditioned stimulus is familiar, it is not easy to establish an aversive conditioning for the stimulus. Taking advantage of this phenomenon, we tried to establish a conditioned taste aversion to MSG or NaCI in two groups of hamsters which drank either of these taste stimuli ad lib for a month. Procedure. Fifteen hamsters were assigned randomly to one of 3 groups, 5 in each group, to examine the effect of taste preexposure on conditioned taste aversions. The 1st group received 0.05 M MSG for 30 days, the 2nd group, 0.03 M NaC1 for 30 days, and the 3rd group, distilled water for 30 days prior to conditioning to NaCI, MSG, and distilled water, respectively. Each liquid was presented during the 30 days of the preexposure period in their drinking tube, which was available ad lib. Generalization of aversion was tested to NaCI, MSG and IMP. Results and discussion. After the preexposure of MSG, the animals could not establish a learned aversion to NaCI as well as MSG by the ordinary one pairing of ingestion of this conditioned stimulus and LiCI

injection. After several pairings, the animals acquired the aversion to NaCI with the suppression ratio of 89 + 8% (mean + S.E.M., n = 5). At this time, however, generalization of aversion occurred to MSG and IMP with the suppression ratios of 85 + 9% and 82 +_ 10%, respectively, but not to other test stimuli including other basic tastes and the amino acids. Essentially the same results were obtained when the animals were conditioned to avoid MSG after a month's preexposure to NaCI. The suppression ratios were 72 _+ 8%, 69 + 3%, and 72 + 6% for NaCI, MSG and IMP, respectively, after the aversion was conditioned to MSG with several trials of the pairings. These results show that the animals were unable to dissociate NaC1 and the umami substances on the basis of their tastes.

Effects of chorda tympani denervation In a further attempt to examine whether or not hamsters can discriminate the tastes of NaCI and umami substances, denervation experiments were performed. Procedure. Twenty-five hamsters were divided into 5 groups of 5 each. Three groups of hamsters received bilateral lesions of the chorda tympani in the middle ear after removing the tympanic membrane and ossicles. The remaining two groups were used as normal control groups, and were conditioned to dis, tilled water. Two of the 3 experimental groups were conditioned to avoid either 0.03 M MSG or 0.3 M MSG after denervation, and the rest was conditioned to avoid 0.03 M MSG before denervation. The animals conditioned to 0.03 M MSG or distilled water were tested for generalization of aversion to sucrose, NaC1, HCI, quinine hydrochloride, MSG, IMP, GMP, MPG, MAG, MCG, and a mixture of 0.024 M MSG and 0.0026 M IMP and the animals conditioned to 0.3 M MSG or distilled water were tested using 0.3 M sucrose, 0.03 M sodium saccharin, 0.3 M NaNO, 0.3 M NaCI, 0.3 M MSG, 0.3 M IMP, 0.3 MPG, 0.3 M MAG, 0.3 M MCG, 1.0 M Ala, 0.3 M Gin, 0.05 M Glu, and a mixture of 0.24 M MSG and 0.026 M IMP. Results and discussion. When the bilateral chorda tympani were cut after acquisition of learned aversion to MSG (or conditioning was before denervation), conditioning effects which had been generalized to sodium salts were completely abolished (Fig. 4). It was also noted that water licking was sup-

153

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NaCl

BB--*j

IMP MSG

=

GMP

R-, i

M÷I MPG MAG

MCG HCI Qui f

-20

iI

o

20

4'0

6'o

Suppression Ratio

8'0

,60

%

Fig. 4. Effects of deafferentation of the chorda tympani on conditioned taste aversion learning acquired preoperatively. Patterns of suppressions of licking across the 11 taste stimuli after conditioning to 0.03 M MSG are shown by open columns, and dotted columns indicating statistically significant suppression of drinking. Solid columns indicate the suppression scores after operation in the same hamsters, Asterisks represent that the lickings were statistically significantly suppressed than those in the control animals. Dotted line indicates the mean suppression ratio for water licking after operation. Each column shows mean + S.E.M. (n = 5). Suc, 0.1 M sucrose; NaCI, 0.03 M NaCI; IMP, 0.03 M IMP; MSG, 0.03 M MSG; GMP, 0.03 M GMP; M + I, 0.024 M MSG + 0.0026 M IMP; MPG, 0.03 M MPG; MAG, 0.03 M MAG; MCG, 0.03 M MCG; HC1, 0.003 M HC1; Qui, 0.001 M quinine hydrochloride.

Since there is a possibility that the lack of conditioning effects after d e n e r v a t i o n of the chorda tympani is attributed to the relatively low concentrations of taste stimuli, we used m o r e c o n c e n t r a t e d chemical solutions to see the conditioning effects after denervation. A s shown in Fig. 5, after aversion was conditioned to 0.3 M M S G in the 3rd group animals, suppression of licking generalized to 0.3 M M S G and high concentrations of sodium salts except 0.03 M sodium saccharin, with suppression ratios below 50%. Licking to M A G was also suppressed at the statistically significant level ( P < 0.05), while intake of a mixture of M S G and I M P was not suppressed at the significant level. It is again shown in the graph that intakes of water and the test stimuli were suppressed by about 20% after d e n e r v a t i o n of the chorda t y m p a ni. The low suppression ratios m a y be due to a certain

( 0.3MMSG) Ala Suc Sacch

L

NaNO3 NaCI MSG

~

IMP MPG

pressed by 26% of the pre-dissection value, and the licking rates to the taste stimuli were similarly suppressed by 19 + 5 % ( m e a n + S . D . , n = 11), ranging from 10 to 26%. A f t e r d e n e r v a t i o n , the animals became cautious and t o o k frequent pauses during 20 s of licking period. T h e suppression of licking rates, therefore, is largely a t t r i b u t e d to these intermission of rhythmical licking. A n o t h e r group of hamsters, which were conditioned to M S G after the bilateral c h o r d a t y m p a n i h a d been sectioned, acquired no l e a r n e d aversions to any of the test stimuli, but the suppression of intake of about 20% of the control values was again d e t e c t e d for all the test stimuli and water as was the case shown in Fig. 4. N o t e that the hamsters that acquired no aversion following c h o r d a t y m p a n i sections h a d received only one conditioning trial.

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20

4'o

6'o

Suppression Ratio

8o

1~o

%

Fig. 5. Patterns of suppression of licking across the 13 taste stimuli after aversion to 0.3 M MSG was conditioned in chorda tympani-denervated hamsters. Each column with the bar represents mean suppression ratio + S.E.M. (n = 5). Note that the concentrations of the test stimuli are high in this experiment; Suc, 0.3 M sucrose; Ala, 1.0 M alanine; Sacch, 0.03 M sodium saccharin;NaNO3, 0.3 M NaNO3;NaCI, 0.3 M NaCI;MSG, 0.3 M MSG; IMP, 0.3 M IMP; M + I, 0.24 M MSG + 0.026 M IMP; MPG, 0,3 M MPG; MAG, 0,3 M MAG; MCG, 0.3 M MCG; Gln, 0.3 M glutamine; Glu, 0.05 M glutamic acid. Dotted columns indicate that the licking rate of a test solution was suppressed by a significant percentage. Dotted line indicates the level of suppression of water licking as compared to licking rate of water in control animals.

154 period of time, during which animals continued to lick, for the ingested solution to diffuse and stimulate the taste buds in the back of the oral cavity.

Effects of glossopharyngeal denervation Procedure. Four hamsters were conditioned to avoid MSG after section of the bilateral glossopharyngeal nerves in the neck under the digastric muscle. The control animals and the test stimuli werethe same as Used in the chorda tympani denervation experiment. Results and discussion. In hamsters with the glossopharyngeal nerves bilaterally denervated, conditioned taste aversion to 0.03 M MSG generalized to other chemicals containing Na ions, but not to other umami substances with different cations (Fig. 6), The generalized suppression ratios ranged from 70 to 87%, which were quite similar to those in intact animals as shown in Fig. 4, indicating that the glossopharyngeal nerve plays little role in the acquisition of the learned aversion to MSG.

(O.03M MSG after GN Cut) Suc

NaCI IMP

MSG GMP M+I

MPG MAG MCG

J-

HCI Qui

-20

~ 0

2'0

1(~0 % Fig, 6. Patterns of suppression of licking across the 11 tastO stimuli after aversion to 0.03 M MSG was conditioned in glossopharyngeal-denervated hamsters, Each eohman with the bar represents m e a n suppression ratio + S.E.M. (n = 4). Abbreviations are the same as those used in Fig. 4. Note that the 40

dO

dO

Suppression Ratio

number of licks to water was not affected by denervation of the glossopharyngealnerve.

ELECTROPHYSIOLOGICAL EXPERIMENTS

Materials and methods A total of 15 golden hamsters (90-120 g) were deeply anesthetized with an intraperitoneal injection of a mixture of sodium pentobarbital (65 mg/kg) and urethan (500 mg/kg); 10 for recording from the chorda tympani and 5 from the glossopharyngeal nerve. The trachea was cannulated and the hypoglossal nerves were cut bilaterally. The chorda tympani, which innervates the taste buds on the anterior part of the tongue, was exposed by the conventional lateral approach 8°, freed from surrounding tissues, and cut just peripheral to its entry to the bulla. The glossopharyngeal nerve, which innervates the taste buds on the posterior part of the tongue, was exposed and cut near its entry to the posterior lacerated foramen. The whole-nerve responses to taste stimulation were recorded differentially against an indifferent electrode attached to the surrounding tissue, displayed on a pen-recorder as integrated responses or pulse counts. A single or a few fibers of the chorda tympani were dissected with a pair of forceps, and their electrical activities were similarly recorded. Impulse discharges were stored on magnetic tape for later analysis. The whole taste nerve responses were summated by use of a summator (or integrator, Nihonkohden, EI-600G) (time constant: 0.5 s). The height from the baseline was measured at the initial peak for dynamic response, and at 15 s after onset of stimulation for steady response. For data analysis of impulse discharges of single chorda tympani fibers, the number of impulses were counted. The background firing rate was computed for each fiber by averaging the number of impulses per 1-s bin during stimulation with distilled water. When the response rate of a fiber during the first 3 s after onset of stimulation exceeded the mean + 2 S.D. of this fiber, the fiber was considered to be responsive. The taste solutions were various concentrations of sucrose, NaC1, HCI, quinine hydrochloride, MSG, IMP, and a mixture of MSG and IMP. The chemical grade for each chemical is the same as described above. Each stimulation was about 3 ml of a test solution passed by gravity flow (10 ml/s) from a burette held about 5 mm above the center of the exposed tongue surface in the case of recording from the chor-

155 da tympani. Taste stimulation of the posterior tongue, for recording from the glossopharyngeal nerve, was performed by pouring taste solutions on both the foliate and the circumvallate papillae after exposing them under a stereoscopic microscope. We also recorded taste responses of the whole chorda tympani to mixtures of MSG and IMP in 3 Wistar male rats (200-300 g). The synergistic mixture effects were compared in rats and in hamsters.

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Results and discussion Whole-nerve responses of the chorda tympani. Fig. 7 shows the representative integrated responses of the whole chorda tympani to MSG, IMP, and NaCI applied to the anterior region of the dorsal tongue, and concentration-response curves obtained from the whole chorda tympani for each of the 3 taste stimuli. The magnitudes of the standard solutions used for the other basic taste stimuli in the following single-fiber analyses are also shown in the graph. IMP, MSG and NaCI generally showed very similar responses, with the following few differences, in terms of their time courses after onset of stimulation, and concentration-response relations. IMP induced larger responses than MSG when compared at the same concentrations. Another difference between the responses to MSG and IMP is that IMP responses showed a slow recovery to the prestimulus background level. An 'off' type response was induced at the onset of water rinsing after IMP stimulation (see inset actual recording in Fig. 7). MSG response, on the other hand, returned to the background level soon after water rinsing just like NaCI response. At concentrations below 0.03 M, IMP responses were larger than NaCI responses, while the reverse was true above 0.03 M. Responses to MSG, IMP and their mixture were recorded to examine the existence of synergistic mixture effect. Whole-nerve responses at the initial peak (dynamic phase) and 15 s after onset of stimulation (steady phase) were compared using 0.1%, 0.3% and 1% solutions containing 6 different proportions of IMP and MSG in each. As graphically represented Fig. 8, the magnitudes of responses to mixtures were much smaller than the sum of those to each component of the mixtures in both initial peak and steady responses. These results obtained in the hamster were in contrast to those in the rat obtained from

/

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i

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Fig. 7. Concentration-response curves obtained f r o m the chorda tympani f o r NaCI, MSG, and I M P . Each value, the mean o f 5 trials, is shown as a relative magnitude of integrated response, where the response to 0.03 M NaC! is taken as 100. The mean relative magnitudes of responses to 0.1 M sucrose, 0.003 M HCI, and 0.001 M quinine are shown by the concentric circles. Inset figures represent examplesof the whole chorda tympani responses to 0.03 M NaCl, 0.03 M IMP, and 0.03 M MSG, when the solutions were applied to the tongue for 20 s as indicated by underlines.

exactly the same experimental procedure in this study, e.g. the ratio (facilitation ratio) of the magnitude of response to the mixture consisting of 80% MSG and 20% IMP to the sum of each component response was 1.1, 2.0 and 1.2 for dynamic responses of 0.1%, 0.3% and 1% solutions, respectively, and were 1.4, 1.9 and 1.5 for steady responses of 0.1%, 0.3% and 1% solutions, respectively. These facilitation ratios are within the same range in rats reported by Sato et al. 52'54.

Whole-nerve responses of the glossopharyngeal nerve. Thresholds for glossopharyngeal responses to taste stimuli applied to both the foliate and circumvallate papillae were much higher than those of the chorda tympani. Responses increased gradually after onset of stimulation. Examples of integrated glossopharyngeal nerve responses are shown in Fig. 9. The largest response was induced by high concentrations of HC1, followed by NaCI and quinine responses. Sucrose responses were smallest even with very high concentration. Essentially no responses were observed for 0.3 M MSG, 0.3 M IMP, and 0.3 M NaCI. Since the mixture of MSG and IMP did not show any good responses, we could not examine the mixture

156

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Fig. 8. The whole chorda tympani responses to MSG, IMP, and the mixture containing MSG and IMP at various proportions. Total concentration of the mixture was set at 0.1, 0.3 and 1% in graphs A, B and C, respectively. Each value is shown as amean relative magnitude of response (n = 4), when the response to 0.03 M NaC1 is taken as 100. The magnitudes of response at the initial peak are shown in the upper graphs (a), and those at the steady state (15 s after onset of stimulation) are shown in the lower graphs (b). Responses to the mixture are shown by filled triangles, and responses to each component (MSG and IMP) are shown by Solid circles and open circles, respectively.

effect of MSG and IMP for the glossopharyngeal nerve responses. Single-fiber responses of the chorda tympani. A total of 25 single fibers, which responded to one or more of the taste stimuli used, were sampled from the chorda tympani. To examine similarity or dissimilarity of the tastes between umami substances and the 4 basic taste stimuli, response patterns across the 25 fibers were examined. When the fibers are arbitrarily arranged from left to right according to their response magnitude to sucrose, and from left to right according to their response magnitude to NaCI, it is clearly shown that the across-fiber patterns are distinct between sucrose and NaCI (Fig. 10). HCI and quinine tended to show large responses in fibers arranged in the middle. These response patterns are very similar with those reported in hamsters by other workers 45"56. The across-fiber patterns for MSG,

IMP and the mixture seem to be similar to those for both sucrose and NaC1. The across-fiber patterns can be quantitatively measured by calculating the correlation coefficients between pairs of responses among taste stimuli. The results are shown as correlation taste profiles in Fig. 11, Responses of all the 3 umami stimuli, especially MSG and the mixture, were highly correlated with NaCI response, but poorly with other taste stimuli. The lower correlation between IMP and NaC1 than between MSG and NaCI may be attributed to the very low concentration (0.2%) of IMP. The mixture effects were examined in single,fiber responses of the hamster chorda tympani. When the 25 fibers were classified into 3 groups according to which taste stimuli the fibers showed the greatest response, 9, 8 and 8 fibers belonged to sucrose-best, NaCl-best and H a - b e s t groups, respectively. The

157

0.1M Sucrose

0.3M Sucrose

1M Sucrose

0.1M NaCI

0.3M NaCI

1M NaCI

0.003M HCI

0.01M HCI

0.03M HCI

0.01M Quinine

0.02M Quinine

0.05M Quinine

0.3M MSG

0.3M IMP

0.15M MSG + 0.15M IMP

Fig. 9. The whole glossopharyngeal nerve responses to 3 concentrations of the 4 basic taste stimuli and umami substances. Stimuli were applied during the periods indicated by solid lines for 30 s.

mean magnitudes of taste responses in each group are shown in Fig. 12. The mean magnitude of response to the mixture was very close to the sum of the mean response magnitudes to the components, MSG and IMP, indicating essentially no synergism. The ratios of responses to the mixture to the sum of the responses to the components (MSG and IMP) were 1.09, 0.85 and 1.08 for sucrose-best, NaCl-best and HCl-best groups, respectively. Although the ratios varied from fiber to fiber, the mean ratios again suggested little synergistic mixture effects in the chorda tympani fibers: 1.07 + 0.98 (mean + S.D.) ranging from 0.10 to 3.41, 0.83 + 0.35 ranging from 0.44 to 1.48 and 1.00 + 0.61 ranging from 0.31 to 2.13, in sucrose-best, NaCl-best and HCl-best groups, respectively. GENERAL DISCUSSION This behavioral study on golden hamsters, using

the conditioned taste aversion technique, does not support the suggested notion 15,39,59,75,76,86 that the taste of umami substances is independent of the classical 4 basic tastes, i.e. the hamster could not discriminate the umami substances (MSG, IMP or their mixture) from the sodium salts including NaCI, sodium saccharin, Na2SO4, NaNO3, and CH3COONa. There are similar results in the Wistar rat 84, and the mouse of ddy strain 26 and BALB/cCrslc strain (Ninomiya, personal communication). Such results may be attributed to taste effects of Na ions commonly included in these chemicals, and may explain the fact that more than half of the Western subjects labeled 'salty' for the taste of MSG 17,5°. The chorda tympani, which innervates the taste buds in the anterior part of the tongue, is very sensitive to electrolytes, especially to Na salts in rats 7,s and hamsters 8,24. Such a strong taste effect of Na ions may overcome the existing taste effects of anions. The stimulating effectiveness of anions in various Na

158 MSG

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MPG and MCG after aversion was conditioned to MSG means that hamsters discriminated these chemicals based on the taste effects of cations (NH4, K, Ca and Na ions). If the taste effectiveness of anions increases even under strong cation influence, such stimuli will elicit taste effects that can be reflected in the conditioned taste aversion learning. Actually, Kasahara and Iwasaki z6 observed that the ddy mouse exhibited a very high suppression score (about 90%) to NaCI after

+ IMP

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Fig. 10. Response profiles of 25 chorda tympani fibers (from A to Y) of hamsters for 7 kinds of taste stimuli. Stimuli are, from the top, 0.1 M sucrose, 0.03 M NaCI, 0.003 M HCI, 0.001 M quinine hydrochloride, 0.8% MSG, 0.2% IMP, and a mixture containing 0.8% MSG and 0.2% IMP. About half of the fibers were arranged in the order of magnitude of response to sucrose from the left, and the remaining half in the order of responsiveness to NaC1 from the right.

salts in the rat, as expressed by the response magnitude in the chorda tympani 23, is in the order of CI > NO 3 _-> saccharin >_--acetate > glutamate, and in the glossopharyngeal nerve 44, saccharin > CI > NO 3 > acetate = glutamate. If these sequences are also true in the hamster, glutamate is one of the least effective anions in both taste nerves. The present finding that suppression of licking did not generalize to MAG,

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159 conditioning to MSG as in the present study, but after conditioning to a mixture of MSG and GMP, the mouse showed moderate suppression ratios (about 50%) to both NaCI and sucrose. Their results indicate that anion effects increased due to the synergistic action of the mixture. According to Ninomiya (personal communication), when the higher concentration (0.3 M) of MSG was used as a conditioned stimulus instead of 0.03 M MSG, the generalization of aversion to NaCI became much larger in SIc:ICR mice. In a human psychophysical study, Yoshida and Saito 86 reported that the taste of MSG approached to the salty taste of NaC1 as the concentration increased. The animals may learn the tastes of electrolytes based on a proportion of strength of stimulating effectiveness between cations and anions in the conditioned taste aversion paradigm. The present finding that the hamster could not discriminate umami substances from NaC1 after conditioning to the mixture or after denervation of the taste nerves suggests that the anion (glutamate) effect is very weak compared to the cation effects in this species. In contrast to synergism well documented in rats 52-55,57,85, synergistic taste responses of the mixture of MSG and IMP could not be shown in hamsters. Sato et al. 56, however, reported some synergistic effects in hamsters by showing that the integrated chorda tympani whole-nerve response to a mixture of 0.27% MSG + 0.03% IMP was 1.4 times larger than that to 0.3% MSG alone, and that to 0.9% MSG + 0.1% IMP was 1.2 times larger than that to 1% MSG. These values, which were obtained from only one experimental animal, are smaller than the comparable values (1.91 and 1.63 times, mean of the 3 experiments) similarly obtained in rats 54. They also analyzed single-unit responses of the hamster chorda tympani, i.e. the mean response to a mixture of 0.27% MSG + 0.03% IMP was 1.54 times larger than that to 0.3% MSG in 12 single fibers. This value is again much smaller than the comparable value (4.80, n = 10) obtained in rats 55. Thus, it is evident that the synergism they showed is much smaller, if at all, in hamsters than in rats. The discrepancy between our resul~ts and theirs may be derived from the difference in calculation for possible synergistic effect, i.e. they compared the mixture response with MSG response only while we compared it with the sum of the responses of individual mixture compo-

nents (MSG and IMP). Our treatment seems to be more reasonable considering the general definition of synergism that the taste response to a mixture is larger than the sum of the taste responses of each component contained in the mixture. Ninomiya and Funakoshi 39, using the conditioned taste aversion paradigm in the SIc:ICR mouse with the chorda tympani or glossopharyngeal nerves sectioned and electrophysiological recordings from each of the taste nerves, suggested that the chorda tympani conveyed the taste of the Na component, and the glossopharyngeal nerves, the taste of the umami component. Since stimulation of the anterior part of the human tongue with MSG did not induce umami, Halpern 17 also suggested a dichotomy of the taste of umami substances, i.e. anterior tongue induces some combinations of 4 basic type tastes, while the posterior part of the tongue may elicit umami. In the present study, aversive conditioning to MSG after denervation of the glossopharyngeal nerve generalized to NaC1 just like in the normal animals, confirming the importance of chorda tympani in transmitting the Na component. However, if the chorda tympani was cut bilaterally, we could not establish the conditioned taste aversion to MSG. Considering this fact together with the poor responsiveness to taste stimuli and the lack of synergism in the glossopharyngeal nerve, the glossopharyngeal responses to MSG may be negligibly small in the hamster. Responses of the glossopharyngeal nerve to taste stimuli applied to the circumvallate and foliate papillae in the mouse 61'62, hamster 18, rat 44,71,81, rabbit 72 and cat 72 are generally characterized by high thresholds, a gradual increase in response magnitudes, and larger responses to HC1 and quinine than in the chorda tympani. We used, therefore, high concentrations of taste stimuli in the chorda tympani-deafferented animals. After conditioning to 0.3 M MSG, the suppression of intake generalized again to sodium salts, although the rate of suppression was as low as 50%. These results suggest that the receptors for umami substances are lacking or very scanty, if at all, in the hamster. Taste effects of NaC1, MSG and IMP, however, were not exactly identical in hamsters. The differences were noted in the following items. (1) Learned aversion to IMP generalized to Gly and Ala besides MSG and NaC1, while aversions to NaC1 or MSG generalized to only NaC1, MSG and IMP (see Fig. 1).

F

160 Ala and Gin weakly generalized to MSG, and Pro and Gly generalized to IMP, but no taste stimuli, except MSG and IMP, generalized to NaCI. (2) Singlefiber analyses showed that MSG and IMP induced good responses in some sucrose-best fibers as well as in NaCl-best fibers. However, a lack of behavioral similarity between sucrose and umami substances, and a low correlation of neural responses between these two kinds of chemicals indicate that umami substances might stimulate other sweetener-sensitive receptor sites than the sucrose receptor. (3) IMP showed a slower recovery to the background level after water rinsing than MSG and NaC1, accompanying an occasional off-type response. Similar slow recovery for MSG as well as IMP was reported in some cortical taste responsive neurons in the hamster 82. This result indicates that umami substances interact with the taste receptor cells more firmly than NaC1 does. If a specific receptor protein is postulated for umami substances to bind as suggested to be for some amino acids including glutamate in carp olfactory cells 49 and bacteria (Escerichia coli) 5, it may be coneluded that the hamster has only a scanty of receptor sites for umami substances, i.e. taste effects of anions are not strong enough to overcome the taste effect of cations (Na, K, N H 4 or Ca ions) contained in umami substances. The lack of evidence for separation of

MSG and IMP from the classical taste tetrahedron, and the lack of a synergistic mixture effect may be disappointing to those w h o believe the uniqueness of umami substances in different species of animals. However, species differences of taste sensitivity are not surprising considering the well-known facts that the cat lacks sweet taste responses s'13, and the sweeteners such as monellin, thaumatin, aspartame, and stevioside are effective in monkeys, but not in rodents, dogs and pigs 11,2°-22'58. Inter-strain differences of taste sensitivity in mice are also well documented 4°-42'61. According to Ninomiya (personal communication), independent responsiveness to umami substances from the conventional 4 basic tastes can be demonstrated invariably by C57BL/6CrSIc strain, but not by all the mice of I C R and C3H/HeSIc stains, and cannot be shown in the BALB/cCrSlc strain.

REFERENCES

7 Beidler, L.M., Properties of chemoreceptors of tongue of rat, J. Neurophysiol., 16 (t953) 595-607. 8 Beidler, L.M., Fishman, I.Y. and Hardiman, C.W., Species differences in taste responses, Am. J. Physiol., 181 (1955) 235-239. 9 Birch, G.G., Structure, chirality, and solution properties of glutamates in relation to taste. In Y. Kawamura and M.R. Kare (Eds.), Umami: A Basic Taste, Dekker, New York, 1987, pp. 173-184. 10 Boudreau, J.C., Mammalian neural taste responses to amino acids and nucleotides. In Y. Kawamura and M.R. Kare (Eds.), Umami: A Basic Taste, Dekker, New York, 1987, pp. 201-217. 11 Brotrwer, J.N., Hellekant, G., Kasahara, Y., van der Wel, H. and Zotterman, Y.. Electrophysiological study of the gustatory effects of the sweet proteins monellin and thaumatin in monkey, guinea pig and rat, Acta Physiol. Scand,, 89 (1973) 550-557. 12 Cagan, R.H., Allosteric regulation of glutamate taste receptor function. In Y. Kawamura and M.R. Fore (Eds.), Umami: A Basic Taste, Dekker, New York, 1987, pp. 155-172. 13 Carpenter, G.A., Species differences in taste preferences,

1 Adachi, A., Study on the taste mechanisms of Na-glutamate and Na-inosinate, J. Physiol. Soc. Jpn., 24 (1962) 607-613 (in Japanese with English summary). 2 Adachi, A., Neurophysiological study on taste effectiveness of seasoning, J. Physiol. Soc. Jpn., 26 (1964) 347-335 (in Japanese with English summary). 3 Adachi, A., Kawamura, Y., Ohara, M. and Ikeda, S., Neurophysiological studies on taste effectiveness of sodium 5'-guanylate as a chemical taste enhancer, Amino Acid Nucleic Acid, 12 (1965) 63-68 (in Japanese with English summary). 4 Adachi, A., Okamoto, J., Hamada, T. and Kawamura, Y., Taste effectiveness of mixtures of sodium 5'-inosinate and various amino acids, J. Physiol. Soc. Jpn., 29 (1967) 65-71 (in Japanese with English summary). 5 Adler, J., Chemoreceptors in bacteria, Science, 166 (1969) 1588-1597. 6 Bartoshuk, L.M., Cain, W.S., Cleveland, T.T., Grossman, L.S., Marks, L.E., Stevens, J.C. and Stoiwijk, J.A.J., Saltiness of monosodium glutamate and sodium intake, J. Am. Med. Ass., 230 (1974) 670.

ACKNOWLEDGEMENTS The Ajinomoto Company, supplied M A G , M C G , MPG, MSG, IMP and GMP. This study was supported by Grants-in-Aid for Special Project Research (61134026) and for Scientific Research (61570822) from the Ministry of Education, Science, and Culture of Japan.

161

J. Comp. Physiol. Psychol., 49 (1956) 139-144. 14 Erickson, R.P., Sensory neural patterns and gustation. In Y. Zotterman (Ed.), Olfaction and Taste L Pergamon, Oxford, 1963, pp. 205-213. 15 Frank, M.E., Sensory physiology of taste and smell discriminations using conditioned food aversion methodology, Ann. N. Y. Acad. Sci., 443 (1985) 89-99. 16 Faurion, A., MSG as one of the sensitivities within a continuous taste space: Electrophysiological and psychophysical studies. In Y. Kawamura and M.R. Kare (Eds.), Umami, A Basic Taste, Dekker, New York, 1987, pp. 387-408. 17 Halpern, B.P., Human judgements of MSG taste: quality and reaction times. In Y. Kawamura and M.R. Kare (Eds.), Umami: A Basic Taste, Dekker, New York, 1987, pp. 327-354. 18 Hanamori, T., Miller, I.J. and Smith, D.V., Taste responsiveness of hamster glossopharyngeal nerve fibers. In S. Roper (Ed.), Abstracts of International Symposium on Olfaction and Taste IX and Association for Chemoreception Sciences, 1986, p. 66. 19 Hanson, H.L., Brushway, M.J. and Lineweaver, H., Monosodium glutamate studies. I. Factors affecting detection of and preference for added glutamate in foods, Food Technol., 14 (1960) 320-327. 20 Hellekant, G., On the gustatory effects of monellin and thaumatin in dog, hamster, pig and rabbit, Chem. Senses Favor, 2 (1976) 97-105. 21 Hellekant, G., Glaser, D., Brouwer, J.N. and van der Wel, H., Gustatory effects of miraculin, monellin and thaumatin in the Saguinus midas tamarin monkey studied with electrophysiological and behavioral techniques, Acta Physiol. Scand., 97 (1976) 241-250. 22 Heilekant, G., Glaser, D., Brouwer, J.N. and van der Wel, H., Gustatory responses in three prosimian and two simian primate species ( Tupaia glis, Nycticebus coucang, Galago senegalensis, Callithrix jacchus jacchus and Saguinus midas niger) to six sweeteners and miraculin and their phylogenetic implications, Chem. Senses, 6 (1981) 165-173. 23 Hiji, Y., Preference for and neural gustatory response to sodium salts in rats, Kumamoto Med. J., 20 (1967) 129-138. 24 Hyman, A.M. and Frank, M., Effects of binary taste stimuli on the neural activity of the hamster chorda tympani, J. Gen. Physiol., 76 (1980) 125-142. 25 Ikeda, K., On a new seasoning, 3". Tokyo Chem. Soc., 30 (1909) 820-836 (in Japanese). 26 Kasahara, T. and lwasaki, K., Preferences for some D- and L-amino acids in mice. In T. Shibuya and S. Saito (Eds.),

Proceedings of the 20th Japanese Symposium on Taste and Smell, 1986, pp. 207-210. 27 Kawamura, Y. and Adachi, A., Single taste nerve responses to the chemical taste enhancers, J. Physiol. Soc. Jpn., 27 (1965) 279-284 (in Japanese with English summary). 28 Kirimura, J., Shimizu, A., Kimizuka, A., Ninomiya, T. and Katsuya, N., The contribution of peptides and amino acids to the taste of foodstuffs, J. Agric. Food Chem., 17 (1969) 689-695. 29 Kodama, S., On an isolation method of inosinic acid, J. Tokyo Chem. Soc., 34 (1913) 751-757 (in Japanese). 30 Kuninaka, A., Studies on taste of ribonucleic acid derivatives, J. Agric. Chem. Soc. Jpn., 34 (1960) 489-492 (in Japanese). 31 Kuninaka, A., Kibi, M. and Sakaguchi, K., History and development of flavor nucleotides, Food Technol., 18 (1964)

287-293. 32 Kurihara, Y., Antisweet activity of gymnemic acid A and its derivatives, Life Sci., 8 (1966) 537-543. 33 Loekhart, E.E. and Gainer, J.M., Effect of monosodium glutamate on taste of pure sucrose and sodium chloride, FoodRes., 15 (1950) 459-464. 34 Maga, J.A., Flavor potentiators, CRC Crit. Rev. Food Sci. Nutr., 18 (1983) 231-312. 35 Matsuo, R., Yamamoto, T., Kiyomitsu, Y. and Kitamura, R., Taste effectiveness of umami substances in hamsters. II. Electrophysiological study. In T. Shibuya and S. Saito (Eds.), Proceedings of the 20th Japanese Symposium on Taste and Smell, 1986, pp. 89-92. 36 Mehren, K.J. and Church, D.C., Influence of taste-modifiers on taste responses of pygmy goats, Anirn. Prod., 22 (1976) 255-260. 37 Mundl, W.J. and Malmo, H.P., Capacitive sensor for lickby-lick recording of drinking, Physiol. Behav, 21 (1979) 781-784. 38 Nachman, M., Learned aversion to the taste of lithium chloride and generalization to other salts, J. Comp. Physiol. Psychol., 56 (1963) 343-349. 39 Ninomiya, Y. and Funakoshi, M., Qualitative discrimination among 'umami' and the four basic taste substances in mice. In Y. Kawamura and M.R. Kare (Eds.), Umami: A Basic Taste, Dekker, New York, 1987, pp. 365-385. 40 Ninomiya, Y., Tonosaki, K. and Funakoshi, M., Gustatory neural responses in the mouse, Brain Research, 244 (1982) 370-373. 41 Ninomiya, Y., Higashi, T., Katsukawa, H., Mizukoshi, T. and Funakoshi, M., Qualitative discrimination of gustatory stimuli in three different strains of mice, Brain Research, 322 (1984) 83-92. 42 Ninomiya, Y., Mizukoshi, T., Higashi, T., Katsukawa, H. and Funakoshi, M., Gustatory neural responses in three different strains of mice, Brain Research, 302 (1984) 305-314. 43 Nowlis, G.H. and Frank, M., Qualities in hamster taste: Behavioral and neural evidence. In J. Le Magnen and P. MacLeod (Eds.), Olfaction and Taste VI, Information Retrieval, London, 1977, pp. 241-248. 44 Ogawa, H., Taste response characteristics in the glossopharyngeal nerve of the rat, Kumamoto Med. J., 25 (1972) 137-147. 45 Ogawa, H., Sato, M. and Yamashita, S., Multiple sensitivity of chorda tympani fibres of the rat and hamster to gustatory and thermal stimuli, J. Physiol. (Lond.), 199 (1968) 223-240. 46 Ohara, I. and Naim, M., Effects of monosodium glutamate on eating and drinking behavior in rats, Physiol. Behav., 19 (1977) 627-634. 47 Ohara, I., Otsuka, S. and Yugari, Y., The influence of cartier of gustatory stimulation on the cephalic phase of canine pancreatic secretion, J. Nutr., 109 (1979) 2098-2105. 48 Ohara, I., Tanaka, Y. and Otsuka, S., Discrimination of monosodium glutamate and sodium chloride solutions by rats, Physiol. Behav., 22 (1979) 877-882. 49 Ohno, T., Yoshii, K. and Kurihara, K., Multiple receptor types for amino acids in the carp olfactory cells revealed by quantitative cross-adaptation method, Brain Research, 310 (1984) 13-21. 50 O'Mahony, M. and Ishii, R., The umami taste concept: Implications for the dogma of four basic tastes. In Y. Kawamura and M.R. Kare (Eds.), Umami: A Basic Taste, Dekker, New York, 1987, pp. 75-93.

162 51 Pritchard, T.C. and Scott, T.R., Amino acids as taste stimuli. II. Quality coding, Brain Research, 253 (1982) 93-104. 52 Sato, M. and Akaike, N., 5'-Ribonucleotides as gustatory stimuli in rats. Electrophysiological studies, Jpn. J. Physiol., 15 (1965) 53-70. 53 Sato, M. and Yamashita, S., 5'-Ribonucleotides as gustatory stimuli in rats. Effects of temperature, Jpn. J. Physiol., 15 (1965) 570-578. 54 Sato, M., Akaike, N. and Yamashita, S., Electrophysiological studies on the effects of ribonucleotides on taste receptors of rats. Effects of addition of ribonucleotides to MSG and of temperature, Amino Acid Nucleic Acid, 11 (1965) 53-61 (in Japanese with English summary). 55 Sato, M., Yamashita, S. and Ogawa, H., Electrophysiological studies on the effects of ribonucleotides on taste receptors of rats. Analysis of single unit response, Amino Acid Nucleic Acid, 13 (1966) 62-69 (in Japanese with English summary). 56 Sato, M., Yamashita, S. and Ogawa, H., Synergistic effect of 5'-ribonucleotides on the response to monosodium glutamate in taste receptors of hamster, Amino Acid Nucleic Acid, 15 (1967) 59-63 (in Japanese with English summary). 57 Sato, M., Yamashita, S. and Ogawa, H., Potentiation of gustatory response to monosodium glutamate in rat chorda tympani fibers by addition of 5'-ribonucleotides, Jpn. J. Physiol., 20 (1970) 444-464. 58 Sato, M., Hiji, Y., Ito, M., Imoto, T. and Saku, C, Properties of sweet taste receptors in macaque monkeys. In Y. Katsuki, M. Sato, S.F. Takagi and Y. Oomura (Eds.), Food Intake and Chemical Senses, University of Tokyo Press, Tokyo, 1977, pp. 187-199. 59 Schiffman, S.S., Mcelroy, A.E. and Erickson, R.P., The range of taste quality of sodium salts, Physiol. Behav., 24 (1980) 217-224. 60 Schiffman, S.S., Sennewald, K. and Gagnon, J., Comparison of taste qualities and thresholds of D- and L-amino acids, Physiol. Behav., 27 (1981) 51-59. 61 Shingai, T. and Beidler, L.M., Interstrain differences in bitter taste responses in mice, Chem. Senses, 10 (1985) 51-55. 62 Shingai, T. and Beidler, L.M., Response characteristics of three taste nerves in mice, Brain Research, 335 (1985) 245-249. 63 Sj6rstr6m, L.B. and Crocker, E.C., The role of monosodium glutamate in the seasoning of certain vegetables, Food Technol., 2 (1948) 317-321. 64 Smith, D.V., Travers, J.B. and Van Buskirk, R.L., Brainstem correlates of gustatory similarity in the hamster, Brain Res. Bull., 4 (1979) 359-372. 65 Steiner, J.E., What the neonate can tell us about umami. In Y. Kawamura and M.R. Kare (Eds.), Umami: A Basic Taste, Dekker, New York, 1987, pp. 41-73. 66 Takasaki, Y. and Torii, K., Effects of water restriction on the development of hypothalamic lesions in weanling rodents given MSG. II. Drinking behaviour and physiological parameters in rats (Rattus norvegicus) and golden hamsters (Mesocricetus auratus), ToxicoL Lett., 16 (1983) 195-210. 67 Tapper, D.N. and Halpern, B.P., Taste stimuli: a behavioral categorization, Science, 161 (1968) 708-710. 68 Torii, K. and Cagan, R.H., Biochemical studies of taste sensation. IX. Enhancement of L-[3H]glutamate binding to bovine taste papillae by 5'-ribonucleotides, Biochim. Biophys. Acta., 627 (1980) 313-323. 69 Torii, K. and Takasaki, Y., Effect of water restriction on the development of hypothalamic lesions in weanling rodents given MSG. I. Drinking behaviour and physiological

parameters in mice (Mus musculus), Toxicol. Left., 16 (1~983) 175-194. 70 Waldern, D.E. and Van Dyk, R.D., Effect of monosodium glutamate in starter rations on feed consumption and performance of early weaned calves, J. Dairy Sci., 54 (1971) 262-265. 71 Yamada, K., Gustatory and thermal responses in the glossopharyngeal nerve of the rat, Jpn. J. Physiol., 16 (1966) 599-611. 72 Yamada, K., Gustatory and thermal responses in the glossopharyngeal nerve of the rabbit and cat, Jpn. J. Physiol., 17 (1967) 94-110. 73 Yamaguchi, S., The synergistic taste effect of monosodium glutamate and disodium 5'-inosinate, J. Food Sci~, 32 (1967) 473-478. 74 Yamaguchi, S., The umami taste. In J.C. Boudreau (Ed,), Food Taste Chemistry, American Chemical Society, Washington, DC, 1979, lap. 33-51. 75 Yamaguchi, S., Fundamental properties of umami in human taste sensation. In Y. Kawamura and M.R. Kate (Eds.), Umami: A Basic Taste, Dekker, New York, 1987, pp. 41-73. 76 Yamaguchi, S. and Kimizuka, A., Psychometric studies on the taste of monosodium glutamate. In L.J. Filler, S. Garattini, M.R. Kare, W.A. Reynolds and R.J. Wurtman (Eds.), Glutamic Acid: Advances in Biochemistry and Physiology, Raven, New York, 1979, pp. 35-54. 77 Yamaguchi, S., Yoshikawa, T., Ikeda, S. and Ninomiya, T., Synergistic taste effects of some new ribonucleotide derivatives, Agric. Biol. Chem., 32 (1968) 797-802. 78 Yamaguchi, S., Yoshikawa, T., Ikeda, S. and Ninomiya, T., The synergistic taste effect of monosodium glutamate and disodium 5'-guanylate, J. Agric. Chem. Soc. Jpn., 42 (1968) 378-381 (in Japanese with English summary), 79 Yamaguchi, S., Yoshikawa, T., Ikeda, S. and Ninomiya, T., Measurement of the relative taste intensity of some L-aamino acids and 5'-nucleotides, J. Food Sci., 36 (1971) 846-849. 80 Yamamoto, T. and Kawamura, Y., A model of neural code for taste quality, Physiol. Behav., 9 (1972) 559-563. 81 Yamamoto, T. and Kawamura, Y., Dual innervation of the foliate papillae of the rat: an electrophysiological study, Chem. Senses Flavor, 1 (1975) 241-244. 82 Yamamoto, T., Asai, K. and Kawamura, Y., Studies on responses of cortical taste neurons to umami substances. In Y. Kawamura and M.R. Kare (Eds.), Umami: A Basic Taste, Dekker, New York, 1987, pp. 441-460. 83 Yamamoto, T., Matsuo, R., Asai, K., Kiyomitsu, Y. and Kitamura, R., Taste effectiveness of umami substances in hamsters. I. Behavioral study. In T. Shibuya and S. Saito (Eds.), Proceedings of the 20th Japanese Symposium on Taste and Smell, 1986, pp. 121-124. 84 Yamamoto, T., Yuyama, N., Kato, Y. and Kawamura, Y., Gustatory responses of cortical neurons in rats. III. Neural and behavioral measures compared, J. Neurophysiol., 53 (1985) 1370-1386. 85 Yamashita, S., Ogawa, H. and Sato, M., The enhancing action of 5'-ribonucleotide on rat gustatory nerve fiber response to monosodium glutamate, Jpn. J. Physiol., 23 (1973) 59-68. 86 Yoshida, M. and Saito, S., Multidimensional scaling of the taste of amino acids, Jpn. Psychol. Res., 11 (1969) 149-166. 87 Yoshii, K., Yokouchi, C. and Kurihara, K., Synergistic effects of 5'-nucleotides on rat taste responses to various amino acids, Brain Research, 367 (1986) 45-51.