C’omp. Bmchem. Physiol. Vol. 13A, No. 3, pp. 361 to 372, 1982 0300-9629/82/110361-12$03.Of)/O

Printed in Great Britain. 0 1982 Pergamon Press Ltd

THE ADAPTATION OF THE FROG TONGUE TO VARIOUS TASTE SOLUTIONS: THE

EFFECT ON GUSTATORY NEURAL RESPONSES TO BITTER STIMULI

KUMIKO SUGIMOTO’* and TOSHIHIDE SATO’

~Department of Physiology, Faculty of Dentistry, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113, Japan and

*Department of Physiology, Nagasaki University School of Dentistry, 7-l Sakamoto-machi, Nagasaki 852, Japan

(Recei~eii 24 February 1982)

Abstract-l. After the frog tongue was adapted for 10 set to various salts and sugars, the initial phasic component of gustatory neural responses to almost all of quinine hydrochloride (Q-HCI), quinine sulfate (Q-H,SO,), brucine, caffeine and picric acid was suppressed.

2. Following 10sec adaptation to acetic acid, the phasic responses to Q-HCI and Q-H,SO, were unchanged, those to brucine and caffeine were enhanced, and that to picric acid was depressed slightly.

3. The response to any one of Q-HCI, Q-H,SO,, brucine and caffeine was suppressed after adaptation to the other three, while those to picric acid and nicotine were unchanged or enhanced after adaptation to another bitter solution.

INTRODUCTION

It is often experienced in our daily life that the subjec- tive taste intensity and quality in response to a taste solution is affected by another taste stimulus to which the tongue has adapted beforehand. Such a phenom- enon has been extensively dealt with in many psycho- physical studies on humans (Dallenbach & Dallen- bath, 1943; Meiselman, 1968; Smith & McBurney, 1969; McBurney, 1969, 1972; McBurney et al., 1972; Mc~urney & Bartoshuk, 1973). In el~ctrophysiologi- cal studies on cats (Wang & Bernard, 1969; I-Iellek- ant, 1969; Bernard, 1972), hamsters (Yinon & Erick- son, 1970) and rats (Smith & Frank, 1972), it has been revealed that gustatory neural activities in response to a taste stimulus are modified after the animal tongue is adapted to various taste solutions.

We have shown that following adaptation of the frog tongue to a bitter solution, such as Q-HCI, the initial phasic gustatory nerve responses to salt, sugar and acid stimuli are potentiated remarkably, but the responses to many bitter stimuli are suppressed (Sato & Sugimoto, 1979; Sugimoto & Sato, 1981). In con- trast to our previous studies, the purpose of the present experiments is to investigate the effect of briefly adapting the frog tongue to various taste sol- utions on the gustatory neural responses to bitter- tasting stimuli, and to clarify the mechanisms under- lying the alteration of bitter responses after chemical adaptation. A brief report of a preliminary experiment has appeared elsewhere (Sugimoto & Sato, 1978).

MATERIALS AND METHODS

Sixty-seven bullfrogs (Rana cutesheiana) weighing 1 IO-330 g were used for the experiments. The animals were anesthetized with an i.p. injection of 50’;‘, (w/v) urethane-

* To whom correspondence should be addressed

Ringer solution (3 g/kg body wt). All experiments were car- ried out at a room temperature of 2t-25’C. To avoid mus- cular contraction of the tongue the hypoglossal nerves and the geniohyoideus and hyoglossal muscles of both sides were cut. The whole glossopharyngeal nerve of either side was dissected free from the surrounding connective tissue and was cut near the hyoid bone. The cut peripheral end of the whole glossopharyngeal nerve was placed on a silver wire recording electrode in situ, and was soaked in a paraf- fin pool to prevent drying. An indifferent silver wire elec- trode was positioned on the muscle near the maxillary inferior bone. The action potentials of whole glossopharyn- geal nerve were amplified with an r-c coupled amplifier and were recorded on a pen-writing recorder as an integrated response curve through an electrical integrator with a time constant of 0.4 sec. Simultaneously, the amplified nerve ac- tivities were monitored on a cathode-ray oscilloscope and were stored on a tape recorder.

The frog tongue was pulled out almost entirely and pinned on a cork plate placed in an acrylic resin chamber. The taste solutions were flowed over the entire tongue sur- face at a flow rate of 0.78 ml/see using the semi-automati- cally controlled stimulator described previously (Sato. 1972a). Unless stated otherwise, after a 10sec application of adapting solution a test taste solution was flowed over the tongue for 10s~ without interposition of any rinsing solution. Before application of any adapting solution the tongue was always pre-adapted to- a froi Ringer solution, which was composed of 113.7 mM N&I. 1.9 mM KCI. 1.1 mM CaCI,, 2.4 mM NaHCO, and 0.1 mM NaH,PO, (pH approx. 7.5). The tongue was rinsed with the Ringer solution immediately after the cessation of test stimulation. The time intervals between successive application of pairs of adapting and test stimuli were not less than 3 min.

The adapting solutions used were: (1) deionized water, which was made with a reagent-grade water making system (Milli-Q2, Millipore Corpn., MA) and had a specific resist- ance of not less than 10 Ma; (2) salt solutions, such as Ringer, NaCI, KCI. NH,CI, CaCI, and MgCI? solutions; (3) sugar solutions, such as sucrose, fructose and glucose solutions: (4) acid solutions, such as HCI, formic acid, ace- tic acid and oxalic acid solutions; (5) bitter solutions, such

361

362 KUMIKO S~GIM~TO and TOSHIHIVE SATO

as Q-KC?. Q-H2S04. brucine, caffeine. picric acid and nicotine solutions.

The test solutions used were: (1) all the bitter solutions mentioned above; (2) a mixture of Q-HCI and NaCI; (3) a mixture of Q-HCl and sucrose. All the taste solutions were made up with deionized water.

The maximum magnitudes of integrated neural re- sponses generated by a 10 set application of test bitter sol- utions after 10sec adaptation to various taste solutions were compared with the control responses after 10sec water adaptation. The maximum neural responses to all bitter stimuli consisted of an initial phasic response except that to nicotine, which consisted of a late gradually increasing response.

RESULTS

Depression of gustutorj3 nrurcd respomes to Q-HCI

after clduptation to sult solutions

Figure 1 illustrates the integrated gustatory neural responses to 1 mM

to water, Ringer. M NaCl M MgClz 10 sec. As be seen in

to 1 mM Q-HCI Ringer (A2) almost the amplitude as after water whereas the response after

M NaCl and 0.1 MgCI, (C2) much smaller the respective response after

(Bl and The mean responses to mM Q-HCl depressed to degrees after

set adaptation 0.3 M of NaCl KCI and M solutions NH,Cl, CaCI, MgCl* compared the control after water tation (striped while the response was affected after adaptation (Fig.

watw, 1 InM mM Wici

Water, 1 mM Q-HCI

0.3M lmhi NaC@-WC1

#ter, 1 mM Q-HCI

0.1 M 1 mM MgCI$-HCI 10

Fig. I, Change in an initial phasic component (arrow head) of integrated gustatory neural responses to 1 mM Q-HCI after 10 see adaptation of the frog tongue to water and salt solutions. Adaptation solutions: water (Al. Bl, Cl). Ringer (At), 0.3 M NaCl (B2) and 0.1 M MgCl, (C2). In this and Figs 5. 8. 13 and 15, adaptation and test stimuli are given underneath the integrated responses. The Q-HCl response was followed by a large phasic response elicited by a Ringer rinse. These records were obtained from two

preparations.

g 1007 1

4 3 g 50-

H L

a ;

Y$ 0’ _I

5) (11) 14) (4) (3) (4)

i

Adapting solution

Fig. 2. Relative amplitudes of initial phasic neural re- sponses to 1 mM Q-HCI following 10 set adaptation to six kinds of salt solutions. Salt adaptation solutions: Ringer. 0.3 M NaCI, 0.3 M KCI. 0.1 M NH,CI, 0.1 M CaCI, and 0.1 M MgCI,. Each column is the mean + SE expressed as a percentage of the control response to Q-H0 (striped column) after f0sec adaptation to water. The numerals

within parentheses indicate the number of preparations.

Adaptation of the tongue to 0.1 M NaCl and 0.1 M KCI, having poor stimulating effects, hardly affected the responses to 1 mM Q-HCI. The suppression of Q-HCI response after 0.1 M divalent salts tended to be greater than that after 0.1 M monovalent salts.

Figures 3A and 3B compare the relative amplitudes of initial phasic neural responses to various concen- trations of Q-HCI after 10sec adaptation to water and 0.3 M NaCl (A) and water and 0.1 M CaCI, (B). It is seen that the phasic Q-HCI responses after adap- tation to either N&i or CaCl: (0) are smaller over all concentrations than those after the control water adaptation (0). The tonic responses to water and Q-HCI after 0.1 M CaCl, adaptation were potentiated remarkably. The magnitude of the tonic response to Q-HCI after 0.1 M CaClz decreased with an increase in Q-HCI concentration. Since the response to water was potentiated after 0.1 M CaCI, adaptation. the in- crease in response to low Q-HCI concentrations after CaCl, was due to a response to water as the solvent of bitter solutions.

Figures 4A and 4B represent the relationship between the concentrations of NaCl (A) and CaCI, (B) adaptation solutions and the relative amplitudes of initial phasic responses to 1 mM Q-HCI. It can be seen that the Q-HCI response decreased steeply when the concentrations of NaCl and CaCl, were raised above 0.1 M and 0.01 M, respectively.

Depression of’ yustutory rwurrrl rrsponst~s to Q-HCI trftrr aduptotion to stc<lar soiutiorts

Figure 5 illustrates the integrated gustatory neural responses to 1 mM Q-HCI following IO set adap- tation to control water and 0.5 M sugar. After adap- tation to 0.5 M sucrose (A2) and 0.5 M glucose (CZ), the initial phasic responses (arrow heads) to 1 mM Q-HC1 were somewhat smaller than the controls (Al,

Gustatory neural responses to bitter stimuli 363

A

_ 2or A

1 v

Fig. 3. Concentration-neural response curves for test Q-HCl solutions after 10sec adaptation to water (0) and salt solution (0). Salt adaptation solution: 0.3 M NaCl (A) and 0.1 M CaCl, (B). Abscissae: water (W) and molar con- centrations of test Q-HCl solutions. Ordinates: Peak amplitudes of initial phasic responses to Q-HCI in arbi- trary units. (A) and (B) were obtained from different

preparations.

w xi3 16’ 10’ 10 Concentrotlon Of Ndl ct.41

------l w lb4 12 d 16’

Fig. 4. Changes in amplitude of initial phasic neural re- sponses to I mM Q-HCl as a function of concentrations of salt adaptation solutions. Adaptation solution applied for 10sec: NaCl (A) and CaCI, (B). The control Q-HCI re- sponse after 10sec adaptation to water (W) is taken as loo:,,. Each point in (A) and (B) is the mean of two

preparations.

A Water, 1 mM

Q-I-ICI

1 ‘I

‘_h,

Water; 1 mM Q-I-ICI

1 v

k-

Water, 1 mM Q-HCI

0.5M 1mM Sucrose,Q-HCI

Ci5M 1mM Fructose,Q-HCI

0.5 M 1 mM Glucose,Q-HCI 1Osec

Fig. 5. Changes in an initial phasic component (arrow head) of integrated neural responses to 1 mM Q-HCI after 10 set adaptation to water and sugar solutions. Adaptation solutions: water (Al, Bl, Cl); 0.5 M sucrose (A2), 0.5 M fructose (B2) and 0.5 M glucose (CZ). All records were

obtained from one preparation.

Cl), while adaptation to 0.5 M fructose did not affect the Q-HCI response (Bl and B2). Application of 0.5 M glucose itself often elicited a large phasic neural response, but 0.5 M sucrose and 0.5 M fructose gener- ally elicited a small gradually increasing response. The results from this and the other similar experi- ments are summarized in Fig. 6. Following adap- tation to 0.5 M sucrose and 0.5 M glucose the mean amplitudes of the initial phasic response to 1 mM Q-HCl were reduced by 27”,, and 24”;, respectively, as compared with the control response after water

Fig. 6. Histograms showing the mean amplitude of initial phasic responses to 1 mM Q-HCl after 10 set adaptation to sugar solutions. Adaptation solutions: 0.5 M sucrose, 0.5 M glucose and 0.5 M fructose. The response is expressed rela- tive to the control response (striped column) after 10sec adaptation to water. The vertical bar indicates SE of the mean. The numerals in parentheses show the number of

preparations.

364 KUMIKO SUGIMOTO and TOSHIRIDE SATO

A

1 ,* / I

W lo-6 K-7 KP VI3

Concentration of Q-HCI (M)

- I

e 2 2 0 i

-010 W

Concentration of sucrose (MI

Fig. 7. A: Concentration-neural response curves for test Q-HCl after 10 set adaptation to water (0) and 0.5 M su- crose (0). Abscissa: water (W) and molar concentration of Q-Ha. Ordinate: peak amplitude of initial phasic re- sponses to Q-Ha in arbitrary units. Data were obtained from one preparation. B: Relative amplitude of initial pha- sic neural response to 1 mM Q-HCl as a function of con- centration of sucrose adaptation solutions. The response to Q-HCl after adaptation to water (W) is taken as 100%. All adaptation periods were 10 sec. Each point shows the mean

of three preparations.

Wateg 1 mM Q-HCI

C 1 v

Watt, 1 mM 1mM 1mM Water.1 mM 1mM 1mM

Q-HCI Acetic&HCI Q-HCI acid ,

f$c Q-HCL

l-G&

Fig. 8. Changes in an initial phasic component (arrow head) of integrated neural responses to I mM Q-HCl after 1Osec adaptation to water or acid. Adaptation solutions: water (AI-Dl). 1 mM HCI (A2), 1 mM formic acid (B2), 1 mM acetic acid (C2) and 1 mM oxalic acid (D2). In record D2 the magnitude of response to 1 mM oxalic acid was depressed due to repeated applications of other acid solutions. All

records were obtained from one preparation.

1mM 1mM HCI, Q-HCI

(striped column), while following 0.5 M fructose adap- tation the Q-HCI response was not reduced signifi- cantly, The rate of Q-HCl response depression after sugar adaptation was considerably smaller than that after salt adaptation.

In Fig. 7A are shown the initial phasic neural re- sponses to various concentrations of Q-HCI after 10sec adaptation to water (0) and 0.5 M sucrose (e). The responses to Q-HCI concentrations over IO-’ M after 0.5 M sucrose were smaller than those after water. Investigation of the relationship between con- centration of adapting sucrose solution and magni- tude of phasic response to 1 mM Q-HCI revealed that the Q-HCl response was distinctly reduced following 10 set adaptation to sucrose concentrations above 0.3 M (Fig. 7B).

Gustator~~ neurul resporwes to Q-UC1 uJter uduptutio~~ to acid solutioi~s

Figure 8 itlustrates an example of the integrated neural responses to 1 mM Q-HCI after 10 set adap- tation of the tongue to control water (AI-D]) and 1 mM solutions of HCl (A2), formic acid (B2). acetic acid (C2) and oxatic acid (D2). This and other similar experiments showed that the initial phasic re- sponse to 1 mM Q-HCl is little affected by adapting the tongue to either inorganic or organic acids at con- centrations of 1 mM, compared with controls (Fig. 9).

Figure 1OA represents the concentration-neural re- sponse curves of test Q-HCI after 10 set adaptation to water (0) and 1 mM acetic acid (0). There is no sig- nificant difference between the two curves except that the water response is enhanced after acetic acid. Figure 1OB indicates the relationship between concen- tration of acetic acid adaptation solution and magni- tude of the initial phasic response to 1 mM Q-HCI. The amplitude of the Q-HCI response was almost constant, regardless of the concentration of acetic acid adaptation solutions.

Water;1 mM 1mM 1mM Q-HCI ;;,icQ-HCI

D 1 V 2 V

Gustatory neural responses to bitter stimuli 365

I (6) (4) (25) (4

Adapting SOlUtiOn

Fig. 9. Relative amplitude of phasic neural responses to I mM Q-HCI following 10sec adaptation to four kinds of acid solutions. Adaptation solutions: t mM HCI, 1 mM formic acid, 1 mM acetic acid and 1 mM oxalic acid. The responses are expressed as a percentage of the control re- sponses (striped column) after 1Osec adaptation to water. Each value represents the mean + SE. The number of

preparations used is shown in the parentheses.

The change in phasic neural response to 1 mM Q-HCl was investigated when the duration of adap- tation in 0.3 M NaCl (a), 0.5 M sucrose (A) and I mM acetic acid (0) was prolonged to 20sec (Fig. 1 I). When the adaptation time in 0.3 M NaCl was only 1 sec. the Q-HCI response was considerably depressed, and the depression was then maintained at an almost constant level up to 20sec of adaptation. With a 3 set adaptation period in 0.5 M sucrose the depression of Q-HCI response reached the maximum, the level remaining constant up to 20sec of adap- tation. In the case of 1 mM acetic acid adaptation, the magnitude of the Q-HCl response was independent of the adapting duration.

There exists the possibility that the depression of the Q-HCI response after salt or sugar adaptation might depend partly upon a momentary mixing of the adaptation and test solutions when the former was switched to the latter. If the depression of the Q-HCl response is due to competitive inhibition at the Q-HCI receptor site between two different molecules in the momentarily mixed solution, the depression of the Q-HCI response after adaptation to salt or sugar should be dependent on the concentration of salt or sugar in the mixture. Mixtures composed of 1 mM Q-HCI and various concentrations of NaCl or su- crose, therefore, were applied to a tongue which had been adapted beforehand to 0.3 M NaCl or 0.5 M su- crose for 1Osec. After adaptation to 0.3 M NaCl the relative amplitudes of the phasic responses to mix-

tures of 1 mM Q-HCl and NaCl below 0.1 M were similar to the amplitude of response to 1 mM Q-Ha alone, but the response to a mixture of 1 mM Q-Ha and 0.3 M NaCl was much larger than the response to 1 mM Q-Ha alone (Fig. 12A). After 0.5 M sucrose adaptation, the phasic responses to mixtures of 1 mM Q-HCI and various concentrations of sucrose showed almost the same amplitude as the response to 1 mM

Q-HCl alone (Fig. 12B). These results indicate that the depression of the Q-HCl response after salt or sugar adaptation may be ascribed not to the momen- tary mixing of the adapting and test solutions but to the process of adaptation itself.

A

After water

OL 1 ;; ’ I 1 J

W lb6 lo” 16’ 103

Concentration of Q-HCI (N)

f

I!

gM f I 6

COnCentrotiOn of acetic acid (M)

Fig. 10. A: Concentration-neural response curves for Q-HCI following 10 set adaptation to water (0) and 1 mM acetic acid (0). Abscissa: water (W) and molar concen- trations of test Q-HCI solutions. Ordinate: peak amplitude of phasic neural response to Q-HCl in arbitrary units. All data were obtained from one preparation. B: Relative amplitude of phasic neural response to 1 mM Q-HCI after 10 set adaptation to various concentrations of acetic acid solutions, The response after 1Osec adaptation to water (W) is taken as 100%. Each point shows the mean value of

4-6 preparations.

KUMIKO SUGIMOTO and TOSHIHIDE SATO

L 1 I 1

0 5 10 is 20 Duration of application of adapting solution (set)

Fig. 11. Change in phasic neural responses to 1 mM Q-HCI as a function of the duration of application of three adap- tation solutions. Adaptation solutions: 1 mM acetic acid (0),0.5 M sucrose (A) and 0.3 M NaCl (0). The response to Q-HCI after pre-adaptation to Ringer is taken as lOOn,,.

Each curve was obtained from a different preparation.

Changes in gustatory neural responses to other bitter solutions after adaptation to salt, sugar or acid sol- utions

Figure 13 illustrates examples of the integrated neural responses to 1 mM brucine (A), 10 mM caffeine (B) and 1 mM picric acid (C) following 10 set adap- tation to water (l), 0.3 M NaCl (2), 0.5 M sucrose (3) and 1 mM acetic acid (4). Following 10 set adaptation to 0.3 M NaCl, 0.1 M CaCl,, 0.5 M sucrose or 1 mM acetic acid, the changes in the amplitudes of initial phasic responses to 1 mM of Q-HISO,+, brucine, picric acid and 10 mM caffeine are summarized in Fig. 14. After adaptation to 0.3 M NaCl and 0.5 M sucrose, the phasic responses to 1 mM Q-H2S0,.

150 r

A After 0.3 M NaCl l

E g 50

% i 3

3 OL41 ’ I I

W 0.01 0.1 1.0

Concentration of NaCl in

mixture hl)

1 mM brucine and 10mM caffeine were significantl) suppressed compared with control responses after water adaptation (striped columns). The phasic re- sponses to 1 mM picric acid was enhanced after adap- tation to NaCl and was depressed slightly after adap- tation to sucrose. After 0.1 M CaCl, adaptation, the responses to 1 mM solutions of Q-H2SOL, brucine and picric acid were depressed to varying degrees. whereas the response to 10mM caffeine was hardly affected, After 1 mM acetic acid adaptation, the re- sponse to 1 mM Q-HISO was hardly affected, the responses to 1 mM brucine and 10 mM caffeine were enhanced, and the response to 1 mM picric acid was depressed slightly.

On the other hand, the magnitudes of the tonic responses to all the bitter solutions tested were con- spicuously increased by adapting the tongue for 10 set to 0.1 M CaCl,.

Gustatory neural response to a bitter so/&ion after adaptation to another bitter solution

All fifteen possible pairs of the six bitter solutions, such as 1 mM Q-HCl, Q-H,SO.+, brucine and picric acid and 10mM caffeine and nicotine, were used as adapting and test solutions alternately. In Fig. 15 are shown examples of integrated neural responses to three pairs of bitter solutions (Q-HCI and brucine: Q-HCI and caffeine; Q-HCI and picric acid). The in- itial phasic response (arrow head) to 1 mM Q-HCI was suppressed after 10 set adaptation to 1 mM bru- tine (A2), 10 mM caffeine (B2) and 1 mM picric acid (C2) compared with controls after water adaptation (Al, Bl, Cl). On the other hand, after 10sec adap- tation to 1 mM Q-HCl, the initial phasic responses (arrow heads) to 1 mM brucine (A4) and 10 mM caf-

6 After 0.5M sucrose

100 .-

1

------•l.-.

50-

OL -0.010 W

Concentration of sucrose in

mixture &I)

Fig. 12. A: Change in magnitude of phasic neural responses to mixtures of 1 mM Q-HCI and NaCl of various concentrations following 10 set adaptation to 0.3 M NaC1. Abscissa: molar concentrations of NaCl in the mixtures. Ordinate-amplitude of phasic neural responses to the mixtures. The response to 1 mM 0-HCI alone after adaptation to 0.3 M NaCl is taken as 100X Each point shows the mean value of 3 preparations. B: Neural responses to mixtures of 1 mM Q-HCI and Sucrose of various concen- trations following 10 set adaptation to 0.5 M sucrose. Ordinate: the responses to the mixtures are expressed as a percentage of the response to 1 mM Q-HCl alone after adaptation to 0.5 M sucrose. All

points were obtained from one preparation.

Gustatory neural responses to bitter stimuli 367

A ’

0.3M 1mM 0.5M 1 mM NaCIprucine Sucraseprucine

1mM 1mM Acetic Brucine acid ,

Water, t mM Brucine

4 1 3

I_r_h_ B -A-l_ -Jk?-JL lmbj tOrnM, Acetc Caffeine

4

Water,lOmM Caffeine

0.3M 1OmM NaQCaffeine

0.5M 1OmM Suffose$affeine

3

1mM 1mM Acetic Picric acid ,acid 10

C

0.5M 1mM Sucrosq;cPi$

Water, 1 mM Picric acid

Fig. 13. Integrated neural responses to bitter solutions after IOsec application of four kinds of adap- tation solution. Arrow heads denote an initial phasic response to a bitter stimulus. Adaptation solutions: water (I), 0.3 M NaCl (2), 0.5 M sucrose (3) and 1 mM acetic acid (4). Bitter test solutions: 1 mM brucine (A), IOmM caffeine (B) and 1 mM picric acid (C). Rinsing with Ringer solution after the cessation of bitter stimulation evoked a large phasic response. Because of repeated application of picric acid the response to the acetic acid adaptation solution was reduced in the record C4. All records were obtained

from one preparation.

lOM"f Caffeine

1iilM Picw acid

250 1

Adapting solution

Fig. 14. Mean amplitude of phasic responses to four bitter solutions following 10 set adaptation to various taste solutions. Adaptation solutions: water, 0.3 M NaCI, 0.1 M CaCI,, 0.5 M sucrose and 1 mM acetic acid. Test solutions: 1 mM Q-H2SO0. I mM brucine, lOmM caffeine and 1 mM picric acid. The responses are expressed as a percentage of the control responses (striped columns) after 10 set adap- tation to water. The vertical bars show SE of the mean. The numerals within parentheses indicate the

number of preparations.

KUMIK~ SUGIMOTO and TOSHIHIUE SATO

1 v

AA

Water, 1 mM Q-HCI

B1 .

_Jk Water, 1 mM

Q-HCL

c1 v

_JJL

Water, 1 mM Q-HCI

ImM 1mM Water, 1 mM Brucine,Q-HCI Brucine

2 3

10mM 1mM Caffeine&I-HCI

2

v

hJJL

1mM 1mM Pii: Q-HCI

Water, 10 mM Caffeine

3

Water, 1 rnM Picric acid

4

L v

1mM 1mM Q-HCI,Brucine

1mM 10mM Q-HCI,Caffeine

1mM 1mM Q-HCI,P&ic

10 set

Fig. 15. Change in integrated neural responses to bitter solutions following 10 set adaptation to different bitter solutions. Arrow heads denote initial phasic responses to bitter test stimuli. The phasic responses to bitter test stimuli after 10 set adaptation to water serve as controls (Al, 3; Bl. 3; Cl, 3). Pairs of bitter solutions: 1 mM Q-HCI-1 mM brucine (A). I mM Q-HCI-10 mM caffeine (B) and 1 mM Q-HCl--1 mM picric acid (C). In records C2-C4, the responses to rinsing with Ringer are truncated. All records were

obtained from one preparation.

feine (B4) were suppressed, but the response to 1 mM picric acid (C4) was little affected. The combined results of this and other similar experiments are sum- marized in Fig. 16. As shown in Fig. 16A, when each of the seven pairs of bitter solutions was alternately applied to the tongue as an adaptation and a test

solution, the initial phasic responses elicited by the individual test stimuli of the pair were characterized by a change in the same direction, i.e. the two re- sponses were decreased or increased together. The amplitudes of the phasic responses were both de- creased to varying degrees in the left six pairs of bitter

a Adapting solution

Fig. 16A.

Gustatory neural responses to bitter stimuli 369

I-

I-

I-

)-

I-

(7)

(4) (4) T T T

Fig. t6B.

Fig. 16. Histograms showing the peak amplitude of phasic neural responses to bitter test stimuli after 1Osec alternate adaptation to paired bitter solutions. Bitter stimuli used for adaptation and test sol- utions: 1 mM Q-HCI, 1 mM Q-H,S04, 1 mM brucine. 1 mM picric acid. LOmM caffeine and 1OmM nicotine. The magnitude of response is expressed as a percentage of the respective control responses after 10 set adaptation to water. The numerals within parentheses indicate the number of preparations. The horizontal dashed line separates depression from enhancement. A: Responses to seven pairs of bitter solutions which are characterized by changes in the same direction (decrease-decrease in six pairs and increase-increase in one pair) following 10sec alternate adaptation between paired solutions. B: Re- sponses to eight pairs of bitter solutions which are characterized by changes in different directions (no change-decrease in six pairs, increase-decrease in one pair and no change-increase in one pair) follow-

ing 10 set alternate adaptation between paired solutions.

solutions. while they were both increased in the right- most pair of picric acid and brucine. In Fig. 16B, the initial phasic responses to the two test stimuli in each of eight pairs of bitter solutions were changed in dif- ferent directions. As seen in the Fig., the responses to picric acid and nicotine were unchanged or enhanced after adaptation to almost all the other bitter sol- utions. On the other hand, after adaptation to nico- tine and picric acid the responses to most and some of

other bitter solutions, respectively, were markedly de- pressed.

The rates of depression and enhancement of the initial phasic gustatory nerve responses due to chemi- cal adaptation were calculated from the results with all fifteen pairs of bitter compounds in Fig. 16. These values are given in Table 1. The closer the value approaches to 1.0, the greater the responses to bitter test stimuli were depressed after bitter adaptation.

Table 1. The average depression and enhancement rates of neural response magnitudes elicited by bitter test solutions after alternate adaptation between paired bitter solutions

1 rnM 1mJ-l I.0 llm 1 rnM 10 rnM

Q-H2SO* Brucine Caffeine Picric acid Nicotine

0.96 0.69

0.53

0.51

0.59

0.58

(0.05)

(-0.15)

-0.90

0.13

(-0.38) 1 r@l Q-HCl

(-0.20) 1 RIM Q-HzSOa

(-0.30) 1 mI4 Brucine

(-0.96) 10 mM Caffeine

-0.35 1 mu Picric acid

The positive values mean depression and the negative enhancement. The values without parentheses were obtained by averaging the values for a pair of test stimuli following IOsec bitter adaptation. The values within parentheses were obtained from the responses to picric acid or nicotine only following 10 set bitter adaptation: adaptation of the frog tongue to picric acid and nicotine causes the taste receptor membrane to be irreversibly inactivated.

T.&F 73,3Ai

370 KUMIKO SUGIMOTO and TOSHIHIUE SATO

As we have stated previously (Sugimoto & Sate, 1978), changes of gustatory neural responses to bitter stimuli after prior adaptation of the frog tongue to a variety of taste solutions can be explained by the fol- lowing three principal factors: (i) similarity or dissimi- larity between the receptor site on the taste receptor membrane for an adaptation stimulus and that for a bitter test stimulus (receptor mechanism I); (ii) chemi- cal modification of the receptor site for a bitter stimu- lus by adaptation solutions (receptor mechanism II); (iii) electrical inactivation of the membrane of the im- pulse firing zone in the gustatory nerve fibre terminal during application of adaptation solutions (neural mechanism).

Concerning the first factor (receptor mechanism I), micro-electrode studies indicate that receptor sites in a taste cell for the four basic taste stimuli (NaC1, Q-HCI, HCI and sucrose) may be different from one another, and that compounds representing similar chemical structures may react with the same receptor site (Sato, 1972b; Ozeki & Sato, 1972; Akaike et al.. 1976). In addition, neurophysiological studies on chordu tr~npani responses in rats (Smith & Frank, 1972) show that the qualitative similarity between taste receptor sites for adaptation and test stimuli can be estimated by measuring the reduction rate of test responses following adaptation of the tongue to adap- tation solutions. We have already obtained evidence which supports the second factor (receptor mechan- ism II), e.g. although a 1 mM Q-HCl adaptation sol- ution itself initiates little neural response at the end of a 10 set application, adaptation of the frog tongue to the Q-HCl causes a remarkable enhancement of the initial phasic neural responses to salts, acids and sugars (Sato, 1975; Sat0 & Sugimoto, 1979; Sugimoto & Sato, 1981). These data indicate that the enhance- ment of the test responses is due to a chemical modifi- cation of the receptor sites for the test stimuli by adaptation of the tongue to Q-Ha. The third factor {neural mechanism) derives from the following histo- logical and physiological properties of gustatory nerve fibres. A single gustatory nerve fibre branches at the bases of lingual papillae and taste buds and there- by innervates many taste cells (Rapuzzi & Casella, 1965; Beidler, 1969; Sato, 1972b). The fibre responds to many taste stimuii with different response magni- tudes (Kusano, 1960) and shows about a 3OOmsec depression of excitability, even after the initiation of an impulse in the periphery (Macdonard & Brodwick, 1973). With these three factors (mechanisms) we are able to interpret changes in taste nerve responses after chemical adaptation in the present experiments.

Bitter response efter salt adaptation

The present experiments indicate that an initial phasic component of the gustatory neural response to Q-HCI is reduced to varying degrees after the frog tongue is adapted to both monovalent and divalent salt solutions for 10 set (Figs l-4). Kusano (1960) has observed that single frog gustatory units sensitive to Q-HCI also respond to divalent and/or monovalent salts. Hence. the mechanism underlying a reduction in the Q-HCl response after salt adaptation can be explained mainly by the third factor (neural mechan-

ism), that the excitability of Q-HCl- and salt-sensitive fibres may be depressed during and after application of salt adaptation stimuli. The fact that only 1 set of NaCl adaptation is enough to suppress the Q-HCI response (Fig, 11) also supports the third factor, apart from the problem of the second factor.

Bitter response c$er sugar adaptation

Following 10sec adaptation of the frog tongue to sugar solutions, the initial phasic responses to all bit- ter solutions examined are depressed (Figs 5, 6 & 14). The problem of inactivation of gustatory nerve fibre terminals induced by an adaptation solution seems to be excluded from the mechanism underlying the de- pression of bitter responses after sugars. because sugar stimulation elicits only a poor neural response in most cases. Based on the supposition of proximity (probably within 3-4A) between some of sweet and bitter receptor sites on the taste receptor membrane (Birch & M~lvaganam, 1976), the depression of the bitter response after sugar adaptation might be inter- preted. to a certain degree, by a steric hindrance or conformational change of the bitter receptor site, which may be induced by binding of a sugar adap- tation molecule to its receptor site. This explanation corresponds to the second factor of adaptation mechanisms.

The suppressing effect of 0.5 M fructose adaptation on the Q-HCl response is smaller than that of 0.5 M sucrose or 0.5 M glucose adaptation (Figs 5 & 6). Shi- mada et af. (1972, 1974) found that the labellar sugar receptor of the flesh-fly treated with p-chloromercuri- benzoate hardly responded to glucose and sucrose but responded normally to fructose. They have proposed that there are two kinds of receptor sites for the two groups of sugar stimuli. which are named pyranose sites and furanose sites, respectively. The difference in the depressant effects on the Q-HCI response between sucrose or glucose adaptation and fructose adaptation is likely to be interpreted by supposing two kinds of sugar receptor sites in frog.

Bitter response after ucid adaptation

Following 10sec acid adaptation, the responses to Q-HCl and Q-H2SOd are not affected, the responses to brucine and caffeine are enhanced, and the re- sponse to picric acid is slightly suppressed (Figs 13 & 14). The lack of change in the quinine response after acid adaptation seems to suggest that the receptor site for quinine is independent of that for acid. In con- trast, Hellekant (1969) has reported that the cat ~~~~d~7 t~~~pa~~ response to Q-HCI is depressed after acid adaptation. The two different results are likely to be a difference between the species. The enhancement of brucine and caffeine responses after acid adap- tation might be explained by a facilitation of binding between brucine or caffeine molecules and their recep- tor sites. which may be changed conformationa1ly by acid: namely, enhancement is due to the second factor of adaptation mechanisms. As has been stated else- where (Sugimoto & Sato, 1981), the depression of the picric acid response after acetic acid might be due to a competitive inhibition at the acid receptor site between acid adaptation moiecules and picric acid molecules when they are mixed.

Gustatory neural responses to bitter stimuli 371

Bitter response ufter bitter uduptation

In our previous paper (Sugimoto & Sato, 1981) we have proposed that the rate of reduction of the frog gustatory neural response to a bitter stimulus follow- ing adaptation to Q-HCl may indicate the degree of similarity between the receptor site for Q-HCI and those for bitter substances: thus, it is determined that the receptor sites for Q-H,SO,, brucine and caffeine are, to varying degrees, similar to the receptor site for Q-HCI, while the receptor sites for picric acid and nicotine are dissimilar to that for Q-HCI. With this conception of receptor mechanism I, we are able to explain the results of changes in bitter responses after bitter adaptation (Figs 1.5 & 16 & Table I). That is, the rate of reduction is large when bitter adaptation and test compounds react with similar receptor sites, but it is small when two compounds react with dis- similar receptor sites. The present results also support a similarity between Q-HCl and each of Q-H2S04, brucine and caffeine and a dissimilarity between Q-HCl and each of picric acid and nicotine. Further- more, the similarity is found in any pair of Q-H,SO,+, brucine and caffeine. The dissimilarity is observed in any pair of each of Q-HCI, Q-H2S04, brucine and caffeine and each of picric acid and nicotine, and in the pair of picric acid and nicotine. From the present experiments we can divide bitter receptor sites into three types: (i) the site for Q-HCl, Q-H2S04, brucine and caffeine; (ii) the site for picric acid; (iii) the site for nicotine. This ensures a complexity of receptor mech- anisms responsible for bitter reception, and supports the proposal by McBurney et al. (1972) that the bitter taste in humans is not coded by a single receptor mechanism. Possibly, the enhancement of bitter re- sponse after bitter adaptation is due to a conforma- tional change of the receptor site during adaptation.

SUMMARY

1. An initial phasic component of frog gustatory neural responses to 1 mM Q-HCl was decreased in amplitude after the tongue was adapted for 1Osec to 0.3 M NaCl or KC1 or 0.1 M NH,Cl, CaClz or MgC& compared with that after water adaptation.

2. The initial phasic neural response to 1 mM Q-HCI was suppressed following 10 set adaptation to 0.5 M sucrose or 0.5 M glucose in comparison with that after adaptation to water, while it was not sup- pressed significantly following adaptation to 0.5 M fructose.

3. After a IO set period of adaptation to 1 mM HCI, formic acid, acetic acid or oxalic acid, the initial pha- sic response to 1 mM Q-HC1 was hardly affected.

4. The suppression of the response to 1 mM Q-HCI appeared when the concentration of NaCl, CaCI, and sucrose in the adaptation solution was raised above 0.1 M, 0.01 M or 0.3 M, respectiveIy.

5. The initial phasic responses to almost all other bitter solutions, such as 1 mM of Q-H2S04, brucine or picric acid and 10mM caffeine, was depressed to varying degrees following adaptation of the tongue for 1Osec to 0.3 M NaCl, 0.1 M CaCl, or 0.5 M sucrose.

6. After 10 set adaptation to 1 mM acetic acid, the

phasic response to 1 mM Q-H&SO4 was unchanged, the responses to 1 mM brucine and 10 mM caffeine were enhanced, and the response to 1 mM picric acid was suppressed slightly.

7. The gustatory neural response to any one of 1 mM Q-HCI, 1 mM Q-HaSO+ 1 mM brucine or 1OmM caffeine was markedly reduced following IOsec adaptation to one of the other three, while those to 1 mM pirric acid or 1OmM nicotine were unchanged or enhanced following adaptation to another bitter solution.

Acknawiedgements-We thank Professor M. Ichioka (Department of Physiology, Faculty of Dentistry, Tokyo Medical and Dental University) for his continuous encour- agement during the present experiment. This study was supported in part by a Grant-in-Aid for Scientific Research (No. 157425) from the Ministry of Education, Science and Culture of Japan.

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